Nutritional Composition of Four Underexploited Wild Fruits in Mali

Malnutrition and food insecurity are major concerns for the Malian authori-ties. The objective of this study was to strengthen the knowledge about the nutritional and physicochemical values of four underutilized edible wild fruits picked at two cities belonging to different bioclimatic zones of Mali. The physicochemical and nutritional parameters were performed using standard methods. The findings revealed that all these parameters varied from one fruit to another, this outcome could be associated with the provenances of the wild fruits (p-value < 0.05). The protein contents varied from 4.53 g/100g by dry matter (DM) for R. sudanica fruits to 5.34 g/100g DM for those of B. aegyptiaca, all these samples were being harvested from Sikasso. The highest concentrations of vitamins are C (150,800 to 151,000 µg/100g DM), E (1,310 to 1,350 µg/100g DM) and A (38 to 40 µg/100g DM) respectively for the fruits of Z. mauritiana, B. aegyptiaca and S. senegalensis. In addition, these fruits would constitute a potential source of minerals such as iron, phosphorus and calcium. Thereby, these fruits are promising raw materials to be used against the malnutrition linked to the micronutrients deficiencies and the management of certain pathologies related to oxidative stress.


Introduction
"Hungry people cannot be educated, cannot be cared for themselves and cannot ensure sustainable economic growth", said Brouwer et al. [1]. This introductory note to the proceedings of the 2nd International Workshop on the Food Pathways to Improve Nutritional Situations in West Africa shows that malnutrition and food insecurity are recurrent problems. This is why nutrition is increasingly recognized as a basic pillar for social and economic development. The efforts to reduce malnutrition and mortality, especially among children, are essential to contribute to achieving the Sustainable Development Goals [2].
Sub-Saharan Africa and Mali, in particular, remain vulnerable to these troubles.
The nutrition indicators show that the situation is alarming. According to the estimates of FAO (2019) [3], the number of undernourished people has started to increase back since 2015, and more than 30% would be in Africa. An in-depth analysis of the FAO statistics on this field revealed that the number of people affected by chronic food insecurity in 2017 was 821 million, i.e., the ninth of the world population. Among them, more than 38 million children under five years old were overweight, 25% of them in Africa [3]. In addition, it is estimated that more than one-third of deaths in children under five years old in Africa are directly or indirectly caused by malnutrition [2]. In Mali, recent studies have shown that 27% of children under 5 years old are chronically malnourished and 10% of them are severely stunted. Nearly ten percent of children under 5 years old (9%) are suffering from severe malnutrition, the same studies also revealed that 19% are underweight [4].
To face these troubles, human has always resorted for many centuries, to wild food products. In recent years, there has been an increasing interest in the scientific world around these wild food products. Many studies carried out in Mali have shown that the Malian flora is very rich in wild food plants [5] [6] [7]. Other studies conducted on some of these edible wild plants have shown that they are rich in micronutrients [8] [9] and also in macronutrients [10] [11]. However, many other food wild species are not explored yet. Among them, there are Balanites aegyptiaca, Saba senegalensis, Ziziphus mauritiana and Raphia sudanica. However, despite the increasing interest in these food wild fruits and according to our knowledge, their nutritional potential is still not well known in Mali and consequently, they are underexploited and under valorized. That is why this study aims to contribute to the fight against malnutrition in Mali through an evaluation of the nutritional composition of these four food wild fruits, namely Balanites aegyptiaca, Saba senegalensis, Ziziphus mauritiana and Raphia sudanica harvested in two different bioclimatic zones of Mali.

Material
The vegetal material was consisted of the wild fruits of Balanites aegyptiaca, Saba collected in the markets of Banamba (from the Sahelian zone) and Sikasso (from the Sudanese zone). The latter (Raphia sudanica) was collected from Sikasso because of its geographical specificity. These sites were chosen taking into account the food security, accessibility and especially the availability of these fruits.

Physicochemical Parameters
 pH and total free acidity The AFNOR methods [12] were used to evaluate the pH and the total free acidity.
Ten grams (10 g) of powered samples were homogenized with 50 mL of distilled water using a magnetic stirrer (Velp Scientifica, Cheshire, Great Britain).
The pH of the homogenate was determined using a digital pH meter (Knick type 647, Interface Technology, Germany) calibrated with the standard buffer solutions (pH 4.0 and pH 7.0) at 25˚C.
After centrifugation of the homogenized solution above, the supernatant was collected and used for the assessment of the total free acidity by titrimetric assay. The suspension was titrated with an alkaline NaOH solution (0.1 N) using the phenolphthalein solution as colored indicator [12]. The total acidity was expressed as a percentage of lactic acid [13] according to the following Formula V: Volume of NaOH used (L); N: Normality of NaOH solution (0.1 N); M: Molecular weight of lactic acid (90 g/mol); W i : Initial sample weight (g).

