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Study of the hard-to-cook property of stored yam tubers (Dioscorea dumetorum) and some determining biochemical factors
Food Research International 38 (2005) 143–149 www.elsevier.com/locate/foodres
Study of the hard-to-cook property of stored yam tubers (Dioscorea dumetorum) and some determining biochemical factors Gabriel Nama Medoua a
a,*
, Israe¨l Lape Mbome a, T. Agbor-Egbe a, C.M.F. Mbofung
b
Centre for Food and Nutrition Research, IMPM, P.O. Box 6163, Yaounde, Cameroon b ENSAI, University of Ngaoundere, P.O. Box 455, Ngaoundere, Cameroon Received 30 August 2004; accepted 20 September 2004
Abstract In an attempt to investigate the hard-to-cook phenomenon in Dioscorea dumetorum tubers during storage, selected physical and chemical characteristics were monitored. A 3 · 5 factorial experiment with storage conditions (15 C, 59% RH; 30 C, 75% RH; 45 C, 86% RH) and storage period (0, 2, 4, 7 and 14 days) as variables was carried out. Changes in cooked hardness, water absorption, water-holding capacity and solid loss of steeped tubers as well as phytate, total phenols and lignin contents were monitored. Water uptake and solid loss of steeped tubers decreased significantly (P 6 0.05) with storage period, suggesting that storage affects cell wall membrane permeability of the tubers. Cooked hardness analyses indicated significant difference (P 6 0.05) between fresh and stored tubers and its rate varied with storage time and storage conditions. Cooked hardness values correlated negatively (r = 0.922–0.857, P 6 0.05, d.f. = 146) with phytate and total phenols. A multiple mechanism for D. dumetorum tubers hardening is presented which includes phytate loss as a minimal contributor to cooked hardness during the first days of storage and total phenol loss via a lignification-like mechanism as a major contributor. 2004 Elsevier Ltd. All rights reserved. Keywords: Dioscorea dumetorum; Hard-to-cook; Phytate; Phenols; Lignification
1. Introduction Dioscorea dumetorum tubers are good sources of protein, carbohydrates and minerals. Their storage under warm, humid conditions encountered in many African countries renders them susceptible to a hardening phenomenon characterized by loss of the ability to soften during cooking. Tuber hardening is a serious handicap and begins within 24 h after harvest (Afoakwa & Sefa-Dedeh, 2002b; Tre`che & Delpeuch, 1979), manifested by the loss of culinary quality which may derive from one or a combination of factors resulting from the normal but inadvertently deleterious reactions leading to textural *
Corresponding author. Tel.: +237 953 8357; fax: +237 231 3495/ +237 222 0019. E-mail address: [email protected] (G.N. Medoua). 0963-9969/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2004.09.005
changes. Despite the fact that several studies (Afoakwa & Sefa-Dedeh, 2002b; Sefa-Dedeh & Afoakwa, 2002; Tre`che, 1989; Tre`che & Delpeuch, 1982) have reported on various changes leading to the hardening of D. dumetorum tubers, the underlying mechanisms causing hardto-cook defect of this yam are not completely elucidated. Two main mechanisms were proposed to explain hard-to-cook defect of vegetables: the so-called pectin– phytate and lignification mechanisms which assume that hardening during storage is due to increased difficulty in achieving cell separation during cooking as a result of phytate hydrolysis and strengthening of cell walls by deposit of a lignin-like material (Kilmer, Seib, & Hoseney, 1994; Stanley & Aguilera, 1985). Interactions or bonds responsible for impaired solubilization of the middle lamella during cooking are thought to include divalent cationic crosslinkages between demethoxylated pectic
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substances and phenolic crosslinkages. The pectin–phytate mechanism assumes that chelating agents (e.g. phytic acid) are enzymatically hydrolysed and the divalent cations released migrate to cell walls where they engage in crosslinking reactions with the demethoxylated pectins in middle lamella. The lignification mechanism assumes that aromatic compounds accumulate at cell wall surfaces where they act as precursors in lignification-like reactions. The objective of this study was to evaluate cooked hardness and its direct manifestation (water absorption, water-holding capacity and solids loss) in D. dumetorum tubers stored under various conditions, as well as some determining biochemical factors (phytate, total phenols and lignin).
2. Materials and methods 2.1. Materials Matured trifoliate yam D. dumetorum tubers (yellow cultivar) were randomly harvested from a farm at Esse in the Centre Province of Cameroon and immediately transported to the laboratory. 2.2. Storage studies Similar quantity of freshly harvested D. dumetorum tubers were stored under three different conditions: low temperature and humidity (15 C, 59% RH), medium temperature and humidity (30 C, 75% RH) and high temperature and humidity (45 C, 86% RH) for a period of 14 days. They were contained in desiccators saturated with aqueous solutions of different salts (NaBr, NaCl and K2CrO4 for 59%, 75% and 86% RH, respectively) and placed in constant temperature chambers (15, 30 and 45 C). During storage, yams were collected at fixed time intervals (day 0, 2, 4, 7 and 14). No growth of mould was observed during the storage period. 2.3. Sample preparation For chemical analyses, the tubers from each storage condition were peeled, chopped into chips of 0.5-cm thickness, dried at 40 C in a ventilated oven for 24 h and stored at 21 C. Prior to analysis, the dried chips were grounded into flour in a Hammer mill (Campsas 82370, Labastide St-Pierre, France) to pass through a 300-lm sieve.
