278 Journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XVII, Issue 3, pag. 278 - 285 IMPROVEMENT OF IN VITRO BIOACCESSIBILITY OF PROVITAMIN A CAROTENOIDS BY FERMENTATION OF TROPICAL LEAFY VEGETABLES * Lessoy ZOUE1 , Sébastien NIAMKE1 1Faculty of Biosciences, Biotechnology Laboratory, Felix Houphouet Boigny University, Abidjan, Côte d’Ivoire, y.lessoy@yahoo.fr *Corresponding author Received 11th July 2018, accepted 14th September 2018 Abstract: The purpose of this study was to assess in vitro bioaccessibility of provitamin A carotenoids in fermented leafy vegetables. These fermented leafy vegetables are often used to prepare dishes in some parts of tropical Africa. Carotenoids were extracted from fermented leafy vegetables and analyzed by using High Performance Liquid Chromatography (HPLC). In order to simulate the human digestion tract, the in vitro bioaccessibility method known as simple, inexpensive and reproductible tool was performed. All leafy vegetables contained considerable amount of provitamin A carotenoids (26.61 ± 1.98 – 305.85 ± 14.38 μg/g dw) with the concentration varying from one source to another. Fermentation of leafy vegetables resulted in losses (24.5 – 78.22%) of provitamin A carotenoid contents. The in vitro bioaccessibility of provitamin A carotenoids of non fermented leafy vegetables ranged from 3.58 to 5.21% while that of fermented leafy vegetables was siginificantly different (P ˂ 0.05) and ranged from 11.51 to 15.32%. The results also showed that fermented leafy vegetables may contribute between 9.48 and 111.31% of the vitamin A daily requirement (RDA) estimated to 550 μg for individuals in developing countries. This study highlighted that fermentation appears as a suitable non thermal processing to improve the bioaccessibility of provitamin A carotenoids of leafy vegetables. Thus, an increased consumption of fermented leafy vegetables could help combat vitamin A deficiency (VAD) which remains a major public health problem in developing countries. Keywords: bioavailability, carotenoids, fermentation processing, leafy vegetables, vitamin A 1. Introduction Dietary carotenoids are fat-soluble pigments found in high concentrations in fruit and vegetables [1]. These bioactive components include carotenes (β-carotene, α-carotene and lycopene) and xanthophylls (zeaxanthin, lutein, α and β- cryptoxanthin) and they may prevent several diseases like cancer, cardiovascular disease and mainly blindness due to vitamin A deficiency (VAD) [2,3]. VAD remains a major public health problem in developing countries where children and pregnant women are usually affected. For these population groups, the largest contribution of vitamin A intake is provided by provitamin A carotenoids from plant foods, which may contribute up to 82% of the total vitamin A [4]. Therefore, food-based strategies for combating vitamin A deficiency in Africa include promotion and consumption of tropical leafy vegetables. Indeed, these plants are available, cheaper and provitamin A carotenoids-rich [5]. Commonly, tropical leafy vegetables are prepared at home by using different cooking methods (boiling, stewing, frying, and blanching) that have been reported to degrade 5 to 78% of β-carotene [6,7]. Considering that important amount of carotenoids needed by individuals may be lost during household cooking, the question of bioaccessibility and http://www.fia.usv.ro/fiajournal mailto:y.lessoy@yahoo.fr Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Lessoy ZOUE , Sébastien NIAMKE, Improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, Food and Environment Safety, Volume XVII, Issue 3 – 2018, pag.278 - 285 279 bioavailability of provitamin A carotenoids from tropical leafy vegetables remains important. The term bioavailability refers to the fraction of an ingested nutrient available for utilization in normal physiological functions and storage. However, bioaccessibility is defined as the amount of an ingested nutrient that is available for absorption after digestion [8]. Many factors may affect the bioavailability of carotenoids and the term SLAMENGHI used to describe these factors was formulated as follow [9]: Species of carotenoid, molecular Linkage, Amount of