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Evaluation of Nutritive and Antioxidant Properties of Blanched Leafy Vegetables Consumed in Northern Côte d’Ivoire

Evaluation of Nutritive and Antioxidant Properties of Blanched Leafy Vegetables Consumed in... Pol. J. Food Nutr. Sci., 2015, Vol. 65, No. 1, pp. 31­38 DOI: 10.1515/pjfns-2015-0003 http://journal.pan.olsztyn.pl Original article Food Quality and Functionality Evaluation of Nutritive and Antioxidant Properties of Blanched Leafy Vegetables Consumed in Northern Côte d'Ivoire Patricia D. Oulai, Lessoy T. Zoué*, Sébastien L. Niamké Laboratoire de Biotechnologies, UFR Biosciences, Université Félix Houphouët-Boigny, 22 BP 582, Abidjan 22, Côte d'Ivoire Key words: antioxidant properties, blanching processing, leafy vegetables, nutritive value The aim of this study was to evaluate the effect of steam blanching processing on the nutritive value and the antioxidant properties of five leafy vegetable species (Amaranthus hybridus, Andasonia digitata, Ceiba patendra, Hibiscus sabdariffa and Vigna unguiculata) that are used for sauce preparation in Northern Côte d'Ivoire. The selected leafy vegetables were subjected to blanching in pressure cooker for 15, 25 and 45 min and the physicochemical properties were determined using AOAC methods. The result of the study revealed that longer time of blanching (higher than 15 min) caused negative impact by reducing nutritive value but positive impact by reducing anti-nutrients and increasing polyphenols. The registered losses (p<0.05) at 15 min were as follow: ash (0.08­10.01%), proteins (0.36­12.03%), vitamin C (19.56­68.67%), carotenoids (18.91­55.48%) oxalates (3.58­21.39%) and phytates (10.51­68.02%). The average increase of polyphenols contents at 15 min of blanching was 1.61 to 30.72%. In addition, a slight increase (0.35­4.16%) of fibres content was observed in the studied blanched leafy vegetables. Furthermore, after 15 min of blanching time the residual contents (p<0.05) of minerals were: calcium (264.88­844.92 mg/100 g), magnesium (49.45­435.43 mg/100 g), potassium (675­1895.41 mg/100 g), iron (14.54­70.89 mg/100 g) and zinc (9.48­36.46 mg/100 g). All these results suggest that the recommended time of domestic blanching must be less than 15 min for the studied leafy vegetables in order to contribute efficiently to the nutritional requirement and to the food security of Ivorian population. INTRODUCTION The growing awareness in recent years of the health promoting has directed increased attention to vegetables as vital components of daily diets [Smith, 2007]. A world vegetable survey showed that 402 vegetable crops are cultivated worldwide, representing 69 families and leafy vegetables which leaves are consumed, are the most (53% of the total) often utilized [Kays, 1995]. The traditional leafy vegetables are rich in micronutrients needed by humans for good health, growth and could contribute significantly to food security, which is a prerequisite for human development. Indeed, Obel-Lawson [2005] reported that an increased consumption of African leafy vegetables (ALVs) can have a positive effect on nutrition, health and economic wellbeing of both rural and urban populations. Socio-economic surveys conducted in various parts of Africa indicate also that ALVs are important commodities in household food and nutrition security [Mnzava, 1997]. Fresh leaves of most ALVs like vegetable amaranths (Amaranthus), slenderleaf (Crotalaria brevidens), spiderplant (Chlorophytum comosum), vegetable cowpeas (Vigna), pumpkin leaves (cucurbits) and jute mallow (Corchorus) contain more than 100% of the recommended daily allowances for vitamins and minerals and 40% proteins for growing children and lactating mothers [Chweya, 1985]. * Corresponding Author: y.lessoy@yahoo.fr; Tel: +(225) 08 98 67 44 The high moisture content of fresh leafy vegetables renders them perishable and seasonal availability limits their utilization all around the year. Hence, there is a need to preserve this nature's store house of nutrients through proper processing techniques for safe storage with efficient nutrient retention [Gupta et al., 2008]. One common processing used before consumption of leafy vegetables is blanching which is a thermal treatment commonly applied to a variety of vegetables before freezing. Its primary purpose is to inactivate enzymes and destroy vegetative microbial cells, allowing stabilization and product quality retention during frozen storage [Canet, 1989]. It is usually carried out in hot water or in steam; this technique is used by indigenous people to reduce or eliminate the bitterness of the vegetables and acid components that are common in leaves. Blanching affords also a series of secondary benefits, due to its washing action, such as elimination of off-flavors that may have been formed during the time between harvesting and processing, and removal of any residual pesticides [Prestamo et al., 1998]. Blanching, however, has some adverse effects, such as pigment modifications, tissues softening and nutrient losses [Murcia et al., 1999; Oboh, 2005]. Among the twenty hundred and seven (207) leafy vegetables widely consumed in tropical Africa, about twenty (20) species of leafy vegetables belonging to 6 botanical families are widely consumed and cultivated by Ivorian populations [PROTA, 2004; Fondio et al., 2007]. Furthermore, the consumption of these leafy vegetables is linked to the region © Copyright by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences © 2015 Author(s). This is an open access article licensed under the Creative Commons Attribution-NonCommercial-NoDerivs License (http://creativecommons.org/licenses/by-nc-nd/3.0/). and ethno-botanical studies have stated that most people in Northern Côte d'Ivoire consume indigenous green leafy vegetables such as Amaranthus hybridus "boronbrou", Andasonia digitata "baobab", Ceiba patendra "fromager", Hibiscus sabdariffa "dah" and Vigna unguiculata "haricot" [Fondio et al., 2007; Soro et al., 2012]. Indeed, dietary habits of these populations include culinary preparation of these leafy vegetables as follow: the mature and freshly leaves are boiled in water for about 30 min in order to reduce bitter taste and then used, after discarding boiled water, for sauce preparation that accompany starchy sorghum paste food commonly named "to". Earlier reports have highlighted the nutritive potential of these fresh leafy vegetables [Oulai et al., 2014] but there is a lack of scientific data with regards to the effect of blanching processing on their physicochemical and nutritive characteristics. Therefore, the purpose of this study is to conduct investigation on the effect of blanching on the nutritive value of these selected leafy vegetables in order to provide necessary information for their wider utilization and contribution to food security of Ivorian populations. MATERIAL AND METHODS Samples collection Leafy vegetables (Amaranthus hybridus, Andasonia digitata, Ceiba patendra, Hibiscus sabdariffa and Vigna unguiculata) were collected fresh and at maturity from cultivated farmlands located at Dabou (latitude: 5°19'14" North; longitude: 4°22'59" West) (Abidjan District). The samples were harvested at the early stage (between one and two weeks of the appearance of the leaves). These plants were previously authenticated by the National Floristic Center (University Felix Houphouët-Boigny, Abidjan-Côte d'Ivoire). Samples processing The fresh leafy vegetables were rinsed with deionized water and the edible portions were separated from the inedible portion. The edible portions were chopped into small pieces (500 g) and allowed to drain at ambient temperature. Each sample was divided into two lots. The first lot (raw, 250 g) was dried in an oven (Memmert, Germany) at 60°C for 72 h [Chima & Igyor, 2007]. The dried leaves were ground with a laboratory crusher (Culatti, France) equipped with a 10 m mesh sieve. Each sample was stored in a clean dry air-tight sample bottle in a refrigerator (4°C) until required for analyses. The second