completely at room temperature. Hardness was estimated through Magness–Taylor flesh penetration test with an 11-mm diameter rod. A maximum penetration of 11 mm was applied at a rate of 20 mm/min and the maximum penetration force parameter register (Force MT), expressed in Newton. 2.4.2. Water absorption and water-holding capacity Water absorption value was determined by steeping 5 chips 1-cm thick in 200 ml distilled water at ambient conditions for 1, 3, 6, 12 and 24 h, after which the water was poured out and excess surface water was removed by blotting with a paper napkin. Water absorption value was expressed as weight of water (g) absorbed per 100 g of tubers. Water-holding capacity of 24 h soaked samples was measured to the moisture amounts of free water according to the centrifugation method of Jauregui, Regenstein, and Baker (1981). About 3 g of sample was wrapped in Whatman No. 3 filter paper, centrifuged at 4000 rpm for 20 min on a desktop centrifuge (Bioblock scientific MLWT.62.1) and expressible moisture was calculated using the following equation: %Expressible water ¼ ððUncentrifuged sample weight Centrifuged sample weightÞ= ðUncentrifuged sample weightÞÞ 100 2.4.3. Solids loss Five slices, 1-cm thick, of tubers were steeped in 200 ml distilled water at ambient conditions for 1, 3, 6, 12 and 24 h. The steep water was collected, concentrated by partially drying in a draft oven at 90 C, freeze-dried and weighed to evaluate solid losses. 2.4.4. Phytate Phytate was determined according to the new chromophore method of Ali, Ponnamperuma, and Youssep (1986). In this method, phytate is extracted with trichloroacetic acid 3% and complexed with FeCl3 1% solution to give ferric phytate. Phytate is then released after reaction of the complex with HCl 0.5 N and NaOH 1.5 N, and is determined colorimetrically in the presence of a chromogenic solution at absorbance 830 nm. 2.4.5. Total phenols Total phenols were determined according to the method of Swain and Hillis (1959) using Folin–Ciocalteu reagent.
2.4. Physico-chemical analyses 2.4.1. Hardness measurements Tuber samples of 1-cm thick, were cooked in boiling water for 30 min on a hot plate and made to cool
2.4.6. Lignin Lignin content was gravimetrically evaluated after acid hydrolysis of the insoluble-alcohol residue under conditions established by Saura-Calixto, Gon˜i, Man˜as,
G.N. Medoua et al. / Food Research International 38 (2005) 143–149
2.4.7. Statistical analyses All measurements were carried out in triplicate. Statistical analyses of data was performed using STATISTICA 6 (data analysis software system, StatSoft, Inc.) and SPSS 10.1 softwares. Comparisons between dependent variables were determined using analysis of variance (ANOVA), Duncan multiple range test, correlation analysis and multiple regression analysis. Statistical significance was defined at P 6 0.05.
Water Absorption (g water/100g yams)
6 5 15˚C, 59% RH
4 3 2 1 0 0
1
3
6
12
24
Steeping time (hours) 6
Water Absorption (g water/100g yams)
and Abia (1991). About 2.5 g of sample were treated four times with 25 ml 1% (v/v) 11 N HCl in methanol for 1 h under continuous stirring and centrifuged at 2000 rpm for 10 min. The residue obtained was then mixed with 100 ml of 12 M sulfuric acid and hydrolysed for 3 h at ambiant temperature under stirring. The solution was then diluted with distilled water to obtain 1 M H2SO4, and heated at 100 C for 2.5 h with continuous shaking, cooled, vacuum filtered through an acid-treated 0.45 lm Millipore HVLP filter, and rinsed with hot distilled water and acetone. The filter containing lignin was air-dried at 60 C overnight and weighed. Results were expressed as g lignin per 100 g sample dry weight.
145
5 30˚C, 75% RH
4 3 2 1 0 0
1
3
6
12
24
Steeping time (hours)
3.1. Water absorption, water-holding capacity and solid loss The amount of water absorbed per unit weight of tubers increased with steeping time (Fig. 1). Generally, the water absorption patterns of tubers stored in the different conditions were characterized by initial period of low water uptake during the first hour, followed by a relatively rapid one. Tubers stored at low temperature and relative humidity (15 C, 59% RH) exhibited higher water uptake after two days of storage (P 6 0.05) compared to those stored at higher temperatures and relative humidities. For all the storage conditions, water uptake decreased significantly with storage time (P 6 0.05). On the other hand, expressible moisture to determine waterholding capacity increased significantly (P 6 0.05) with tuber storage duration (Fig. 2), suggesting that cellular water uptake decrease with tuber storage. Tubers stored at low temperature and relative humidity (15 C, 59% RH) exhibited lower rate of expressible moisture increase compared to those stored at higher temperatures and relative humidities. Decrease in water uptake observed in this study may be due to microstructural differences of the stored tubers. Previous studies (Afoakwa & Sefa-Dedeh, 2001; Sealy, Renaudin, Gallant, Bouchet, & Brillouet, 1985; Tre`che & Agbor-Egbe, 1996) associated post-harvest hardening of D. dumetorum tubers to increases in the
5
45˚C, 86% RH
4 3 2 1 0 0
1
3
6
12
24
Steeping time (hours)
Fig. 1. Water absorption during hydration of samples stored for: n = 0 day, d = 2 days, = 4 days, n = 7 days, s = 14 days.