carotenoids in a meal, Matrix in which the carotenoid is incorporated, Effectors of absorption and bioconversion, Nutrient status of the host, Genetic factors, Host- related factors, and mathematical Interactions. Absorption of carotenoids involves the release from the food matrix, dispersion and solubilization into mixed bile salt micelles, uptake by intestinal mucosal cells and circulation through the lymphatic system into the bloodstream [10]. For green leafy vegetables, the main factor that affects the bioaccessibility of carotenoids is the leaf matrix which may hinder the release of the carotenoids during digestion [11]. In order to reduce this matrix effect, traditional processing methods such as boiling, blanching and pounding are commonly used in households [12]. Some reports also indicated that boiling or blanching leafy vegetables resulted in 2 to 12% bioaccessibility of β-carotene [11]. Another scarce processing method used to reduce the leaf matrix effect is fermentation. Indeed, some populations of West and Central Africa ferment leafy vegetables for 3 to 4 days to prepare dishes with a distinct flavor. In addition, fermentation can enhance the nutritional quality of leafy vegetables by producing beneficial by-products. To the best of our knowledge, the bioaccessibility of provitamin A carotenoids in such relishes is not documented and little information were reported about the ability of such fermented tropical leafy vegetables to supply daily vitamin A requirement. In vitro model method is known as simple, inexpensive and reproductible tool to stimulate the human digestion tract [13]. Therefore, the aim of this study was to assess the impact of fermentation on the bioaccessibility of provitamin A carotenoids from tropical leafy vegetables by using in vitro model. 2. Materials and methods 2.1 Chemicals The solvents (methanol, dichloroethane, methyltertiary-butyl diethyl ether and formic acid) were all HPLC grade and purchased from Fisher Scientific. The purified carotenoids standards (lutein, all- trans-β-carotene, 13-cis-β-carotene, 9-cis- β-carotene, α-carotene, β-apo-8-carotenal) and enzymes (porcine pepsin, pancreatin, bile extract) were from Sigma-Aldrich. 2.2 Fermentation processing of leafy vegetables Twelve (12) leafy vegetables widely consumed in Côte d’Ivoire were selected (Table 1). All the leaves were collected at maturity from a periurban farmland (latitude: 5°19′14″ North; Longitude: 4°22′59″West) located in Abidjan District (Côte d’Ivoire). The collected leaves were authenticated by National Floristic Center (University Felix Houphouët-Boigny, Abidjan). The leaves were washed several times with distilled water, drained at ambient temperature, cut into small pieces and separated into two portions of 250 g each. The first portion was wrapped in clean papaya leaves for 4 days to induce natural fermentation in a covered plastic box. Afterwards, the fermented (F) leaves were oven-dried (50°C/3 days) and ground Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Lessoy ZOUE , Sébastien NIAMKE, Improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, Food and Environment Safety, Volume XVII, Issue 3 – 2018, pag.278 - 285 280 into a powder with a laboratory crusher. The second portion was not subjected to the fermentation step but was used as control (non-fermented NF leaves). All the dried and powdered samples were stored at -18°C in airtight containers for further experimentation. Table 1. Common and local names of selected tropical leafy vegetables Leafy vegetables Common name Local name Abelmoschus esculentus Okra Gombo Amaranthus hybridus Green amaranth Boronbrou Basella alba Indian spinach Epinard Celosia argentea Common cockscomb Soko Colocasia esculenta Taro Taro Corchorus olitorius Jew’s mallow Kplala Hibiscus sabdariffa Roselle Dah Ipomea batatas Sweet potato Patate Manihot esculenta Cassava Manioc Myrianthus arboreus Bush pineapple Tikliti Solanum melongena Eggplant Aubergine Talinum triangulare Ceylon spinach Mamichou 2.3 Carotenoid analysis 2.3.1 Carotenoid extraction Total carotenoids of F and NF leaves were extracted using a previously reported method [14] with slight modifications. Powdered samples (0.05 g) were