lot (250 g) was steam-blanched for 15, 25 and 45 min in a pressure cooker. The blanched samples were cooled, drained at ambient temperature and subjected to the same treatment used for raw samples. Chemicals All solvents (n-hexane, petroleum ether, acetone, ethanol and methanol) were purchased from Merck. Standards used (gallic acid, -carotene) and reagents (metaphosphoric acid, Folin-Ciocalteu, DPPH) were purchased from Sigma-Aldrich. All chemicals used in the study were of analytical grade. Nutritive properties Proximate analysis Proximate analysis was performed using the AOAC [1990] methods. The moisture content was determined by the difference of weight before and after drying the sample (10 g) in an oven (Memmert, Germany) at 105°C until constant weight. Ash fraction was determined by the incineration of dried sample (5 g) in a muffle furnace (Pyrolabo, France) at 550°C for 12 h. The percentage residue weight was expressed as ash content. For crude fibres, 2 g of sample were weighed into separate 500 mL round bottom flasks and 100 mL of 0.25 mol/L sulphuric acid solution was added. The mixture obtained was boiled under reflux for 30 min. Thereafter, 100 mL of 0.3 mol/L sodium hydroxide solution was added and the mixture was boiled again under reflux for 30 min and filtered through Whatman paper. The insoluble residue was then incinerated, and weighed for the determination of crude fibres content. Proteins were determined through the Kjeldhal method and the lipid content was determined by Soxhlet extraction using hexane as a solvent. Carbohydrates and calorific value were calculated using the following formulas [FAO, 2002]: Carbohydrates: 100 ­ (% moisture + % proteins + % lipids + % ash + % fibres) Calorific value: (% proteins x 2.44) + (% carbohydrates x 3.57) + (% lipids x 8.37) The results of ash, fibres, proteins, lipids and carbohydrates contents were expressed on dry matter basis. Mineral analysis Minerals contents were determined by the ICP-MS (inductively coupled argon plasma mass spectrometer) method described by CEAEQ [2013].The dried powdered samples (5 g) were burned to ashes in a muffle furnace (Pyrolabo, France). The ashes obtained were dissolved in 10 mL of HCl/ HNO3 and transferred into 100 mL flasks and the volume was made up using deionized water. The mineral composition of each sample was determined using an Agilent 7500c argon plasma mass spectrometer. Calibrations were performed using external standards prepared from a 1000 ppm single stock solution made up with 2% nitric acid. Anti-nutritional factors determination Oxalates content was determined by using the method described by Day & Underwood [1986]. One (1) g of dried powdered sample was weighed into 100 mL conical flask. A quantity of 75 mL of sulphuric acid (3 mol/L) was added and stirred for 1 h with a magnetic stirrer. The mixture was filtered and 25 mL of the filtrate was titrated while hot against KMnO4 solution (0.05 mol/L) to the end point. Phytates contents were determined using the Wade's reagent colorimetric method [Latta & Eskin, 1980]. A quantity (1 g) of dried powdered sample was mixed with 20 mL of hydrochloric acid (0.65 N) and stirred for 12 h with a magnetic stirrer. The mixture was centrifuged at 12,000 rpm for 40 min. An aliquot (0.5 mL) of supernatant was added with 3 mL P.D. Oulai et al. TABLE 1. Proximate composition of raw and blanched leafy vegetables consumed in Northern Côte d'Ivoire. Ash (%) Fibres (%) Proteins (%) H. sabdariffa Raw 15 min 25 min 45 min 10.30±0.10a 9.62±0.15b 9.29±0.14c 8.99±0.03c 14.27±1.70a 14.32±1.50a 14.63±2.30a 15.50±2.47a 14.47±0.10a 13.22±0.16b 13.13±0.01b 12.83±0.04b A. hybridus Raw 15 min 25 min 45 min 8.59±1.34a 7.93±0.00a 7.60±0.03a 6.91±1.41b 17.80±0.30a 17.17±0.71a 17.25±0.35a 17.50±1.06a 13.25±0.13a 13.00±0.03a 12.29±0.06b 12.25±0.03b A. digitata Raw 15 min 25 min 45 min 10.97±0.40 Lipids (%) Carbohydrates (%) Calorific value (kcal/100 g) 4.75±0.15b 6.20±0.28a 6.40±0.14a 6.80±0.42a 56.21±1.78a 56.64±1.79a 56.56±2.59a 56.47±2.94a 275.71±0.55a 286.37±3.63a 287.52±8.05a 289.82±6.85a 2.15±0.01b 2.60±0.14b 3.18±0.03a 3.60±0.14a 58.21±1.78a 59.30±0.54a 59.28±0.30a 59.74±2.30a 305.19±7.73a 265.17±3.17b 269.23±1.45b 273.30±9.47b 12.56±0.45 18.08±0.10a 17.25±0.07b 14.88±0.06c 13.57±0.10d V unguiculata . 2.18± 0.03b 2.39±0.06b 2.99±0.03a 3.20±0.28a 56.23±1.25b 56.85±0.43b 59.23±0.26a 59.16±1.76a 267.03±4.00a 265.04±0.88a 272.80±1.30a 271.09±3.67a 10.76±0.05a 9.63±0.11b 9.32±0.08c 12.75±0.35a 13.27±0.25a 14.75±1.30a Raw 15 min 25 min 45 min 11.17±0.25 18.00±0.92 21.96±0.30a 21.88±0.03a 19.69±0.01b 19.26±0.01b C. patendra 4.23±0.25c 4.33±0.47c 4.68±0.35b 5.55±0.18a 44.64±1.72a 43.88±3.83a 45.80±2.60a 45.66±1.91a 248.35±10.33a 246.27±9.71a 250.73±6.28a 256.46±8.38a 11.16±0.86a 10.58±0.25b 10.19±0.22b 18.75±2.47a 19.25±2.47a 19.34±2.12a Raw 15 min 25 min 45 min 25.67±1.12a 23.10±0 42a 22.48±0.46b 22.20±0.00b 31.50±1.50a 31.94±1.33a 32.75±0.71a 33.52±0.78a 15.20±0.05a 13.37±0.10b 12.25±0.14c 11.38±0.10d 1.39±0.22d 2.59±0.83c 3.91±0.71b 4.69±0.64a 26.30±0.11a 29.00±2.69a 28.61±1.10a 28.21±1.51a 142.61±7.74c 157.83±2.37b 164.76±2.34a 167.73±0.17a of Wade's reagent. The reaction mixture was incubated for 15 min and absorbance was measured at 490 nm by using a spectrophotometer (PG Instruments, England). Phytates content was estimated using a calibration curve of sodium phytate (10 mg/mL) as a standard. Antioxidant properties Vitamin C and carotenoids determination Vitamin C contained in the analysed samples was determined by titration [Pongracz et al., 1995]. About 10 g of ground fresh leaves were soaked for 10 min in 40 mL metaphosphoric acid-acetic acid (2%, w/v). The mixture was centrifuged at 3000 rpm for 20 min and the supernatant obtained was diluted and adjusted with 50 mL of bi-distilled water. Ten (10) mL of this mixture were titrated to the end point with dichlorophenol-indophenol (DCPIP) 0.5 g/L. Carotenoids were extracted and quantified following the method described by Rodriguez-Amaya [2001]. Two (2) g of ground fresh leaves were mixed three times with 50 mL of acetone until loss of pigmentation. The mixture obtained was filtered and total carotenoids were extracted with 100 mL of petroleum ether. Absorbance of extracted fraction was then read at 450 nm by using a spectrophotometer (PG Instruments, England). Total carotenoids content was subsequently estimated using a calibration curve of -carotene (1 mg/mL) as a standard. Polyphenols determination Polyphenols were extracted and determined using Folin­ ­Ciocalteu's reagent [Singleton et al., 1999]. A quantity (1 g) of dried powdered sample was soaked in 10 mL of methanol 70% (w/v) and centrifuged at 1000 rpm for 10 min. An aliquot (1 mL) of supernatant was oxidized with 1 mL of Folin­Cio- TABLE 2. Mineral composition of raw and blanched leafy vegetables consumed in Northern Côte d'Ivoire. Mineral Ca Mg P H. sabdariffa Raw 15 min 25 min 45 min 402.21±0.55a 272.61±5.30b 270.64±5.53c 255.25±1.03d 295.93±0.41a 64.51±1.32b 59.34±0.24c 59.08±1.15c 407.59±0.00a 213.47±8.76b 189.16±3.87c 183.47±0.74c 816.19±1.12a 698.45±4.28b 647.88±2.63c 528.88±1.27d 102.08±0.14a 30.59±0.12b 26.66±0.52c 25.84±0.53c K Fe Na Zn 23.46±0.03a 22.31±0.09a 21.15±0.43a 18.34±0.36b 26.06±0.04a 9.85±0.20b 9.70±0.04b 9.20±0.18c A. hybridus Raw 15 min 25 min 45 min 932.60±0.55a 844.92±0.00b 830.30±3.35c 702.57±4.38d 497.75±0.49a 435.43±1.76b 387.91±0.00c 292.49±9.69d 368.69±0.00a 366.18±0.82a 343.36±7.00b 241.40±0.00c 1989.32±2.12a 1895.41±7.65b 1501.63±0.00c 1005.65±2.23d 77.88±0.05a 70.89±0.00b 66.49±0.27c 46.36±9.46d 94.39±0.04a 94.38±0.00a 94.31±0.38a 61.70±2.59b 31.73±0.04a 30.01±0.12a 24.86±0.00b 18.49±3.77c A. digitata Raw 15 min 25 min 45 min 496.26±2.20 264.36±1.17 761.63±0.00a 174.99±0.78b 94.66±0.86c 38.79±0.27d 1856.90±8.23a 1469.25±1.10b 1355.82±2.37c 1119.30±4.96d 106.27±0.47a 14.54±0.06b 14.41±0.10b 13.47±0.12c 37.13±0.12a 36.91±0.25a 33.49±0.31b 28.27±0.13c 22.61±0.10a 12.25±0.08b 11.79±0.05b 11.68±0.11b 445.81±1.98b 408.99±3.73c 401.78±2.76c 49.45±0.45b 48.75±0.34b 45.08±0.20c V unguiculata . Raw 15 min 25 min 45 min 439.54±0.56 341.34±0.18 309.04±0.00a 295.80±2.89a 289.63±0.36b 148.86±1.94b 718.11±0.91a 675.00±0.84b 566.82±9.56c 477.64±1.04d 91.45±0.12a 54.92±0.07b 45.62±3.18c 41.68±0.88c 33.32±0.02a 21.74±1.52b 20.55±0.43b 15.15±0.02c 40.83±0.04a 36.46±0.05b 24.03±0.51c 20.81±1.45d 424.71±2.64b 423.30±0.53b 402.33±8.46c 265.18± 0.33b 209.60±4.41c 208.14±4.53c C. patendra Raw 15 min 25 min 45 min 997.02±0.55a 264.88±7.86b 258.28±0.00b 240.21±4.99c 773.55±0.43a 136.09±0.00b 106.08±2.20c 104.51±1.92c 570.85±2.11a 145.76±3.45b 89.86±0.00c 74.36±0.79d 1585.58±0.87a 921.92±0.00b 787.84±4.47c 694.08±4.42d 219.84±0.12a 20.60±0.38b 19.81±0.00b 17.36±0.36c 42.69±0.02a 26.73±0.49b 18.32±0.38c 15.55±0.00d 35.68±0.02a 9.48±0.17b 8.45±0.00c 7.57±0.16d calteu's reagent and neutralized by 1 mL of 20% (w/v) sodium carbonate. The reaction mixture was incubated for 30 min at ambient temperature and absorbance was measured at 745 nm by using a spectrophotometer (PG Instruments, England). The polyphenols content was obtained using a calibration curve of gallic acid (1 mg/mL) as a standard. Antioxidant activity Antioxidant activity assay was carried out using the 2,2-diphenyl-1-pycrilhydrazyl (DPPH) spectrophotometric method [Choi et al., 2002]. About 1 mL of 0.3 mmol/L DPPH solution in ethanol was added to 2.5 mL of sample solution (1 g of dried powdered sample mixed in 10 mL of methanol and filtered through Whatman No. 4 filter paper) and was allowed to react for 30 min at room temperature. Absorbance values were measured with a spectrophotometer (PG Instruments, England) set at 415 nm. The average absorbance val- ues were converted to percentage antioxidant activity using the following formula: Antioxidant activity (%) = 100 ­ [(Abs of sample ­ Abs of blank) x 100/Abs positive control] Statistical analysis All the analyses were performed in triplicate and data were analyzed using EXCELL and STATISTICA 7.1 (StatSoft). Differences between means were evaluated by Duncan's test. Statistical significant difference was stated at p<0.05. RESULTS AND DISCUSSION Proximate composition of the selected leafy vegetables is presented in Table 1. The physicochemical parameters generally differed significantly (p<0.05) from a blanching P.D. Oulai et al. TABLE 3. Anti-nutritional factors/mineral ratios of raw and blanched leafy vegetables consumed in Northern Côte d'Ivoire. Phytates/Ca Phytates/Fe Oxalates/Ca a b c a b d a c d b c d d a b c c H. sabdariffa H. hybridus A. digitata V. unguiculata C. pentadra a b c (A) Raw 15 min 25 min 45 min Oxalates (mg/100g) H. sabdariffa Raw 15 min 25 min 45 min 0.21±0.01a 0.10±0.01b 0.03±0.00c 0.01±0.00c 0.85±0.10a 0.90±0.01a 0.29±0.01b 0.10±0.01c 3.26±0.10a 0.10±0.00b 0.03±0.00c 0.01±0.00c 100 90 a (B) Raw 15 min a b a a b c d b c d b c a d b c d c d 25 min 45 min 80 70 60 50 40 30 20 10 0 H. sabdariffa H. hybridus A. digitata V. unguiculata C. pentadra A. hybridus Raw 15 min 25 min 45 min 0.03±0.00 0.41±0.01 0.07±0.00 0.01±0.00a 0.01±0.00a 0.01±0.00a 0.15±0.01b 0.15±0.01b 0.16±0.01b 0.01±0.00a 0.01±0.00a 0.01±0.00a A. digitata Raw 15 min 25 min 45 min 0.04±0.00a 0.04±0.00a 0.03±0.00 0.02±0.00 a a 0.19±0.01d 1.22±0.01a 0.94±0.01 b c 1.57±0.01a 0.04±0.00b 0.03±0.00 FIGURE 1. Oxalates (A) and phytates (B) contents of raw and blanched leafy vegetables consumed in Northern Côte d'Ivoire. Data are represented as Means ± SD (n=3). Means with no common letter (a, b, c, d) differ significantly (p<0.05) for each leafy vegetable. 0.69±0.01 0.02±0.00b V unguiculata . Raw 15 min 25 min 45 min 0.04±0.00a 0.03±0.00a 0.02±0.00a 0.02±0.00a 0.19±0.01b 0.23±0.01a 0.23±0.01a 0.22±0.01a 1.66±0.10a 0.03±0.00b 0.02±0.00b 0.02±0.00b C. patendra Raw 15 min 25 min 45 min 0.04±0.00b 0.09±0.00a 0.07±0.00a 0.07±0.00a 0.17±0.01c 1.12±0.01a 0.95±0.01b 1.02±0.10b 0.78±0.01a 0.09±0.00b 0.07±0.00b 0.07±0.00b time of a leafy vegetable to another. The ash content after 15 min of blanching ranged from 7.93±0.00% (A. hybridus) to 23.10±0.42% (C. patendra) with a decrease rate at 15 min ranged from 0.08 to 10.01%. After 15 min of blanching, the ash losses increased in the order: V unguiculata (0.08%) > A. digi. tata (1.91%) > H. sabdariffa (6.60%) > A. hybridus (7.68%) > C. patendra (10.01%). As concerns protein contents, blanching processing caused 0.36 to 12.03% reduction after 15 min. These protein losses increased in the order: V unguiculata . (0.36%) > A. hybridus (1.88%) > A. digitata (4.59%) > H. sabdariffa (8.64%) > C. patendra (12.03%). This reduced protein content could be attributed to the severity of thermal process during blanching which leads to protein degradation [Lund, 1997]. The ash and protein content losses are lower than that (9.78­28.0%) reported for 15 min cooked Nigerian leafy veg- etables [Lola, 2009]. This fact could be explained by the agronomic cultural conditions and the period of leafy vegetables harvesting. In spite of the ash losses levels, the studied leafy vegetables may be considered as good sources of minerals when compared to values obtained for cereals and tubers [Antia et al., 2006]. Moreover, the leaves of Vigna unguiculata revealed the higher retention (99.70­99.92%) of ash and proteins after 15 min of blanching. All selected leafy vegetables highlighted a slight increase (p>0.05) in their crude fibres content (0.35­4.16%) at 15 min of blanching. Indeed, the increased temperature during blanching leads to breakage of weak bonds between polysaccharides and the cleavage of glycosidic linkages, which may result in solubilization of the dietary fibres [Svanberg et al., 1997]. With regard to their fibres content at 15 min (12.75­31.94%), adequate intake of blanched leafy vegetables could lower the risk of constipation, diabetes, colon and breast cancer [Ishida et al., 2000]. The relatively low values of lipids contents at 15 min of blanching (2.39­6.20%) corroborate the findings of many authors which showed that leafy vegetables are poor sources of fat [Ejoh et al., 1996]. The estimated calorific values agreed with general observation that vegetables have low energy values due to their low crude fat and relatively high level of moisture [Sobowale et al., 2011]. Mineral composition of blanched leafy vegetables used in this study is shown in Table 2. The residual contents of minerals after 15 min of blanching were significantly different (p<0.05): calcium (264.88­844.92 mg/100 g), magnesium (49.45­435.43 mg/100 g), potassium (675­1895.41 mg/100 g), iron (14.54­70.89 mg/100 g) and zinc (9.48­ ­36.46 mg/100 g). With regard to these values, the consumption of 15 min blanched leafy vegetables could cover at least 50% of the standard mineral requirements for human. Indeed, these standard requirements are: calcium (1000 mg/ Phytates (mg/100g) 90 a a 15 min b a b c c d b b b d b c d b c d a 25 min 45 min (A) Vitamin C (mg/100g) Polyphenols (mg/100g) 80 70 60 50 40 30 20 10 0 H. sabdariffa A. hybridus a 350 300 250 200 150 100 50 0 b b b a a Raw a a a (A) b a a Raw 15 min b a c b c c b a 25 min 45 min A. digitata V. unguiculata C. pentadra H. sabdariffa A. hybridus A. digitata V. unguiculata C. pentadra Antioxydant activity (%) Carotenoids (mg/100g) (B) a a a b b c c d H. sabdariffa A. hybridus b b b a c b b b d a b b b A. digitata V. unguiculata C. pentadra b Raw 15 min 25 min 45 min c b a a b a c a b a b a a a b b a a (B) Raw 15 min 25 min 45 min c d H. sabdariffa A. hybridus A. digitata V. unguiculata C. pentadra FIGURE 2. Vitamin C (A) and carotenoids (B) contents of raw and blanched leafy vegetables consumed in Northern Côte d'Ivoire. Data are represented as Means ± SD (n=3). Means with no common letter (a, b, c, d) differ significantly (p<0.05) for each leafy vegetable. FIGURE 3. Polyphenols contents (A) and antioxidant activity (B) of raw and blanched leafy vegetables consumed in Northern Côte d'Ivoire. Data are represented as Means ± SD (n=3). Means with no common letter (a, b, c, d) differ significantly (p<0.05) for each leafy vegetable. day); magnesium (400 mg/day), iron (8 mg/day) and zinc (6 mg/day) [FAO, 2004]. Calcium and phosphorus