*
24.5
Expressible water (%)
3. Results and discussion
Water Absorption (g water/100g yams)
6
23.5
22.5
21.5 0
2
4
7
14
Storage time (days)
Fig. 2. Expressible water of D. dumetorum tubers during storage at: = 15 C, 59% RH, n = 30 C, 75% RH, m = 45 C, 86% RH.
¤
thickness of the cell wall membranes. According to these observations, it could be deduced from the results of this study that thickening of cell wall membrane of
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D. dumetorum tubers during storage significantly slows down the capacity of the tubers to absorb water. A similar pattern to water absorption was observed for solid loss (Fig. 3) which was significantly affected (P 6 0.05) by storage conditions. Results from this study suggest that storage and the resulting cell wall membrane thickening affected exchanges across the membrane. Decreases in tuber membrane permeability with storage could justify their failure to soften during cooking. For Stanley and Aguilera (1985), failure of cells to separate during cooking could be explained by a resistance to water entrance and movement across the cell membranes. Our results also suggest that storage of D. dumetorum tubers at low temperature and humidity delay cell wall membrane thickening process, whereas storage at high temperature and relative humidity accelerate the phenomenon.
Solids loss (%)
0.12 15˚C,59% RH
0.1 0.08 0.06 0.04 0.02
3.2. Cooked hardness D. dumetorum tubers kept at the three storage conditions developed the hard-to-cook defect, represented by significant increase (P 6 0.05) of cooked hardness with storage time (Fig. 4). This textural modification due to post-harvest hardening phenomenon was explain at the microstructural level by a significant increase in cell wall membrane thickening in the middle lamella during tuber storage, accompanied by additional deposit of membrane constituents (Afoakwa & Sefa-Dedeh, 2001; Tre`che & Delpeuch, 1982). For samples stored at low temperature and relative humidity (15 C, 59% RH), cooked hardness values were significantly lower than for the samples stored at higher temperatures and relative humidities. This suggests that post-harvest hardening of D. dumetorum tubers is under the dependence of temperature and relative humidity. Hence, storage at low temperature and relative humidity could be used to delay post-harvest hardening phenomenon of D. dumetorum tubers. Variance analysis (ANOVA) showed that storage conditions and storage duration have a significant effect (P < 0.05) on the cooked hardness of D. dumetorum tubers (Table 1). 3.3. Phytate
0 0
1
3
6
12
24
Steeping time (hours)
30˚C, 75%RH
0.1 0.08
90
0.06 0.04 0.02 0 0
1
3
6
12
24
Steeping time (hours)
Cooked hardness (N)
Solids loss (%)
0.12
Data obtained for phytate levels indicate that there was a significant difference in the rate of phytate loss under the different storage conditions (Fig. 5). Duncan multiple range test revealed that the rates fell into three
Solids loss (%)
0.12
75 60 45 30 15 0 0
45˚C, 86% RH
0.1
2
4
7
14
Storage time (days)
0.08
Fig. 4. Cooked texture of D. dumetorum tubers during storage at: = 15 C, 59% RH, n = 30 C, 75% RH, m = 45 C, 86% RH.
0.06
¤
0.04 0.02
Table 1 F values for cooked hardness of D. dumetorum tubers
0 0
1
3
6
12
24
Steeping time (hours) Fig. 3. Solids loss during steeping of D. dumetorum tubers stored for: n = 0 day, d = 2 days, = 4 days, n = 7 days, s = 14 days.
*
Variables
Cooked hardness
Storage time Storage conditions
274.073* 6.211*
*
Significant at P < 0.05.
147
320
680
Total phenols (mg/100 g dry matter)
Phytate (mg/100 g dry matter)
G.N. Medoua et al. / Food Research International 38 (2005) 143–149
630 580 530 480 430
300 280 260 240 220 200
380 2
0
4
7
0
14
2
¤
categories in relation to the storage conditions: (15 C, 59% RH), (30 C, 75% RH) and (45 C, 86% RH) (P < 0.05). The rate of phytate loss for tubers stored at low temperature and relative humidity (15 C, 59% RH) was significantly lower than for tubers stored at higher storage conditions. A general trend, therefore, was observed between rate of phytate loss and development of cooked hardness. There was a significant correlation (r = 0.922, P < 0.01, d.f. = 146) between these two parameters. This implies that phytate loss is a contributor factor in the hardening process of D. dumetorum tubers. The relationship between hard-to-cook defect and phytate content has been reported with significant correlations by several authors in several species and varieties of legumes (Hincks & Stanley, 1986; Kumar, Venkataraman, Jaya, & Krishnamurth, 1978; Longe, 1983; Mattson, 1946; Mattson, Akerberg, Erickson, KoulterAnderson, & Vahtras, 1950; Moscoso, Bourne, & Hood, 1984). 3.4. Total phenols and lignin content Total phenol contents of the tubers decreased significantly (P 6 0.05) with storage time (Fig. 6) and hence, with hardening of tubers. Furthermore, storage at low temperature, low humidity (15 C, 59% RH) slowed down the rate of decrease in total phenols loss during storage. A general trend was seen between total phenol levels and cooked hardness of tubers. Correlation coefficient observed between these two parameters was significant (r = 0.857, P < 0.01, d.f. = 146), indicating that total phenols play a significant role in the development of the hard-to-cook defect. Similar variations have been reported by other researchers (Deshpende & Cheryan, 1985; Hincks & Stanley, 1986; Kadam, Kute, Lawande, & Salunkhe, 1982) who observed a decrease of polyphenols content during storage of beans, with a concomitant increase of hard-to-cook defect. Changes in phenolic compounds
7
14
Fig. 6. Total phenol losses during storage of D. dumetorum tubers at: = 15 C, 59% RH, n = 30 C, 75% RH, m = 45 C, 86% RH.