mixed with 5 mL ethanol containing butylated hydroxytoluene (0.1%, w/v) and heated in a water bath at 85°C for 5 min. Then, 400 μL KOH in water (80% w/v) was added for saponification and the suspension was mixed using a vortex for 20 s and heated in a water bath at 85°C for 5 min. The tubes containing the reaction mixture were placed in ice after introducing 3 mL deionized water and carotenoids were extracted three times with 4 mL hexanes. β-apo-8’-carotenal was used as an internal standard and was added after saponification to account for mechanical losses. The combined extracts were dried under nitrogen and reconstituted in 1 mL 50:50 methanol-dichloroethane. 2.3.2 HPLC analysis For the carotenoid identification and quantification, 25 μL extract was injected into an HPLC system (Waters Corporation; Milford, MA, USA) consisting of a 717 autosampler, 1525 binary pump, and a 2996 photo-diode array detector (PDA). The column used was a C30 YMC carotenoid column (4·6 x 250mm, 3mm). The HPLC solvent gradient included methanol-water (92:8, v/v) with 10 mM ammonium acetate (solvent A) and 100 % methyltertiary-butyl ether (solvent B). Samples were analyzed at 1 mL/min with a 30-min linear gradient from 70 to 40% solvent A. Lutein, β-carotene (including all-trans, 13-cis, and 9-cis), and α-carotene were identified and quantified using HPLC-purified standards. Chromatograms were generated at 450 nm. 2.4 In vitro bioaccessibility of provitamin A carotenoids Determination of in vitro bioaccessibility of provitamin A carotenoids was carried out according to an in vitro digestion method [15] with slight modification. About 0.5 g of powdered vegetable sample was mixed with 10 mL ascorbic acid (1%, w/v). For the digestion, 5 mL of a 0.5% Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Lessoy ZOUE , Sébastien NIAMKE, Improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, Food and Environment Safety, Volume XVII, Issue 3 – 2018, pag.278 - 285 281 (w/v) porcine pepsin solution in HCl 0.1M with physiological amounts of calcium (3.6 mmol), magnesium (1.5 mmol), sodium (49 mmol), potassium (12 mmol) and phosphate (6.4 mmol) were added. pH was adjusted to 2.0 with HCl 2 M and nitrogen gas was blown into the flask and the scraw capped test tube was incubated at 37°C in a shaking water bath at 250 rpm for 1 h. After the gastric digestion phase, the pH value was adjusted to 5 with NaOH 1M and 3 mL of pancreatin-bile solution (4 g/L bile extract and 25 g/L pancreatin dissolved in 0.1M NaHCO3) was added. The pH value was further increased to 7.5 and the mixture was incubated again for 30 min. After intestinal digestion phase, the sample was centrifuged at 5000 g for 20 min. The supernatant was collected and mixed with 5 mL of 25% (w/v) NaCl and 5 mL of ethanol stabilised with 0.1% (w/v) BHT. Afterwards, carotenoids were extracted two times by centrifugation (5000 g; 20 min) with 5 mL hexane and the combined extracts were dried under nitrogen before reconstitution in 1 mL 50:50 methanol-dichloroethane. The released provitamin A carotenoids were analyzed and quantified by HPLC as described in 2.3.2. 2.5 Retinol activity equivalent determination The calculation of retinol activity equivalent (RAE) of fermented leafy vegetables after in vitro bioaccessibility was based on the bioconversion factor defined as 2 μg accessible β-carotene to 1 μg of retinol (2:1)[16] . 2.6 Statistical analysis Values were reported as means ± SD. Data were analyzed using XLStat 2016.2 (Addinsoft, NY, USA). Carotenoid composition and bioaccessibility values were compared using one-way ANOVA. Differences among processed leaves and treatment groups were determined using least significant differences (LSD) at P ˂ 0.05. 