 Humidity
The humidity content was determined by differential weighing according to the French standard NF V 03-707 [14]. Ten grams (10 g) grams of sample powder were weighed in crucible before (W 1 ) and after (W 2 ) passing in an oven at 105˚C

Nutritional Composition  Total protein contents
The protein's levels were assessed by the determination of the total nitrogen according to the Kjeldhal method (NF V03-050, 1970) [16]. Thus, 1 g of sample powder dissolved in 5 mL of concentrated sulfuric acid was mineralized using a mineralizer (Velp Scientifica, Cheshire, Great Britain) in the presence of Kjeltabs catalyst. The obtained mineralized sample was purified by distillation (Velp Scientifica UDK 159, Cheshire, Great Britain). The nitrogen was then quantified by titration with HCl, 1 N. The total protein content was deduced from the nitrogen content using a conversion coefficient of 6.25 [16].  Fat contents The fat content was assessed by the gravimetric method after Soxhlet extraction using petroleum ether according to the standard ISO 659, (1998) [17]. The extraction was carried out hot (70˚C -75˚C) by soaking 5 g of powder followed by rinsing with petroleum ether. After 6 -8 hours of extraction, the petroleum ether was evaporated using an evaporator (Bucchi CCT C21-901, Merck, Schnelldorf, Germany). After drying in an oven, the fat content was expressed as percentage of dry matter (% DM) using the following Formula (4) [17]: W 0 : Weight of the empty balloon (g); W 1 : Weight of the balloon after extraction and drying (g); W s : Initial sample weight (g).

 Total and reducing sugar contents
The total and the reducing sugar were estimated by spectrophotometric method.

Preparation of extracts
In a test tube, 1 g of the sample was dispersed in 10 mL of 25% dimethyl sulfoxide (DMSO) in water (v/v). The mixture was incubated in a boiling water bath for 15 min. A volume of 0.1 mL was then taken and made up to 100 mL with distilled water.

Quantification
The total sugars were assayed using the colorimetric method described by Fox & Robyt [18]. A volume of 0.5 mL of the previously prepared extract was added to 0.5 mL of 5% phenol solution. After homogenization, 2 mL of 75% H 2 SO 4 was added. After homogenization, the tubes were placed in a boiling water bath To quantify the reducing sugars, the colorimetric method with a DNS solution as reagent was used [19]. A volume of 0.1 mL of the prepared extract was diluted with 0.5 mL of distilled water. To this solution, 1 mL of DNS reagent was added. After homogenization, the tubes were placed in a boiling water bath (100˚C) for 5 min, then cooled on ice and in the dark for 15 min. After returning to the ambient temperature (25˚C -30˚C), the absorbance was read at 540 nm. The concentrations of reducing sugars of our extracts were deduced from a standard curve of D-glucose established in the same conditions as the extracts.
The values of total and reducing sugars were expressed as percentage of dry matter (% DM).  Energy value The energy value was calculated according to the Atwater coefficients [20]. Energy value (Kcal/100g DM) = % proteins × 4 Kcal + % carbohydrates × 4 Kcal + % lipids × 9 Kcal  Mineral contents The determination of mineral elements (Na, K, Ca, P, Fe, Mg, Mn, Zn, Cu, Ni, Pb, Co) was carried out by flame atomic absorption spectrometry according to AOAC (2012) [21].
A quantity of 0.5 g of sample was mineralized by progressive heating from 100 to 300˚C in the presence of nitric acid solution (5%) for 90 min. After an ultrasonication treatment for 30 min at 80˚C, the mineralized material was centrifuged (Ohaus® centrifuge) at 1000 rpm for 5 min. The absorbance was read using an atomic emission spectrometer (Perkin Elmer Optima 8000, Waltham, USA). The mineral concentrations were deduced from the standard ranges established under the same conditions as the extracts.  Vitamins contents Preparation of the sample The extraction was carried out according to the method described by Kini et al. [8] with the wild fruit species. To 0.5 g of sample were added 10 mL of a solution of 10% KOH in methanol-water mixture (1: 1; v/v). To avoid oxidation processes (with fat-soluble vitamins: A and E) during saponification, 0.025 g of ascorbic acid was added. The mixture is then brought to reflux in a water bath at 70˚C for 30 min. After cooling, the mixture was extracted with 3 × 5 mL of hexane. The hexane based phases were combined and dried over anhydrous sodium sulfate and then evaporated. The dry residue obtained was dissolved in HPLC grade methanol for analysis.
Quantification A High Performance Liquid Chromatography (HPLC) method was used to quantify the content of vitamin A (β-carotene or provitamin A), vitamin E (α-tocopherol) and vitamin C. The HPLC (Agilent) was equipped with a 20 µL injection valve, an Alumina-C18 column and a visible UV detector. The mobile phase consisted of an acetonitrile/methanol (80:20 v/v) mixture with a flow rate of 1 mL/min. The standards used were trans-retinol for vitamin A, α-tocopherol for