¤
exhibited during post-harvest storage have been attributed variously to polymerization/condensation reactions that produce insoluble high molecular weight polymers (Kadam et al., 1982); attachment to carbohydrate matrix (Salunkhe, Jadhav, Kadam, & Chavan, 1982); and binding to protein (Bresani, Elias, Wolzak, Hagerman, & Butler, 1983). Data obtained for lignin indicate that there was a significant increase of lignin levels with storage (Fig. 7). Furthermore, low temperature and humidity storage of the tubers slowed down the rate of increase in the lignin levels. Similar increases in lignin during storage of D. dumetorum tubers were reported by previous studies (Afoakwa & Sefa-Dedeh, 2002a; Tre`che, 1989) and were also attributed to the hardening phenomenon. In this study, there was a significant correlation (r = 0.811, P < 0.01, d.f. = 146) between total phenol levels and lignin content during storage, suggesting that phenolic compounds play a significant role in the hard-to-cook defect of tubers via lignification of meddular lamella. From the results obtained in this study, it could be suggested that the changes in total phenols content is in part due to their increased polymerization during storage and that a possible lignification-like mechanism is functioning to restrict cell separation on cooking.
Lignin (g/100g dry matter)
Fig. 5. Changes in phytate levels during storage of D. dumetorum tubers at: = 15 C, 59% RH, n = 30 C, 75% RH, m = 45 C, 86% RH.
4
Storage time (days)
Storage time (days)
6.3 5.3 4.3 3.3 2.3 0
2
4
7
14
Storage time (days)
Fig. 7. Changes in lignin contents during storage of D. dumetorum tubers at: = 15 C, 59% RH, n = 30 C, 75% RH, m = 45 C, 86% RH.
¤
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2
R (%) or cooked hardness (N)
Lignin is a product of the oxidation of polyphenolic compounds. The process of lignification can occur either enzymatically (mediated by cell wall bound peroxidase and laccase-like polyphenol oxidases) or, at least partially non-enzymatically (Blouin, Zarins, & Cherry, 1982; Mayer & Staples, 2002). Multiple regression analysis performed to relate phytate and total phenols to the cooked hardness at the different storage periods is presented in Fig. 8 and Table 2. It was found that when both variables are included about 100% of the total variation was accounted for at each storage period. During the two first days of storage, changes were predominantly due to phytate while in the latter days total phenols were the major contributors. Similar observations were made by Hincks and Stanley (1986) for black beans. Our results indicate that the transition begins after 2 days of storage, which is around the time that cooked hardness is accelerated. It would appear that phytate levels cause only minor differences in cooked hardness of tubers, which may escape consumer differentiation. With time, as total phenols become polymerized or bound, in what is believed to be associated with lignification-like mechanism, a major change in cooked hardness results, completely overriding the minor effects of phytate.
100 80 60 40 20 0 0
2
4
7
14
Storage time (days)
Fig. 8. Changes in R2 contribution to cooked hardness with storage time for two independent variables: m = phytate, d = total phenols, s = typical tubers hardening curve.
Table 2 Coefficients in the multiple regression equation linking cooked hardness with total phenols and phytate Cooked hardness Intercept Phytate Total phenols R2 (adj) F-stata P
151.9940 0.5040 0.6150 0.9328 1035.407 0.0001
a F-stat is for test of significant difference between calculated and experimental data.
4. Conclusion Cell wall membrane permeability of D. dumetorum tubers decreased significantly after harvest as shown by water absorption, water-holding capacity and solid loss decrease, indicating thickening of the cell walls. Changes in cooked hardness of tubers during storage was associated with phytate loss and total phenol losses due to their possible participation in a lignification-like mechanism. Phytate caused only minor changes in cooked hardness of the tubers while total phenols caused major changes which completely override those of phytate. In this respect, it could be suggested that D. dumetorum tuber hardening occurs following multiple mechanisms and that storage at low temperature and relative humidity (15 C, 59% RH) leads to slowing down of the cooked hardness phenomenon.