3. Results and discussion 3.1 Carotenoid profile Carotenoid contents and their chromatographic profile are given in Table 2 and Fig. 1, respectively. The identified carotenoids were lutein and provitamin A carotenoids (13-cis-β-carotene, α-carotene, all-trans-β-carotene, and 9-cis-β-carotene). All leafy vegetables contained considerable amount of provitamin A carotenoids (26.61 ± 1.98 – 305.85 ± 14.38 μg/g dw) with concentration varying from one source to another. Many factors affect carotenoid content of leafy vegetables, such as variety, location, cultivation, and post-harvest handling practices [17]. Considering non fermented (NF) leafy vegetables, the best sources of provitamin A carotenoids were A. esculentus (305.85 ± 14.38 μg/g dw), C. olitorius (200.56 ± 16.21 μg/g dw), M. esculenta (233.20 ± 13.13 μg/g dw) and S. melongena (220.71 ± 2.23 μg/g dw). Fermentation of leafy vegetables resulted in losses (24.5 – 78.22%) of provitamin A carotenoids contents. It’s important indicating that the traditional fermentation of leafy vegetables involves lactic acid bacteria as main fermenting microorganisms [18]. The losses of carotenoids in this study is not surprising since carotenoids degradation occurs during lactic acid fermentation of vegetables as indicated by some authors [19]. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Lessoy ZOUE , Sébastien NIAMKE, Improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, Food and Environment Safety, Volume XVII, Issue 3 – 2018, pag.278 - 285 282 Fig. 1. Chromatrogram of carotenoids from selected tropical leafy vegetables (a): lutein; (b): 13-cis-β-carotene; (c): α-carotene; (d): all-trans-β-carotene; and (e): 9-cis-β-carotene Table 2. Total provitamin A contents of fermented (F) and non fermented (NF) leafy vegetables Leafy vegetables Total provitamin A carotenoids (μg/g dw) Non fermented (NF) Fermented (F) A. esculentus 305.85±14.38a 193.90±3.07 b A. hybridus 129.41±2.41a 33.49±0.27 b B. alba 83.07±3.01a 62.71±6.55 b C. argentea 148.46±5.55a 32.33±1.48 b C. esculenta 189.78±3.23a 80.40±3.71 b C. olitorius 200.56±16.21a 122.88±3.21 b H. sabdariffa 26.61±1.98a 17.16±4.23 b I. batatas 156.97±1.90a 48.85±8.92 b M. esculenta 233.20±13.13a 140.78±1.88 b M. arboreus 73.02±8.98a 38.11±0.64 b S. melongena 220.71±2.23a 128.86±3.10 b T. triangulare 178.25±1.62a 45.98±0.43 b Data are presented as means of triplicate analyses ± SD. Means with the same superscript letter in the same line for a single vegetable are not different at P > 0.05 3.2 In vitro bioaccessibility Bioaccessibility of food components such as vitamins is an important feature to assess their role in human health. The in vitro bioaccessibility model has been developed as simple, inexpensive, and reproducible tools to study the human digestion of different food components (ascorbic acid, carotenoids, chlorophylls, polyphenols) [20]. The in vitro bioaccessibility of provitamin A carotenoids of non fermented leafy vegetables ranged from 3.58 to 5.21% while that of fermented leafy vegetables was siginificantly different (P ˂ 0.05) and ranged from 11.51 to 15.32% (Table 3). The results for provitamin A carotenoids bioaccessibility of non fermented leafy Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Lessoy ZOUE , Sébastien NIAMKE, Improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, Food and Environment Safety, Volume XVII, Issue 3 – 2018, pag.278 - 285 283 vegetables were consistent since less than 5% provitamin A carotenoids is generally released from unprocessed food matrix as indicated by some authors [21]. Indeed, the first step in digestion of carotenoid comprises disintegration of the food matrix in order to release carotenoids [22]. In green leaves, carotenoids components are organised in pigment–protein complexes located in cell chloroplasts, which have to be accessed before making carotenes available [23]. Contrary to non-fermented leaves, fermentation processing increased bioaccessibility of provitamin A carotenoids. The values of bioaccessibility of fermented leaves were similar to those (6-29%) of cooked or blanched leafy vegetables without oil [11,15]. Moreover, accessible percentage of provitamin A carotenoids from fermented leafy vegetables were slightly lower than the factor 1/6 (16,7%) proposed by the US Institute of Medicine (IOM) for the bioaccessibility of all-trans-β-carotene from vegetable food items [16]. Thus, fermentation of leafy vegetables appears as a suitable non thermal technique to improve the bioaccessibility of provitamin A carotenoids. Table 3. In vitro bioaccessibility of fermented (F) and non fermented (NF) leafy vegetables Leafy vegetables Bioaccessibility (%) Non fermented (NF) Fermented (F) A. esculentus 4.55±0.01a 12.63±0.90 b A. hybridus 5.21±0.02a 14.33±0.01 b B. alba 5.05±0.01a 11.51±0.10 b C. argentea 4.05±0.03a 13.38±0.01 b C. esculenta 4.16±0.78a 12.11±0.69 b C. olitorius 4.34±0.78a 15.32±0.49 b H. sabdariffa 4.31±0.05a 12.16±0.01 b I. batatas 4.69±0.01a 14.58±0.02 b M. esculenta 5.03±0.17a 13.89±0.56b M. arboreus 5.02±0.15a 14.21±0.05 b S. melongena 3.58±0.20a 12.76±0.35b T. triangulare 4.85±0.02a 13.72±0.05 b Data are presented as means of triplicate analyses ± SD. Means with the same superscript letter in the same line for a single vegetable are not different at P > 0.05 3.3 Contribution to vitamin A requirements Assuming that the minimum intake of fermented leafy vegetable relish in a single meal is 50 g dry matter, the corresponding amount of accessible provitamin A carotenoids from the results of in vitro digestion was between 104.33 and 1224.47 μg (Table 4). The conversion factor adopted by IOM [16] assumes that one- sixth (1/6) of the total carotene content is absorbed into the mucosa, and that one- half (1/2) of absorbed β-carotene is converted to retinol. These conversion factors were used to estimate how much fermented leafy vegetables might contribute to the daily requirement (RDA) of vitamin A. In this study, the in vitro digestion data were used instead of the one-sixth (1/6) factor with assumption that 100% of the carotenes released after in vitro digestion is absorbed into the mucosa Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 3 – 2018 Lessoy ZOUE , Sébastien NIAMKE, Improvement of in vitro bioaccessibility of provitamin a carotenoids by fermentation of tropical leafy vegetables, Food and Environment Safety, Volume XVII, Issue 3 – 2018, pag.278 - 285 284 [23]. Considering the one-half (1/2) factor of accessible provitamin A carotenoids, the retinol activity equivalent (RAE) varied from 52.16 μg (fermented H. sabadariffa leaves) to 612.23 μg for fermented A. esculentus leaves. The contribution to the RDA of retinol was calculated and the results showed that fermented leafy vegetables may contribute between 9.48 and 111.31% the RDA estimated to 550 μg vitamin A for individuals in developing countries [24]. Therefore, increasing consumption of fermented leafy vegetables could help alleviate vitamin A deficiency (VAD) which remains a major public health problem in developing countries. Table 4. Contribution of fermented leafy vegetables to daily vitamin A requirement Fermented leafy vegetables Accessible provitamin A carotenoids (μg) Retinol activity equivalent (μg) Contribution to daily vitamin A requirement (%) A. esculentus 1224.47 612.23 111.31 A. hybridus 239.95 119.97 21.81 B. alba 360.89 180.44 32.80 C. argentea 216.28 108.14 19.66 C. esculenta 486.82 243.41 44.25 C. olitorius 941.26 470.63 85.56 H. sabdariffa 104.33 52.16 9.48 I. batatas 356.11 178.05 32.37 M. esculenta 977.71 488.85 88.88 M. arboreus 270.77 135.38 24.61 S. melongena 822.12 411.06 74.73 T. triangulare 315.42 157.71 28.67 4. Conclusion In this study, the fermented leafy vegetables showed relatively important in vitro bioaccessibility of provitamin A carotenoids. Thus, they may contribute to the daily requirement of vitamin A. These findings could be useful in dietary intervention programmes to alleviate vitamin A deficiency (VAD) in developing countries. However, several food- and host-related factors may influence vitamin A bioavailability at different points. Thus, in vitro methodology for provitamin A bioaccessibility should be validated in different in vivo conditions. 5. Acknowledgments The authors thank the US Government for the Fulbright fellowship granted to the first author LZ at Wisconsin-Madison University, USA. 6. References [1]. LIU R.H., Health-Promoting Components of Fruits and Vegetables in the Diet, Advances in Nutrition, 4:384–392, (2013). [2]. KRINSKY N.I., JOHNSON E.J., Carotenoid actions and their relation to health and disease, Molecular Aspects of Medicine, 26:459–516, (2005). [3]. ZAMPATTI S., RICCI F., CUSUMANO A., MARSELLA L.T., NOVELLI G., GIARDINA E., Review of nutrient actions on age-related macular degeneration, Nutrition Research, 34:95–105, (2014). [4]. 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