play an important role in growth and maintenance of bones, teeth and muscles [Turan et al., 2003]. As concerns magnesium, this mineral is known to prevent cardiomyopathy, muscle degeneration, growth retardation, congenital malformations and bleeding disorders [Chaturvedi et al., 2004]. Iron plays an important role in the prevention of anemia while zinc is important for vitamin A and vitamin E metabolism [Martorell & Trowbridge, 2002; FAO, 2004]. To predict the bioavailability of calcium and iron, anti-nutrients to nutrients ratios of blanched leafy vegetables were calculated (Table 3). The calculated [phytates]/[Ca] and [oxalates]/[Ca] ratios in all the blanched species were below the critical level of 2.5 which is known to impair calcium bioavailability [Hassan et al., 2007; Umar et al., 2007]. The effect of blanching on anti-nutritional factors (oxalates and phytates) contents is depicted in Figure 1. The losses (p<0.05) at 15 min were 3.58­21.39% and 10.51­68.02% for oxalates and phytates, respectively. Oxalates losses increased in the order: A. digitata (3.59%) > A. hybridus (8.33%) >V . unguiculata (11.87%) > C. patendra (20.70%) > H. sabdariffa (21.39%) while phytates losses increased in the order: A. digitata (10.51%) > V unguiculata (26.89%) > C. patendra . (39.85%) > A. hybridus (65.89%) > H. sabdariffa (68.02%). These percentage losses are similar to those (7­56%) of phytates obtained for blanched leafy vegetables from Thailand [Samsub et al., 2008]. The reductions in oxalates and phytates contents during blanching could improve the health status of consumers. Indeed, oxalates and phytates chelate divalent cations such as calcium, magnesium, zinc and iron, thereby reducing their bioavailability [Sandberg, 2002]. In view of the highest oxalates and phytates losses (21.39 and 68.02%) of Hibiscus sabdariffa leaves, blanching process- ing may appear as a detoxification procedure by removing these anti-nutritional factors [Ekop & Eddy, 2005]. Blanching of the studied leafy vegetables also resulted in a decrease of carotenoids and vitamin C contents (Figure 2). For carotenoids, losses at 15 min were estimated to 18.91 to 55.48%. Carotenoids losses increased in the order: A. hybridus (18.91%) > C. patendra (23.41%) > H. sabdariffa (33.61%) > A. digitata (43.19%) > V. unguiculata (55.48%). The decrease in carotenoids contents could be attributed to the oxidation and isomerization of -carotene [Speek et al., 1986]. Carotenoids are considered as sources of provitamin A in plants and their amount determines their bioavailability in a human diet [Rodriguez-Amaya, 2001]. Therefore, consumption of blanched leafy with fat added, could contribute significantly to improving the vitamin A status of children [Takyi, 1999]. For vitamin C content, a significant reduction (19.56­68.67%) was highlighted at 15 min during blanching processing (Figure 2). Vitamin C losses increased in the order: H. sabdariffa (19.57%) > A. digitata (46.58%) > V. unguiculata (61.13%) > C. patendra (62.5%) > A. hybridus (68.67%). This decrease in vitamin C is lower than that (60­ ­90%) reported for blanched Indian leafy vegetables [Gupta et al., 2008] but agrees with earlier findings of Oboh [2005] on some Nigerian vegetables who reported 47.5­82.4% loss in vitamin C content during blanching. It is important to note that ascorbic acid is a heat-labile and water-soluble antioxidant that promotes absorption of soluble iron by chelating or by maintaining the iron in the reduced form [Yamaguchi et al., 2001]. With regard to the decrease of vitamin C, consumption of blanched leafy vegetables may be supplemented with other sources of vitamin C such as tropical fruits to cover the daily demand for humans (40 mg/day) as recommended by the Food Agriculture Organization [FAO, 2004]. The significant reduction of antioxidants components such P.D. Oulai et al. 4. Canet W., Quality and stability of frozen vegetables. 1989, in: Developments in Food Preservation (ed. S. Thorne), vol. 5, New York: Elsevier Science Publishing Inc., p. 50. 5. CEAEQ, Détermination des métaux. Méthode par spectrométrie de masse à source ionisante au plasma d'argon. MA 200 ­ Met 1.2, Rev 4. Quebec, 2013, p. 24. 6. Chaturvedi V.C., Shrivastava R., Upreti R.K., Viral infections and trace elements: A complex trace element. Current Sci., 2004, 87, 1536­1554. 7. Chima C.E., Igyor M.A., Micro-nutriments and anti-nutritional content of select tropical vegetables grown in south-east, Nigeria. Nig. Food J., 2007, 25, 111­116. 8. Choi C.W., Kim S.C., Hwang S.S., Choi B.K., Ahn H.J., Lee M.Z., Park S.H., Kim S.K., Antioxidant activity and free radical scavenging capacity between Korean medicinal plant and flavonoids by assay guided comparison. Plant Sci., 2002, 163, 1161­1168. 9. Chweya J.A., Identification and nutritional importance of indigenous green leafy vegetables in Kenya. Acta Hort., 1985, 153, 99­108. 10. Day R.A., Underwood A.L., Quantitative Analysis. 1986, 5th ed. Prentice Hall, p. 701. 11. Dewanto V., Wu X., Adom K.K., Liu R.H., Thermal processing enhances the nutritional value of tomatoes by increasing total anti- oxidant activity. J. Agric. Food Chem., 2002, 50, 3010­3014. 12. Ejoh A.R., Tchouanguep M.F., Fokou E., Nutrient composition of the leaves and flowers of Colocasia esculenta and the fruits of Solanum melongena. Plant Food Human Nutr., 1996, 49, 107­112. 13. Ekop A.S., Eddy N.O., Comparative studies of the level of toxicants in the seed of Indian almond (Terminalia catappa) and African walnut (Coula edulis). Chem. Class J., 2005, 2, 74­76. 14. FAO, Food and Agriculture Organization. Food energy-methods of analysis and conversion factors. FAO Ed, Rome, 2002, p. 97. 15. FAO, Human vitamin and mineral requirements. FAO Ed, 2004, p. 361. 16. Fondio L., Kouamé C., N'zi J.C., Mahyao A., Agbo E., Djidji A.H., Survey of indigenous leafy vegetable in the urban and periurban areas of Côte d'Ivoire. Acta Hort., 2007, 752, 287­289. 17. Gupta S., Lakshimi J.A., Prakash J., Effect of different blanching treatments on ascorbic acid retention in green leafy vegetables. Nat. Prod. Radiance, 2008, 7, 111­116. 18. Hassan L.G., Umar K.J., Umar Z., Anti-nutritive factors in Tribulus terrestris (Linn) leaves and predicted calcium and zinc bioavailability. J. Trop. Biosci., 2007, 7, 33­36. 19. Ishida H., Suzuno H., Sugiyama N., Innami S., Todokoro T., Maekawa A., Nutritional evaluation of chemical component of leaves stalks and stems of sweet potatoes (Ipomea batatas). Food Chem., 2000, 68, 359­367. 20. Kays S.J., Dias J.S., Common names of commercially cultivated vegetables of the world in 15 languages. Econom Bot., 1995, 49, 115­52. 21. Latta M., Eskin M., A simple and rapid colorimetric method for phytate determination. Journal of Agriculture and Food Chem., 1980, 28, 1313­1315. 22. Lund D.B., Effects of heat processing on nutrients. 1997, in: Nutritional Evaluation of Food Processing (eds. R. Harries, E. Karmas). The AVI Publishing Co. Inc Westport, pp. 205­203. 