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G.N. Medoua et al. / Food Research International 38 (2005) 143–149 Longe, O. G. (1983). Varietal differences in chemical characteristics related to cooking quality of cowpea. Journal of Food Processing and Preservation, 7, 143–150. Mattson, S. (1946). The cookability of yellow peas: a colloid-chemical and biochemical study. Acta Agriculture Suecana, 2, 185–231. Mattson, S., Akerberg, E., Erickson, E., Koulter-Anderson, E., & Vahtras, K. (1950). Factors determining the composition and cookability of peas. Acta Agriculture Scandinavia, 1, 40–61. Mayer, A. M., & Staples, R. C. (2002). Laccas: new functions for old enzyme. Phytochemistry, 60, 551–565. Moscoso, W., Bourne, M. C., & Hood, L. F. (1984). Relationships between the hard-to-cook phenomenon in red kidney beans and water absorption, puncture force, pectin, phytic acid, and minerals. Journal of Food Science, 49, 1577–1583. Salunkhe, D. K., Jadhav, S. J., Kadam, S. S., & Chavan, J. K. (1982). Chemical, biochemical and biological significance of polyphenols in cereals and legumes. CRC Critial Reviews in Food Science and Nutrition, 17, 277–305. Saura-Calixto, F., Gon˜i, I., Man˜as, E., & Abia, R. (1991). Klanson lignin, condensed tannins and resistant protein as dietary fiber constituents: determination in grape pomaces. Food Chemistry, 39, 299–309. Sealy, L., Renaudin, S., Gallant, D. J., Bouchet, B., & Brillouet, J. M. (1985). Ultrastructural study of yam tuber as related to postharvest hardness. Food Microstructure, 4, 173–181.
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Study of the hard-to-cook property of stored yam tubers (Dioscorea dumetorum) and some determining biochemical factors Gabriel Nama Medoua a
a,*
, Israe¨l Lape Mbome a, T. Agbor-Egbe a, C.M.F. Mbofung
b
Centre for Food and Nutrition Research, IMPM, P.O. Box 6163, Yaounde, Cameroon b ENSAI, University of Ngaoundere, P.O. Box 455, Ngaoundere, Cameroon Received 30 August 2004; accepted 20 September 2004
Abstract In an attempt to investigate the hard-to-cook phenomenon in Dioscorea dumetorum tubers during storage, selected physical and chemical characteristics were monitored. A 3 · 5 factorial experiment with storage conditions (15 C, 59% RH; 30 C, 75% RH; 45 C, 86% RH) and storage period (0, 2, 4, 7 and 14 days) as variables was carried out. Changes in cooked hardness, water absorption, water-holding capacity and solid loss of steeped tubers as well as phytate, total phenols and lignin contents were monitored. Water uptake and solid loss of steeped tubers decreased significantly (P 6 0.05) with storage period, suggesting that storage affects cell wall membrane permeability of the tubers. Cooked hardness analyses indicated significant difference (P 6 0.05) between fresh and stored tubers and its rate varied with storage time and storage conditions. Cooked hardness values correlated negatively (r = 0.922–0.857, P 6 0.05, d.f. = 146) with phytate and total phenols. A multiple mechanism for D. dumetorum tubers hardening is presented which includes phytate loss as a minimal contributor to cooked hardness during the first days of storage and total phenol loss via a lignification-like mechanism as a major contributor. 2004 Elsevier Ltd. All rights reserved. Keywords: Dioscorea dumetorum; Hard-to-cook; Phytate; Phenols; Lignification
1. Introduction Dioscorea dumetorum tubers are good sources of protein, carbohydrates and minerals. Their storage under warm, humid conditions encountered in many African countries renders them susceptible to a hardening phenomenon characterized by loss of the ability to soften during cooking. Tuber hardening is a serious handicap and begins within 24 h after harvest (Afoakwa & Sefa-Dedeh, 2002b; Tre`che & Delpeuch, 1979), manifested by the loss of culinary quality which may derive from one or a combination of factors resulting from the normal but inadvertently deleterious reactions leading to textural *
Corresponding author. Tel.: +237 953 8357; fax: +237 231 3495/ +237 222 0019. E-mail address: [email protected] (G.N. Medoua). 0963-9969/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2004.09.005
changes. Despite the fact that several studies (Afoakwa & Sefa-Dedeh, 2002b; Sefa-Dedeh & Afoakwa, 2002; Tre`che, 1989; Tre`che & Delpeuch, 1982) have reported on various changes leading to the hardening of D. dumetorum tubers, the underlying mechanisms causing hardto-cook defect of this yam are not completely elucidated. Two main mechanisms were proposed to explain hard-to-cook defect of vegetables: the so-called pectin– phytate and lignification mechanisms which assume that hardening during storage is due to increased difficulty in achieving cell separation during cooking as a result of phytate hydrolysis and strengthening of cell walls by deposit of a lignin-like material (Kilmer, Seib, & Hoseney, 1994; Stanley & Aguilera, 1985). Interactions or bonds responsible for impaired solubilization of the middle lamella during cooking are thought to include divalent cationic crosslinkages between demethoxylated pectic
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substances and phenolic crosslinkages. The pectin–phytate mechanism assumes that chelating agents (e.g. phytic acid) are enzymatically hydrolysed and the divalent cations released migrate to cell walls where they engage in crosslinking reactions with the demethoxylated pectins in middle lamella. The lignification mechanism assumes that aromatic compounds accumulate at cell wall surfaces where they act as precursors in lignification-like reactions. The objective of this study was to evaluate cooked hardness and its direct manifestation (water absorption, water-holding capacity and solids loss) in D. dumetorum tubers stored under various conditions, as well as some determining biochemical factors (phytate, total phenols and lignin).