Statistical analysis
The data obtained were processed using an Excel® spreadsheet version 2013. The statistical analysis was performed using Minitab 18.1 software (Minitab Inc., PA, USA). All measurements were performed in triplicates, and the values were expressed as mean values ± standard deviation. The differences between samples were assessed using the one-way ANOVA via Fischer test. The differences between the samples were considered significant at p < 0.05.

Physicochemical Parameters
The results related to the physicochemical characteristics are presented in Table   1. Table 1 reveals that all these physicochemical parameters varied from one fruit to another (p-value < 0.05). This same trend is observed in the areas of origin (Banamba and Sikasso). The pH and the total free acidity varied from one fruit to another (p-value < 0.05). On the other hand, no significant difference was observed between these two parameters in the ecological zones (p-value > 0.05).
The pH varied respectively from 2.80 ± 0.19 for Saba of Banamba to 3.92 ± 0.21 for Balanites of Sikasso. The lowest total acidity was recorded in Balanites of Sikasso with 0.85% ± 0.05% lactic acid equivalents and the highest in Saba of Banamba with 2.38% ± 0.08% lactic acid equivalents ( Table 1). The fruits of Saba senegalensis are the richest one in water with contents ranging from 19.03% ± 0.67% to 21.66% ± 0.18% (Table 1).

Macronutrients
The nutritional composition of our fruits is written down in Table 2.
The data from Table 2 show that all the performed nutritional parameters varied from one fruit to another (p-value < 0.05). Excepted the fruits of Balanites, there is also a zonal variation for all these nutritional parameters. The protein contents of our samples have varied from 4.53 ± 0.25 g/100g DM for Raphia fruits to 5.34 ± 0.52 g/100g DM for those of Balanites all harvested in Sikasso (   (Table 3). Furthermore for all the performed vitamins, it appears an inter-fruit variation (p-value < 0.05) without significant variation between the geographical areas (p-value > 0.05), except the fruits of S. senegalensis about the vitamin C.   Mineral element contents Table 4 summarizes the contents of mineral elements. It shows that the concentrations of mineral elements are different from one fruit species to another and sometimes from one area to another. These wild fruits contain appreciable quantities of mineral especially iron (Fe), phosphorus (P) and calcium (Ca).

Physicochemical Parameters
The physicochemical parameters of the four wild fruits from Banamba and Sikasso were evaluated. The results (Table 1) showed that these physicochemical parameters have been varied according to the fruits and according to the provenance (p-value < 0.05). The assessment of pH and titratable acidity are important parameters in the quality control of any foodstuffs. These parameters indicate the evolution of the medium acidity, according to the metabolism of the microorganisms. The results of Table 1 showed that all the investigated fruits are acidic (pH < 7). So a pH value from 3 to 6 is very favorable to the development of yeasts and molds [13]. In this way, our obtained data denote the preserving difficulties of our fruits because of their favorable pH to microbial activity. Our different samples are quite rich in water with the contents greater than 10% (Table 1); which would pose a conservation problem. It is known that for the humidity greater than 10%, the degradation of certain constituents by enzymatic hydrolysis becomes favorable [24]. That would explain the low medium and long terms availability of these fruits on the markets. Therefore, it would be important to develop processes for transforming these fruits for a better conservation.