23. Lola A., The effect of boiling on the nutrients and anti-nutrients in two non-conventional vegetables. Pak. J. Nutr., 2009, 8, 1430­ ­1433. as carotenoids and vitamin C in the studied leafy vegetables could also be due to the preliminary oven-drying treatment used. Indeed, higher temperatures affect negatively carotenoids and vitamin C by leading to their partial destruction [Rice-Evans & Miller, 1995]. This justifies the traditional practices which consist in drying leafy vegetables at ambient temperature in order to preserve their antioxidant properties. The effect of blanching on polyphenols content and antioxidant activity of the selected leafy vegetables is depicted in Figure 3. It was observed a high increase of polyphenols contents varying from 1.61 to 30.72%. The percent gain in the total phenol content during blanching may be due to the breakdown of tough cell walls and release of phenolic compounds trapped in the fibres of green leafy vegetables [Oboh & Rocha, 2007]. This result agrees with earlier report by Dewanto et al. [2002], where ferulic acid, a phenolic component found in the cell wall of grains such as corn, wheat and oats, doubled after 10 min of blanching. The content in polyphenols which is linked to the antioxidant activity of the studied blanched leafy vegetables could be advantageous for lower cellular oxidative stress, which has been implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis [Rice-Evans & Miller, 1995; Amic et al., 2003]. CONCLUSION African leafy vegetables (ALVs) contain significant levels of nutrients that are essential for human health. The result of this study revealed that blanching at 15, 25 and 45 min decreased considerably the nutritional value of these leafy vegetables. Nevertheless, the losses in anti-nutrients (oxalates, phytates) might have asserted a beneficial effect on the bioavailability of minerals like calcium, iron and zinc. Thus, the study suggests that the recommended time of domestic blanching must be less than 15 min for the studied leafy vegetables in order to contribute efficiently to the nutritional requirement and to the food security of Ivorian population. ACKNOWLEDGEMENT This work was completely conducted at Félix Houphouet-Boigny University (Biotechnology Laboratory, Biosciences Department). This work was also supported by a PhD financial support from the Higher Education and Scientific Research Ministry of Côte d'Ivoire. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Polish Journal of Food and Nutrition Sciences de Gruyter

Evaluation of Nutritive and Antioxidant Properties of Blanched Leafy Vegetables Consumed in Northern Côte d’Ivoire

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Pol. J. Food Nutr. Sci., 2015, Vol. 65, No. 1, pp. 31­38 DOI: 10.1515/pjfns-2015-0003 http://journal.pan.olsztyn.pl Original article Food Quality and Functionality Evaluation of Nutritive and Antioxidant Properties of Blanched Leafy Vegetables Consumed in Northern Côte d'Ivoire Patricia D. Oulai, Lessoy T. Zoué*, Sébastien L. Niamké Laboratoire de Biotechnologies, UFR Biosciences, Université Félix Houphouët-Boigny, 22 BP 582, Abidjan 22, Côte d'Ivoire Key words: antioxidant properties, blanching processing, leafy vegetables, nutritive value The aim of this study was to evaluate the effect of steam blanching processing on the nutritive value and the antioxidant properties of five leafy vegetable species (Amaranthus hybridus, Andasonia digitata, Ceiba patendra, Hibiscus sabdariffa and Vigna unguiculata) that are used for sauce preparation in Northern Côte d'Ivoire. The selected leafy vegetables were subjected to blanching in pressure cooker for 15, 25 and 45 min and the physicochemical properties were determined using AOAC methods. The result of the study revealed that longer time of blanching (higher than 15 min) caused negative impact by reducing nutritive value but positive impact by reducing anti-nutrients and increasing polyphenols. The registered losses (p<0.05) at 15 min were as follow: ash (0.08­10.01%), proteins (0.36­12.03%), vitamin C (19.56­68.67%), carotenoids (18.91­55.48%) oxalates (3.58­21.39%) and phytates (10.51­68.02%). The average increase of polyphenols contents at 15 min of blanching was 1.61 to 30.72%. In addition, a slight increase (0.35­4.16%) of fibres content was observed in the studied blanched leafy vegetables. Furthermore, after 15 min of blanching time the residual contents (p<0.05) of minerals were: calcium (264.88­844.92 mg/100 g), magnesium (49.45­435.43 mg/100 g), potassium (675­1895.41 mg/100 g), iron (14.54­70.89 mg/100 g) and zinc (9.48­36.46 mg/100 g). All these results suggest that the recommended time of domestic blanching must be less than 15 min for the studied leafy vegetables in order to contribute efficiently to the nutritional requirement and to the food security of Ivorian population. INTRODUCTION The growing awareness in recent years of the health promoting has directed increased attention to vegetables as vital components of daily diets [Smith, 2007]. A world vegetable survey showed that 402 vegetable crops are cultivated worldwide, representing 69 families and leafy vegetables which leaves are consumed, are the most (53% of the total) often utilized [Kays, 1995]. The traditional leafy vegetables are rich in micronutrients needed by humans for good health, growth and could contribute significantly to food security, which is a prerequisite for human development. Indeed, Obel-Lawson [2005] reported that an increased consumption of African leafy vegetables (ALVs) can have a positive effect on nutrition, health and economic wellbeing of both rural and urban populations. Socio-economic surveys conducted in various parts of Africa indicate also that ALVs are important commodities in household food and nutrition security [Mnzava, 1997]. Fresh leaves of most ALVs like vegetable amaranths (Amaranthus), slenderleaf (Crotalaria brevidens), spiderplant (Chlorophytum comosum), vegetable cowpeas (Vigna), pumpkin leaves (cucurbits) and jute mallow (Corchorus) contain more than 100% of the recommended daily allowances for vitamins and minerals and 40% proteins for growing children and lactating mothers [Chweya, 1985]. * Corresponding Author: y.lessoy@yahoo.fr; Tel: +(225) 08 98 67 44 The high moisture content of fresh leafy vegetables renders them perishable and seasonal availability limits their utilization all around the year. Hence, there is a need to preserve this nature's store house of nutrients through proper processing techniques for safe storage with efficient nutrient retention [Gupta et al., 2008]. One common processing used before consumption of leafy vegetables is blanching which is a thermal treatment commonly applied to a variety of vegetables before freezing. Its primary purpose is to inactivate enzymes and destroy vegetative microbial cells, allowing stabilization and product quality retention during frozen storage [Canet, 1989]. It is usually carried out in hot water or in steam; this technique is used by indigenous people to reduce or eliminate the bitterness of the vegetables and acid components that are common in leaves. Blanching affords also a series of secondary benefits, due to its washing action, such as elimination of off-flavors that may have been formed during the time between harvesting and processing, and removal of any residual pesticides [Prestamo et al., 1998]. Blanching, however, has some adverse effects, such as pigment modifications, tissues softening and nutrient losses [Murcia et al., 1999; Oboh, 2005]. Among the twenty hundred and seven (207) leafy vegetables widely consumed in tropical Africa, about twenty (20) species of leafy vegetables belonging to 6 botanical families are widely consumed and cultivated by Ivorian populations [PROTA, 2004; Fondio et al., 2007]. Furthermore, the consumption of these leafy vegetables is linked to the region © Copyright by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences © 2015 Author(s). This is an open access article licensed under the Creative Commons Attribution-NonCommercial-NoDerivs License (http://creativecommons.org/licenses/by-nc-nd/3.0/). and ethno-botanical studies have stated that most people in Northern Côte d'Ivoire consume indigenous green leafy vegetables such as Amaranthus hybridus "boronbrou", Andasonia digitata "baobab", Ceiba patendra "fromager", Hibiscus sabdariffa "dah" and Vigna unguiculata "haricot" [Fondio et al., 2007; Soro et al., 2012]. Indeed, dietary habits of these populations include culinary preparation of these leafy vegetables as follow: the mature and freshly leaves are boiled in water for about 30 min in order to reduce bitter taste and then used, after discarding boiled water, for sauce preparation that accompany starchy sorghum paste food commonly named "to". Earlier reports have highlighted the nutritive potential of these fresh leafy vegetables [Oulai et al., 2014] but there is a lack of scientific data with regards to the effect of blanching processing on their physicochemical and nutritive characteristics. Therefore, the purpose of this study is to conduct investigation on the effect of blanching on the