2. Materials and methods 2.1. Materials Matured trifoliate yam D. dumetorum tubers (yellow cultivar) were randomly harvested from a farm at Esse in the Centre Province of Cameroon and immediately transported to the laboratory. 2.2. Storage studies Similar quantity of freshly harvested D. dumetorum tubers were stored under three different conditions: low temperature and humidity (15 C, 59% RH), medium temperature and humidity (30 C, 75% RH) and high temperature and humidity (45 C, 86% RH) for a period of 14 days. They were contained in desiccators saturated with aqueous solutions of different salts (NaBr, NaCl and K2CrO4 for 59%, 75% and 86% RH, respectively) and placed in constant temperature chambers (15, 30 and 45 C). During storage, yams were collected at fixed time intervals (day 0, 2, 4, 7 and 14). No growth of mould was observed during the storage period. 2.3. Sample preparation For chemical analyses, the tubers from each storage condition were peeled, chopped into chips of 0.5-cm thickness, dried at 40 C in a ventilated oven for 24 h and stored at 21 C. Prior to analysis, the dried chips were grounded into flour in a Hammer mill (Campsas 82370, Labastide St-Pierre, France) to pass through a 300-lm sieve.
completely at room temperature. Hardness was estimated through Magness–Taylor flesh penetration test with an 11-mm diameter rod. A maximum penetration of 11 mm was applied at a rate of 20 mm/min and the maximum penetration force parameter register (Force MT), expressed in Newton. 2.4.2. Water absorption and water-holding capacity Water absorption value was determined by steeping 5 chips 1-cm thick in 200 ml distilled water at ambient conditions for 1, 3, 6, 12 and 24 h, after which the water was poured out and excess surface water was removed by blotting with a paper napkin. Water absorption value was expressed as weight of water (g) absorbed per 100 g of tubers. Water-holding capacity of 24 h soaked samples was measured to the moisture amounts of free water according to the centrifugation method of Jauregui, Regenstein, and Baker (1981). About 3 g of sample was wrapped in Whatman No. 3 filter paper, centrifuged at 4000 rpm for 20 min on a desktop centrifuge (Bioblock scientific MLWT.62.1) and expressible moisture was calculated using the following equation: %Expressible water ¼ ððUncentrifuged sample weight Centrifuged sample weightÞ= ðUncentrifuged sample weightÞÞ 100 2.4.3. Solids loss Five slices, 1-cm thick, of tubers were steeped in 200 ml distilled water at ambient conditions for 1, 3, 6, 12 and 24 h. The steep water was collected, concentrated by partially drying in a draft oven at 90 C, freeze-dried and weighed to evaluate solid losses. 2.4.4. Phytate Phytate was determined according to the new chromophore method of Ali, Ponnamperuma, and Youssep (1986). In this method, phytate is extracted with trichloroacetic acid 3% and complexed with FeCl3 1% solution to give ferric phytate. Phytate is then released after reaction of the complex with HCl 0.5 N and NaOH 1.5 N, and is determined colorimetrically in the presence of a chromogenic solution at absorbance 830 nm. 2.4.5. Total phenols Total phenols were determined according to the method of Swain and Hillis (1959) using Folin–Ciocalteu reagent.
2.4. Physico-chemical analyses 2.4.1. Hardness measurements Tuber samples of 1-cm thick, were cooked in boiling water for 30 min on a hot plate and made to cool
2.4.6. Lignin Lignin content was gravimetrically evaluated after acid hydrolysis of the insoluble-alcohol residue under conditions established by Saura-Calixto, Gon˜i, Man˜as,
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2.4.7. Statistical analyses All measurements were carried out in triplicate. Statistical analyses of data was performed using STATISTICA 6 (data analysis software system, StatSoft, Inc.) and SPSS 10.1 softwares. Comparisons between dependent variables were determined using analysis of variance (ANOVA), Duncan multiple range test, correlation analysis and multiple regression analysis. Statistical significance was defined at P 6 0.05.
Water Absorption (g water/100g yams)
6 5 15˚C, 59% RH
4 3 2 1 0 0
1
3
6
12
24
Steeping time (hours) 6
Water Absorption (g water/100g yams)
and Abia (1991). About 2.5 g of sample were treated four times with 25 ml 1% (v/v) 11 N HCl in methanol for 1 h under continuous stirring and centrifuged at 2000 rpm for 10 min. The residue obtained was then mixed with 100 ml of 12 M sulfuric acid and hydrolysed for 3 h at ambiant temperature under stirring. The solution was then diluted with distilled water to obtain 1 M H2SO4, and heated at 100 C for 2.5 h with continuous shaking, cooled, vacuum filtered through an acid-treated 0.45 lm Millipore HVLP filter, and rinsed with hot distilled water and acetone. The filter containing lignin was air-dried at 60 C overnight and weighed. Results were expressed as g lignin per 100 g sample dry weight.
145
5 30˚C, 75% RH
4 3 2 1 0 0
1
3
6
12
24
Steeping time (hours)
3.1. Water absorption, water-holding capacity and solid loss The amount of water absorbed per unit weight of tubers increased with steeping time (Fig. 1). Generally, the water absorption patterns of tubers stored in the different conditions were characterized by initial period of low water uptake during the first hour, followed by a relatively rapid one. Tubers stored at low temperature and relative humidity (15 C, 59% RH) exhibited higher water uptake after two days of storage (P 6 0.05) compared to those stored at higher temperatures and relative humidities. For all the storage conditions, water uptake decreased significantly with storage time (P 6 0.05). On the other hand, expressible moisture to determine waterholding capacity increased significantly (P 6 0.05) with tuber storage duration (Fig. 2), suggesting that cellular water uptake decrease with tuber storage. Tubers stored at low temperature and relative humidity (15 C, 59% RH) exhibited lower rate of expressible moisture increase compared to those stored at higher temperatures and relative humidities. Decrease in water uptake observed in this study may be due to microstructural differences of the stored tubers. Previous studies (Afoakwa & Sefa-Dedeh, 2001; Sealy, Renaudin, Gallant, Bouchet, & Brillouet, 1985; Tre`che & Agbor-Egbe, 1996) associated post-harvest hardening of D. dumetorum tubers to increases in the
5
45˚C, 86% RH
4 3 2 1 0 0
1
3
6
12
24
Steeping time (hours)
Fig. 1. Water absorption during hydration of samples stored for: n = 0 day, d = 2 days, = 4 days, n = 7 days, s = 14 days.
*
24.5
Expressible water (%)
3. Results and discussion
Water Absorption (g water/100g yams)
6
23.5
22.5
21.5 0
2
4
7
14
Storage time (days)
Fig. 2. Expressible water of D. dumetorum tubers during storage at: = 15 C, 59% RH, n = 30 C, 75% RH, m = 45 C, 86% RH.
¤
thickness of the cell wall membranes. According to these observations, it could be deduced from the results of this study that thickening of cell wall membrane of
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D. dumetorum tubers during storage significantly slows down the capacity of the tubers to absorb water. A similar pattern to water absorption was observed for solid loss (Fig. 3) which was significantly affected (P 6 0.05) by storage conditions. Results from this study suggest that storage and the resulting cell wall membrane thickening affected exchanges across the membrane. Decreases in tuber membrane permeability with storage could justify their failure to soften during cooking. For Stanley and Aguilera (1985), failure of cells to separate during cooking could be explained by a resistance to water entrance and movement across the cell membranes. Our results also suggest that storage of D. dumetorum tubers at low temperature and humidity delay cell wall membrane thickening process, whereas storage at high temperature and relative humidity accelerate the phenomenon.
Solids loss (%)
0.12 15˚C,59% RH
0.1 0.08 0.06 0.04 0.02
3.2. Cooked hardness D. dumetorum tubers kept at the three storage conditions developed the hard-to-cook defect, represented by significant increase (P 6 0.05) of cooked hardness with storage time (Fig. 4). This textural modification due to post-harvest hardening phenomenon was explain at the microstructural level by a significant increase in cell wall membrane thickening in the middle lamella during tuber storage, accompanied by additional deposit of membrane constituents (Afoakwa & Sefa-Dedeh, 2001; Tre`che & Delpeuch, 1982). For samples stored at low temperature and relative humidity (15 C, 59% RH), cooked hardness values were significantly lower than for the samples stored at higher temperatures and relative humidities. This suggests that post-harvest hardening of D. dumetorum tubers is under the dependence of temperature and relative humidity. Hence, storage at low temperature and relative humidity could be used to delay post-harvest hardening phenomenon of D. dumetorum tubers. Variance analysis (ANOVA) showed that storage conditions and storage duration have a significant effect (P < 0.05) on the cooked hardness of D. dumetorum tubers (Table 1). 3.3. Phytate
0 0
1
3
6
12
24
Steeping time (hours)
30˚C, 75%RH
0.1 0.08
90
0.06 0.04 0.02 0 0
1
3
6
12
24
Steeping time (hours)
Cooked hardness (N)
Solids loss (%)
0.12
Data obtained for phytate levels indicate that there was a significant difference in the rate of phytate loss under the different storage conditions (Fig. 5). Duncan multiple range test revealed that the rates fell into three
Solids loss (%)
0.12
75 60 45 30 15 0 0
45˚C, 86% RH
0.1
2
4
7
14
Storage time (days)
0.08
Fig. 4. Cooked texture of D. dumetorum tubers during storage at: = 15 C, 59% RH, n = 30 C, 75% RH, m = 45 C, 86% RH.
0.06
¤
0.04 0.02
Table 1 F values for cooked hardness of D. dumetorum tubers
0 0
1
3
6
12
24
Steeping time (hours) Fig. 3. Solids loss during steeping of D. dumetorum tubers stored for: n = 0 day, d = 2 days, = 4 days, n = 7 days, s = 14 days.
*
Variables
Cooked hardness
Storage time Storage conditions
274.073* 6.211*
*
Significant at P < 0.05.