nutritive value of these selected leafy vegetables in order to provide necessary information for their wider utilization and contribution to food security of Ivorian populations. MATERIAL AND METHODS Samples collection Leafy vegetables (Amaranthus hybridus, Andasonia digitata, Ceiba patendra, Hibiscus sabdariffa and Vigna unguiculata) were collected fresh and at maturity from cultivated farmlands located at Dabou (latitude: 5°19'14" North; longitude: 4°22'59" West) (Abidjan District). The samples were harvested at the early stage (between one and two weeks of the appearance of the leaves). These plants were previously authenticated by the National Floristic Center (University Felix Houphouët-Boigny, Abidjan-Côte d'Ivoire). Samples processing The fresh leafy vegetables were rinsed with deionized water and the edible portions were separated from the inedible portion. The edible portions were chopped into small pieces (500 g) and allowed to drain at ambient temperature. Each sample was divided into two lots. The first lot (raw, 250 g) was dried in an oven (Memmert, Germany) at 60°C for 72 h [Chima & Igyor, 2007]. The dried leaves were ground with a laboratory crusher (Culatti, France) equipped with a 10 m mesh sieve. Each sample was stored in a clean dry air-tight sample bottle in a refrigerator (4°C) until required for analyses. The second lot (250 g) was steam-blanched for 15, 25 and 45 min in a pressure cooker. The blanched samples were cooled, drained at ambient temperature and subjected to the same treatment used for raw samples. Chemicals All solvents (n-hexane, petroleum ether, acetone, ethanol and methanol) were purchased from Merck. Standards used (gallic acid, -carotene) and reagents (metaphosphoric acid, Folin-Ciocalteu, DPPH) were purchased from Sigma-Aldrich. All chemicals used in the study were of analytical grade. Nutritive properties Proximate analysis Proximate analysis was performed using the AOAC [1990] methods. The moisture content was determined by the difference of weight before and after drying the sample (10 g) in an oven (Memmert, Germany) at 105°C until constant weight. Ash fraction was determined by the incineration of dried sample (5 g) in a muffle furnace (Pyrolabo, France) at 550°C for 12 h. The percentage residue weight was expressed as ash content. For crude fibres, 2 g of sample were weighed into separate 500 mL round bottom flasks and 100 mL of 0.25 mol/L sulphuric acid solution was added. The mixture obtained was boiled under reflux for 30 min. Thereafter, 100 mL of 0.3 mol/L sodium hydroxide solution was added and the mixture was boiled again under reflux for 30 min and filtered through Whatman paper. The insoluble residue was then incinerated, and weighed for the determination of crude fibres content. Proteins were determined through the Kjeldhal method and the lipid content was determined by Soxhlet extraction using hexane as a solvent. Carbohydrates and calorific value were calculated using the following formulas [FAO, 2002]: Carbohydrates: 100 ­ (% moisture + % proteins + % lipids + % ash + % fibres) Calorific value: (% proteins x 2.44) + (% carbohydrates x 3.57) + (% lipids x 8.37) The results of ash, fibres, proteins, lipids and carbohydrates contents were expressed on dry matter basis. Mineral analysis Minerals contents were determined by the ICP-MS (inductively coupled argon plasma mass spectrometer) method described by CEAEQ [2013].The dried powdered samples (5 g) were burned to ashes in a muffle furnace (Pyrolabo, France). The ashes obtained were dissolved in 10 mL of HCl/ HNO3 and transferred into 100 mL flasks and the volume was made up using deionized water. The mineral composition of each sample was determined using an Agilent 7500c argon plasma mass spectrometer. Calibrations were performed using external standards prepared from a 1000 ppm single stock solution made up with 2% nitric acid. Anti-nutritional factors determination Oxalates content was determined by using the method described by Day & Underwood [1986]. One (1) g of dried powdered sample was weighed into 100 mL conical flask. A quantity of 75 mL of sulphuric acid (3 mol/L) was added and stirred for 1 h with a magnetic stirrer. The mixture was filtered and 25 mL of the filtrate was titrated while hot against KMnO4 solution (0.05 mol/L) to the end point. Phytates contents were determined using the Wade's reagent colorimetric method [Latta & Eskin, 1980]. A quantity (1 g) of dried powdered sample was mixed with 20 mL of hydrochloric acid (0.65 N) and stirred for 12 h with a magnetic stirrer. The mixture was centrifuged at 12,000 rpm for 40 min. An aliquot (0.5 mL) of supernatant was added with 3 mL P.D. Oulai et al. TABLE 1. Proximate composition of raw and blanched leafy vegetables consumed in Northern Côte d'Ivoire. Ash (%) Fibres (%) Proteins (%) H. sabdariffa Raw 15 min 25 min 45 min 10.30±0.10a 9.62±0.15b 9.29±0.14c 8.99±0.03c 14.27±1.70a 14.32±1.50a 14.63±2.30a 15.50±2.47a 14.47±0.10a 13.22±0.16b 13.13±0.01b 12.83±0.04b A. hybridus Raw 15 min 25 min 45 min 8.59±1.34a 7.93±0.00a 7.60±0.03a 6.91±1.41b 17.80±0.30a 17.17±0.71a 17.25±0.35a 17.50±1.06a 13.25±0.13a 13.00±0.03a 12.29±0.06b 12.25±0.03b A. digitata Raw 15 min 25 min 45 min 10.97±0.40 Lipids (%) Carbohydrates (%) Calorific value (kcal/100 g) 4.75±0.15b 6.20±0.28a 6.40±0.14a 6.80±0.42a 56.21±1.78a 56.64±1.79a 56.56±2.59a 56.47±2.94a 275.71±0.55a 286.37±3.63a 287.52±8.05a 289.82±6.85a 2.15±0.01b 2.60±0.14b 3.18±0.03a 3.60±0.14a 58.21±1.78a 59.30±0.54a 59.28±0.30a 59.74±2.30a 305.19±7.73a 265.17±3.17b 269.23±1.45b 273.30±9.47b 12.56±0.45 18.08±0.10a 17.25±0.07b 14.88±0.06c 13.57±0.10d V unguiculata . 2.18± 0.03b 2.39±0.06b 2.99±0.03a 3.20±0.28a 56.23±1.25b 56.85±0.43b 59.23±0.26a 59.16±1.76a 267.03±4.00a 265.04±0.88a 272.80±1.30a 271.09±3.67a 10.76±0.05a 9.63±0.11b 9.32±0.08c 12.75±0.35a 13.27±0.25a 14.75±1.30a Raw 15 min 25 min 45 min 11.17±0.25 18.00±0.92 21.96±0.30a 21.88±0.03a 19.69±0.01b 19.26±0.01b C. patendra 4.23±0.25c 4.33±0.47c 4.68±0.35b 5.55±0.18a 44.64±1.72a 43.88±3.83a 45.80±2.60a 45.66±1.91a 248.35±10.33a 246.27±9.71a 250.73±6.28a 256.46±8.38a 11.16±0.86a 10.58±0.25b 10.19±0.22b 18.75±2.47a 19.25±2.47a 19.34±2.12a Raw 15 min 25 min 45 min 25.67±1.12a 23.10±0 42a 22.48±0.46b 22.20±0.00b 31.50±1.50a 31.94±1.33a 32.75±0.71a 33.52±0.78a 15.20±0.05a 13.37±0.10b 12.25±0.14c 11.38±0.10d 1.39±0.22d 2.59±0.83c 3.91±0.71b 4.69±0.64a 26.30±0.11a 29.00±2.69a 28.61±1.10a 28.21±1.51a 142.61±7.74c 157.83±2.37b 164.76±2.34a 167.73±0.17a of Wade's reagent. The reaction mixture was incubated for 15 min and absorbance was measured at 490 nm by using a spectrophotometer (PG Instruments, England). Phytates content was estimated using a calibration curve of sodium phytate (10 mg/mL) as a standard. Antioxidant properties Vitamin C and carotenoids determination Vitamin C contained in the analysed samples was determined by titration [Pongracz et al., 1995]. About 10 g of ground fresh leaves were soaked for 10 min in 40 mL metaphosphoric acid-acetic acid (2%, w/v). The mixture was centrifuged at 3000 rpm for 20 min and the supernatant obtained was diluted and adjusted with 50 mL of bi-distilled water. Ten (10) mL of this mixture were titrated to the end point with dichlorophenol-indophenol (DCPIP) 0.5 g/L. Carotenoids were extracted and quantified following the method described by Rodriguez-Amaya [2001]. Two (2) g of ground fresh leaves were mixed three times with 50 mL of acetone until loss of pigmentation. The mixture obtained was filtered and total carotenoids were extracted with 100 mL of petroleum ether. Absorbance of extracted fraction was then read at 450 nm by using a spectrophotometer (PG Instruments, England). Total carotenoids content was subsequently estimated using a calibration curve of -carotene (1 mg/mL) as a standard. Polyphenols determination Polyphenols were extracted and determined using Folin­ ­Ciocalteu's reagent [Singleton et al., 1999]. A quantity (1 g) of dried powdered sample was soaked in 10 mL of methanol 70% (w/v) and centrifuged at 1000 rpm for 10 min. An aliquot (1 