147
320
680
Total phenols (mg/100 g dry matter)
Phytate (mg/100 g dry matter)
G.N. Medoua et al. / Food Research International 38 (2005) 143–149
630 580 530 480 430
300 280 260 240 220 200
380 2
0
4
7
0
14
2
¤
categories in relation to the storage conditions: (15 C, 59% RH), (30 C, 75% RH) and (45 C, 86% RH) (P < 0.05). The rate of phytate loss for tubers stored at low temperature and relative humidity (15 C, 59% RH) was significantly lower than for tubers stored at higher storage conditions. A general trend, therefore, was observed between rate of phytate loss and development of cooked hardness. There was a significant correlation (r = 0.922, P < 0.01, d.f. = 146) between these two parameters. This implies that phytate loss is a contributor factor in the hardening process of D. dumetorum tubers. The relationship between hard-to-cook defect and phytate content has been reported with significant correlations by several authors in several species and varieties of legumes (Hincks & Stanley, 1986; Kumar, Venkataraman, Jaya, & Krishnamurth, 1978; Longe, 1983; Mattson, 1946; Mattson, Akerberg, Erickson, KoulterAnderson, & Vahtras, 1950; Moscoso, Bourne, & Hood, 1984). 3.4. Total phenols and lignin content Total phenol contents of the tubers decreased significantly (P 6 0.05) with storage time (Fig. 6) and hence, with hardening of tubers. Furthermore, storage at low temperature, low humidity (15 C, 59% RH) slowed down the rate of decrease in total phenols loss during storage. A general trend was seen between total phenol levels and cooked hardness of tubers. Correlation coefficient observed between these two parameters was significant (r = 0.857, P < 0.01, d.f. = 146), indicating that total phenols play a significant role in the development of the hard-to-cook defect. Similar variations have been reported by other researchers (Deshpende & Cheryan, 1985; Hincks & Stanley, 1986; Kadam, Kute, Lawande, & Salunkhe, 1982) who observed a decrease of polyphenols content during storage of beans, with a concomitant increase of hard-to-cook defect. Changes in phenolic compounds
7
14
Fig. 6. Total phenol losses during storage of D. dumetorum tubers at: = 15 C, 59% RH, n = 30 C, 75% RH, m = 45 C, 86% RH.
¤
exhibited during post-harvest storage have been attributed variously to polymerization/condensation reactions that produce insoluble high molecular weight polymers (Kadam et al., 1982); attachment to carbohydrate matrix (Salunkhe, Jadhav, Kadam, & Chavan, 1982); and binding to protein (Bresani, Elias, Wolzak, Hagerman, & Butler, 1983). Data obtained for lignin indicate that there was a significant increase of lignin levels with storage (Fig. 7). Furthermore, low temperature and humidity storage of the tubers slowed down the rate of increase in the lignin levels. Similar increases in lignin during storage of D. dumetorum tubers were reported by previous studies (Afoakwa & Sefa-Dedeh, 2002a; Tre`che, 1989) and were also attributed to the hardening phenomenon. In this study, there was a significant correlation (r = 0.811, P < 0.01, d.f. = 146) between total phenol levels and lignin content during storage, suggesting that phenolic compounds play a significant role in the hard-to-cook defect of tubers via lignification of meddular lamella. From the results obtained in this study, it could be suggested that the changes in total phenols content is in part due to their increased polymerization during storage and that a possible lignification-like mechanism is functioning to restrict cell separation on cooking.
Lignin (g/100g dry matter)
Fig. 5. Changes in phytate levels during storage of D. dumetorum tubers at: = 15 C, 59% RH, n = 30 C, 75% RH, m = 45 C, 86% RH.
4
Storage time (days)
Storage time (days)
6.3 5.3 4.3 3.3 2.3 0
2
4
7
14
Storage time (days)
Fig. 7. Changes in lignin contents during storage of D. dumetorum tubers at: = 15 C, 59% RH, n = 30 C, 75% RH, m = 45 C, 86% RH.
¤
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2
R (%) or cooked hardness (N)
Lignin is a product of the oxidation of polyphenolic compounds. The process of lignification can occur either enzymatically (mediated by cell wall bound peroxidase and laccase-like polyphenol oxidases) or, at least partially non-enzymatically (Blouin, Zarins, & Cherry, 1982; Mayer & Staples, 2002). Multiple regression analysis performed to relate phytate and total phenols to the cooked hardness at the different storage periods is presented in Fig. 8 and Table 2. It was found that when both variables are included about 100% of the total variation was accounted for at each storage period. During the two first days of storage, changes were predominantly due to phytate while in the latter days total phenols were the major contributors. Similar observations were made by Hincks and Stanley (1986) for black beans. Our results indicate that the transition begins after 2 days of storage, which is around the time that cooked hardness is accelerated. It would appear that phytate levels cause only minor differences in cooked hardness of tubers, which may escape consumer differentiation. With time, as total phenols become polymerized or bound, in what is believed to be associated with lignification-like mechanism, a major change in cooked hardness results, completely overriding the minor effects of phytate.
100 80 60 40 20 0 0
2
4
7
14
Storage time (days)
Fig. 8. Changes in R2 contribution to cooked hardness with storage time for two independent variables: m = phytate, d = total phenols, s = typical tubers hardening curve.
Table 2 Coefficients in the multiple regression equation linking cooked hardness with total phenols and phytate Cooked hardness Intercept Phytate Total phenols R2 (adj) F-stata P
151.9940 0.5040 0.6150 0.9328 1035.407 0.0001
a F-stat is for test of significant difference between calculated and experimental data.
4. Conclusion Cell wall membrane permeability of D. dumetorum tubers decreased significantly after harvest as shown by water absorption, water-holding capacity and solid loss decrease, indicating thickening of the cell walls. Changes in cooked hardness of tubers during storage was associated with phytate loss and total phenol losses due to their possible participation in a lignification-like mechanism. Phytate caused only minor changes in cooked hardness of the tubers while total phenols caused major changes which completely override those of phytate. In this respect, it could be suggested that D. dumetorum tuber hardening occurs following multiple mechanisms and that storage at low temperature and relative humidity (15 C, 59% RH) leads to slowing down of the cooked hardness phenomenon.
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