mL) of supernatant was oxidized with 1 mL of Folin­Cio- TABLE 2. Mineral composition of raw and blanched leafy vegetables consumed in Northern Côte d'Ivoire. Mineral Ca Mg P H. sabdariffa Raw 15 min 25 min 45 min 402.21±0.55a 272.61±5.30b 270.64±5.53c 255.25±1.03d 295.93±0.41a 64.51±1.32b 59.34±0.24c 59.08±1.15c 407.59±0.00a 213.47±8.76b 189.16±3.87c 183.47±0.74c 816.19±1.12a 698.45±4.28b 647.88±2.63c 528.88±1.27d 102.08±0.14a 30.59±0.12b 26.66±0.52c 25.84±0.53c K Fe Na Zn 23.46±0.03a 22.31±0.09a 21.15±0.43a 18.34±0.36b 26.06±0.04a 9.85±0.20b 9.70±0.04b 9.20±0.18c A. hybridus Raw 15 min 25 min 45 min 932.60±0.55a 844.92±0.00b 830.30±3.35c 702.57±4.38d 497.75±0.49a 435.43±1.76b 387.91±0.00c 292.49±9.69d 368.69±0.00a 366.18±0.82a 343.36±7.00b 241.40±0.00c 1989.32±2.12a 1895.41±7.65b 1501.63±0.00c 1005.65±2.23d 77.88±0.05a 70.89±0.00b 66.49±0.27c 46.36±9.46d 94.39±0.04a 94.38±0.00a 94.31±0.38a 61.70±2.59b 31.73±0.04a 30.01±0.12a 24.86±0.00b 18.49±3.77c A. digitata Raw 15 min 25 min 45 min 496.26±2.20 264.36±1.17 761.63±0.00a 174.99±0.78b 94.66±0.86c 38.79±0.27d 1856.90±8.23a 1469.25±1.10b 1355.82±2.37c 1119.30±4.96d 106.27±0.47a 14.54±0.06b 14.41±0.10b 13.47±0.12c 37.13±0.12a 36.91±0.25a 33.49±0.31b 28.27±0.13c 22.61±0.10a 12.25±0.08b 11.79±0.05b 11.68±0.11b 445.81±1.98b 408.99±3.73c 401.78±2.76c 49.45±0.45b 48.75±0.34b 45.08±0.20c V unguiculata . Raw 15 min 25 min 45 min 439.54±0.56 341.34±0.18 309.04±0.00a 295.80±2.89a 289.63±0.36b 148.86±1.94b 718.11±0.91a 675.00±0.84b 566.82±9.56c 477.64±1.04d 91.45±0.12a 54.92±0.07b 45.62±3.18c 41.68±0.88c 33.32±0.02a 21.74±1.52b 20.55±0.43b 15.15±0.02c 40.83±0.04a 36.46±0.05b 24.03±0.51c 20.81±1.45d 424.71±2.64b 423.30±0.53b 402.33±8.46c 265.18± 0.33b 209.60±4.41c 208.14±4.53c C. patendra Raw 15 min 25 min 45 min 997.02±0.55a 264.88±7.86b 258.28±0.00b 240.21±4.99c 773.55±0.43a 136.09±0.00b 106.08±2.20c 104.51±1.92c 570.85±2.11a 145.76±3.45b 89.86±0.00c 74.36±0.79d 1585.58±0.87a 921.92±0.00b 787.84±4.47c 694.08±4.42d 219.84±0.12a 20.60±0.38b 19.81±0.00b 17.36±0.36c 42.69±0.02a 26.73±0.49b 18.32±0.38c 15.55±0.00d 35.68±0.02a 9.48±0.17b 8.45±0.00c 7.57±0.16d calteu's reagent and neutralized by 1 mL of 20% (w/v) sodium carbonate. The reaction mixture was incubated for 30 min at ambient temperature and absorbance was measured at 745 nm by using a spectrophotometer (PG Instruments, England). The polyphenols content was obtained using a calibration curve of gallic acid (1 mg/mL) as a standard. Antioxidant activity Antioxidant activity assay was carried out using the 2,2-diphenyl-1-pycrilhydrazyl (DPPH) spectrophotometric method [Choi et al., 2002]. About 1 mL of 0.3 mmol/L DPPH solution in ethanol was added to 2.5 mL of sample solution (1 g of dried powdered sample mixed in 10 mL of methanol and filtered through Whatman No. 4 filter paper) and was allowed to react for 30 min at room temperature. Absorbance values were measured with a spectrophotometer (PG Instruments, England) set at 415 nm. The average absorbance val- ues were converted to percentage antioxidant activity using the following formula: Antioxidant activity (%) = 100 ­ [(Abs of sample ­ Abs of blank) x 100/Abs positive control] Statistical analysis All the analyses were performed in triplicate and data were analyzed using EXCELL and STATISTICA 7.1 (StatSoft). Differences between means were evaluated by Duncan's test. Statistical significant difference was stated at p<0.05. RESULTS AND DISCUSSION Proximate composition of the selected leafy vegetables is presented in Table 1. The physicochemical parameters generally differed significantly (p<0.05) from a blanching P.D. Oulai et al. TABLE 3. Anti-nutritional factors/mineral ratios of raw and blanched leafy vegetables consumed in Northern Côte d'Ivoire. Phytates/Ca Phytates/Fe Oxalates/Ca a b c a b d a c d b c d d a b c c H. sabdariffa H. hybridus A. digitata V. unguiculata C. pentadra a b c (A) Raw 15 min 25 min 45 min Oxalates (mg/100g) H. sabdariffa Raw 15 min 25 min 45 min 0.21±0.01a 0.10±0.01b 0.03±0.00c 0.01±0.00c 0.85±0.10a 0.90±0.01a 0.29±0.01b 0.10±0.01c 3.26±0.10a 0.10±0.00b 0.03±0.00c 0.01±0.00c 100 90 a (B) Raw 15 min a b a a b c d b c d b c a d b c d c d 25 min 45 min 80 70 60 50 40 30 20 10 0 H. sabdariffa H. hybridus A. digitata V. unguiculata C. pentadra A. hybridus Raw 15 min 25 min 45 min 0.03±0.00 0.41±0.01 0.07±0.00 0.01±0.00a 0.01±0.00a 0.01±0.00a 0.15±0.01b 0.15±0.01b 0.16±0.01b 0.01±0.00a 0.01±0.00a 0.01±0.00a A. digitata Raw 15 min 25 min 45 min 0.04±0.00a 0.04±0.00a 0.03±0.00 0.02±0.00 a a 0.19±0.01d 1.22±0.01a 0.94±0.01 b c 1.57±0.01a 0.04±0.00b 0.03±0.00 FIGURE 1. Oxalates (A) and phytates (B) contents of raw and blanched leafy vegetables consumed in Northern Côte d'Ivoire. Data are represented as Means ± SD (n=3). Means with no common letter (a, b, c, d) differ significantly (p<0.05) for each leafy vegetable. 0.69±0.01 0.02±0.00b V unguiculata . Raw 15 min 25 min 45 min 0.04±0.00a 0.03±0.00a 0.02±0.00a 0.02±0.00a 0.19±0.01b 0.23±0.01a 0.23±0.01a 0.22±0.01a 1.66±0.10a 0.03±0.00b 0.02±0.00b 0.02±0.00b C. patendra Raw 15 min 25 min 45 min 0.04±0.00b 0.09±0.00a 0.07±0.00a 0.07±0.00a 0.17±0.01c 1.12±0.01a 0.95±0.01b 1.02±0.10b 0.78±0.01a 0.09±0.00b 0.07±0.00b 0.07±0.00b time of a leafy vegetable to another. The ash content after 15 min of blanching ranged from 7.93±0.00% (A. hybridus) to 23.10±0.42% (C. patendra) with a decrease rate at 15 min ranged from 0.08 to 10.01%. After 15 min of blanching, the ash losses increased in the order: V unguiculata (0.08%) > A. digi. tata (1.91%) > H. sabdariffa (6.60%) > A. hybridus (7.68%) > C. patendra (10.01%). As concerns protein contents, blanching processing caused 0.36 to 12.03% reduction after 15 min. These protein losses increased in the order: V unguiculata . (0.36%) > A. hybridus (1.88%) > A. digitata (4.59%) > H. sabdariffa (8.64%) > C. patendra (12.03%). This reduced protein content could be attributed to the severity of thermal process during blanching which leads to protein degradation [Lund, 1997]. The ash and protein content losses are lower than that (9.78­28.0%) reported for 15 min cooked Nigerian leafy veg- etables [Lola, 2009]. This fact could be explained by the agronomic cultural conditions and the period of leafy vegetables harvesting. In spite of the ash losses levels, the studied leafy vegetables may be considered as good sources of minerals when compared to values obtained for cereals and tubers [Antia et al., 2006]. Moreover, the leaves of Vigna unguiculata revealed the higher retention (99.70­99.92%) of ash and proteins after 15 min of blanching. All selected leafy vegetables highlighted a slight increase (p>0.05) in their crude fibres content (0.35­4.16%) at 15 min of blanching. Indeed, the increased temperature during blanching leads to breakage of weak bonds between polysaccharides and the cleavage of glycosidic linkages, which may result in solubilization of the dietary fibres [Svanberg et al., 1997]. With regard to their fibres content at 15 min (12.75­31.94%), adequate intake of blanched leafy vegetables could lower the risk of constipation, diabetes, colon and breast cancer [Ishida et al., 2000]. The relatively low values of lipids contents at 15 min of blanching (2.39­6.20%) corroborate the findings of many authors which showed that leafy vegetables are poor sources of fat [Ejoh et al., 1996]. The estimated calorific values agreed with general observation that vegetables have low energy values due to their low crude fat and relatively high level of moisture [Sobowale et al., 2011]. Mineral composition of blanched leafy vegetables used in this study is shown in Table 2. The residual contents of minerals after 15 min of blanching were significantly different (p<0.05): calcium (264.88­844.92 mg/100 g), magnesium (49.45­435.43 mg/100 g), potassium (675­1895.41 mg/100 g), iron (14.54­70.89 mg/100 g) and zinc (9.48­ ­36.46 mg/100 g). With regard to these values, the consumption of 15 min blanched leafy vegetables could cover at least 50% of the standard mineral requirements for human. Indeed, these standard requirements are: calcium (1000 mg/ Phytates (mg/100g) 90 a a 15 min b a b c c d b b b d b c d b c d a 25 min 45 min (A) Vitamin C (mg/100g) Polyphenols (mg/100g) 80 70 60 50 40 30 20 10 0 H. sabdariffa A. hybridus a 350 300 250 200 150 100 50 0 b b b a a Raw a a a (A) b a a Raw 15 min b a c b c c b a 25 min 45 min A. digitata V. unguiculata C. pentadra H. sabdariffa A. hybridus A. digitata V. unguiculata C. pentadra Antioxydant activity (%) Carotenoids (mg/100g) (B) a a a b b c c d H. sabdariffa A. hybridus b b b a c b b b d a b b b A. digitata V. unguiculata C. pentadra b Raw 15 min 25 min 45 min c b a a b a c a b a b a a a b b a a (B) Raw 15 min 25 min 45 min c d H. sabdariffa A. hybridus A. digitata V. unguiculata C. pentadra FIGURE 2. Vitamin C (A) and carotenoids (B) contents of raw and blanched leafy vegetables consumed in Northern Côte d'Ivoire. Data are represented as Means ± SD (n=3). Means with no common letter (a, b, c, d) differ significantly (p<0.05) for each leafy vegetable. FIGURE 3. Polyphenols contents (A) and antioxidant activity (B) of raw and blanched leafy vegetables consumed in Northern Côte d'Ivoire. Data are represented as Means ± SD (n=3). Means with no common letter (a, b, c, d) differ significantly (p<0.05) for each leafy vegetable. day); magnesium (400 mg/day), iron (8 mg/day) and zinc (6 mg/day) [FAO, 2004]. Calcium and phosphorus play an important role in growth and maintenance of bones, teeth and muscles [Turan et al., 2003]. As concerns magnesium, this mineral is known to prevent cardiomyopathy, muscle degeneration, growth retardation, congenital malformations and bleeding disorders [Chaturvedi et al., 2004]. Iron plays an important role in the prevention of anemia while zinc is important for vitamin A and vitamin E metabolism [Martorell & Trowbridge, 2002; FAO, 2004]. To predict the bioavailability of calcium and iron, anti-nutrients to nutrients ratios of blanched leafy vegetables were calculated (Table 3). The calculated [phytates]/[Ca] and [oxalates]/[Ca] ratios in all the blanched species were below the critical level of 2.5 which is known to impair calcium bioavailability [Hassan et al., 2007; Umar et al., 2007]. The effect of blanching on anti-nutritional factors (oxalates and phytates) contents is depicted in Figure 1. The losses (p<0.05) at 15 min were 3.58­21.39% and 10.51­68.02% for oxalates and phytates, respectively. Oxalates losses increased in the order: A. digitata (3.59%) > A. hybridus (8.33%) >V . unguiculata (11.87%) > C. patendra (20.70%) > H. sabdariffa (21.39%) while phytates losses increased in the order: A. digitata (10.51%) > V unguiculata (26.89%) > C. patendra . (39.85%) > A. hybridus (65.89%) > H. sabdariffa (68.02%). These percentage losses are similar to those (7­56%) of phytates obtained for blanched leafy vegetables from Thailand [Samsub et al., 2008]. The reductions in oxalates and phytates contents during blanching could improve the health status of consumers. Indeed, oxalates and phytates chelate divalent cations such as calcium, magnesium, zinc and iron, thereby reducing their bioavailability [Sandberg, 2002]. In view of the highest oxalates and phytates losses (21.39 and 68.02%) of Hibiscus sabdariffa leaves, blanching process- ing may appear as a detoxification procedure by removing these anti-nutritional factors [Ekop & Eddy, 2005]. Blanching of the studied leafy vegetables also resulted in a decrease of carotenoids and vitamin C contents (Figure 2). For carotenoids, losses at 15 min were estimated to 18.91 to 55.48%. Carotenoids losses increased in the order: A. hybridus (18.91%) > C. patendra (23.41%) > H. sabdariffa (33.61%) > A. digitata (43.19%) > V. unguiculata (55.48%). The decrease in carotenoids contents could be attributed to the oxidation and isomerization of -carotene [Speek et al., 1986]. Carotenoids are considered as sources of provitamin A in plants and their amount determines their bioavailability in a human diet [Rodriguez-Amaya, 2001]. Therefore, consumption of blanched leafy with fat added, could contribute significantly to improving the vitamin A status of children [Takyi, 1999]. For vitamin C content, a significant reduction (19.56­68.67%) was highlighted at 15 min during blanching processing (Figure 2). Vitamin C losses increased in the order: H. sabdariffa (19.57%) > A. digitata (46.58%) > V. unguiculata (61.13%) > C. patendra (62.5%) > A. hybridus (68.67%). This decrease in vitamin C is lower than that (60­ ­90%) reported for blanched Indian leafy vegetables [Gupta et al., 2008] but agrees with earlier findings of Oboh [2005] on some Nigerian vegetables who reported 47.5­82.4% loss in vitamin C content during blanching. It is important to note that ascorbic acid is a heat-labile and water-soluble antioxidant that promotes absorption of soluble iron by chelating or by maintaining the iron in the reduced form [Yamaguchi et al., 2001]. With regard to the decrease of vitamin C, consumption of blanched leafy vegetables may be supplemented with other sources of vitamin C such as tropical fruits to cover the daily demand for humans (40 mg/day) as recommended by the Food Agriculture Organization [FAO, 2004]. The significant reduction of antioxidants components such P.D. Oulai et al. 4. Canet W., Quality and stability of frozen vegetables. 1989, in: Developments in Food Preservation (ed. S. Thorne), vol. 5, New York: Elsevier Science Publishing Inc., p. 50. 5. CEAEQ, Détermination des métaux. Méthode par spectrométrie de masse à source ionisante au plasma d'argon. MA 200 ­ Met 1.2, Rev 4. Quebec, 2013, p. 24. 6. 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Nutr., 2009, 8, 1430­ ­1433. as carotenoids and vitamin C in the studied leafy vegetables could also be due to the preliminary oven-drying treatment used. Indeed, higher temperatures affect negatively carotenoids and vitamin C by leading to their partial destruction [Rice-Evans & Miller, 1995]. This justifies the traditional practices which consist in drying leafy vegetables at ambient temperature in order to preserve their antioxidant properties. The effect of blanching on polyphenols content and antioxidant activity of the selected leafy vegetables is depicted in Figure 3. It was observed a high increase of polyphenols contents varying from 1.61 to 30.72%. The percent gain in the total phenol content during blanching may be due to the breakdown of tough cell walls and release of phenolic compounds trapped in the fibres of green leafy vegetables [Oboh & Rocha, 2007]. This result agrees with earlier report by Dewanto et al. [2002], where ferulic acid, a phenolic component found in the cell wall of grains such as corn, wheat and oats, doubled after 10 min of blanching. The content in polyphenols which is linked to the antioxidant activity of the studied blanched leafy vegetables could be advantageous for lower cellular oxidative stress, which has been implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis [Rice-Evans & Miller, 1995; Amic et al., 2003]. CONCLUSION African leafy vegetables (ALVs) contain significant levels of nutrients that are essential for human health. The result of this study revealed that blanching at 15, 25 and 45 min decreased considerably the nutritional value of these leafy vegetables. Nevertheless, the losses in anti-nutrients (oxalates, phytates) might have asserted a beneficial effect on the bioavailability of minerals like calcium, iron and zinc. Thus, the study suggests that the recommended time of domestic blanching must be less than 15 min for the studied leafy vegetables in order to contribute efficiently to the nutritional requirement and to the food security of Ivorian population. ACKNOWLEDGEMENT This work was completely conducted at Félix Houphouet-Boigny University (Biotechnology Laboratory, Biosciences Department). This work was also supported by a PhD financial support from the Higher Education and Scientific Research Ministry of Côte d'Ivoire.

Journal

Polish Journal of Food and Nutrition Sciences – de Gruyter

Published: Mar 1, 2015

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