Nutritional Composition
The performed nutritional parameters are presented in Table 2. It appears that these parameters are function of species and provenances of fruits (p-value < 0.05). Protein contents Found in all living things, the proteins are biopolymers of amino acids essential for growth [25]. The protein contents of our samples have varied significantly from one fruit to another (p-value = 0.001 < 0.05); from 4.53 ± 0.25 g/100g DM for Raphia to 5.34 ± 0.52 g/100g DM for Balanites from Sikasso (Table 2) ± 0.05% DM. Our protein contents are higher than those obtained by Tiendrebeogo et al. [26] with the fruits of Saba (3.7 to 6.3 g/kg DM) harvested in Burkina Faso. A study has shown that plant protein isolates are used as additives in food products due to their functional properties (emulsifying capacity, water retention capacity, etc.) [27]. This is why these proteins are currently coveted to replace valuably those of animal origin which are not accessible to all because of their high cost.
Carbohydrates levels The carbohydrates are the most important constituents of the most eatable fruits. They are also responsible for the sweetness of the food [13]. According to the data of Table 2, the total sugars contents of the fruits have varied from 53.93 ± 0.61 g/100g DM for B. aegyptiaca to 71.02 ± 1.95 g/100g DM for Z. mauritiana. As for reducing sugars, the highest levels have been observed in the fruits of S. senegalensis (p-value = 0.45E−10 < 0.05). The total sugars contents of our fruits are higher than those obtained by Makalao et al. (2015) [10] which were 10.92% and 13.05% respectively for Z. mauritiana and B. aegyptiaca collected in Tchad. Likewise, these levels are higher than those obtained with the fruits of S. senegalensis (104.50 ± 0.7 to 158.8 ± 1.5 g/kg DM) collected in Burkina Faso [26]. On the opposite a higher amount was obtained by Achaglinkame et al. (2019) [9] from B. aegyptiaca with 73.63% ± 0.38%. This richness of our wild fruits in carbohydrates would make them a potential source of calories and would promote their transformation, especially into jams and juices [13] for a better availability and accessibility.
Fat contents The fats are components of cell structures, reserve substances and constitute a cell energy source. Our investigated wild fruits showed relatively low fat contents (  [9]. Considering this low fat content, these wild fruits could be recommended to the obese and diabetic patients or people in overweight. Energy values Our results show that the calorific power of all our fruits is low ( Table 2) comparatively to the recommended daily allowances. Therefore, they could be recommended in the diet of diabetic and overweight people; because a relationship between the calorific power and these pathologies was reported [26].
Vitamins contents Appreciate amounts of vitamins A, C and E have been recorded in our wild fruit extracts. For all the vitamins measured, there were an inter-fruit variation (p-value < 0.05) but no significant variation between the zones (p-value > 0.05), except for the fruits of S. senegalensis for the vitamin C (Table 3). Our levels of vitamin C are higher than those obtained by Makalao et al. [10] which were 8.57 mg/100g and 137.56 ± 0.26 mg/100g respectively for the fruits of B. aegyptiaca and Z. mauritiana. Others studies showed similar quantities of vitamin A from B. aegyptiaca fruits: Achaglinkame et al. [9] recorded 0.84 ± 0.03 mg/100g DM and 0.25 -0.81 mg/g DM were obtained by Murthy et al. [28]. On the other side, higher amounts of vitamin C were obtained by Achaglinkame et al. (2019) [9] with 105.97 ± 2.57 mg/100g DM from B. aegyptiaca. Likewise, our contents were low than those obtained by Cisse et al. (2019) [29] with the fruits of S. senegalensis which were 154 to 198 mg/100g. As for vitamin E, our data were close to those of Kini et al. [8] who noted 31 µg/100g and 103 µg/100g respectively for the fruits of B. aegyptiaca and Z. mauritiana. The same authors registered 1.559 µg/100g of vitamin A for the fruit juice of S. senegalensis. In human trials, some supplementation experiments had shown the potential benefits of vitamins (C and E) combined to other compounds: reduction in adiposity, lowering of triglycerides and cholesterol levels; betterment of insulin sensitivity [30] and reduction inflammatory response [31] [32]. Hence, our wild fruits would constitute a significant source of vitamins and could contribute to reduce the anomalies linked to certain vitamin deficiencies and to fight against the pathologies linked to oxidative stress.
Mineral contents Table 4 summarizes the contents of mineral elements which are micronutrients involved in a wide range of physiological functions such as mineralization, the control of hydromineral balance, the enzymatic, hormonal, nervous and immune systems. For example potassium (K), in equilibrium with sodium, is an essential element for the activity of nervous and muscle cells. In our study, the highest potassium (P) contents were 358.10 mg/100g DM for Balanites of Sikasso and the lowest 152.00 mg/100g DM for Raphia (Table 4). The data reveal also that our samples could be a hopeful source of phosphorus (P) with the amounts altered from 1141.00 to 2312.00 mg/100g DM ( Iron (Fe) is an essential component in the composition of hemoglobin in red blood cells, in muscle myoglobin and in many enzymatic reactions. The very promising data were obtained for iron: from 20.57 mg/100g DM for Balanites to 51.16 mg/100g DM for Saba (Table 4). Based on this richness in iron, these wild fruits could be recommended in the fight against anemia, especially in the pregnant women in whom a drug supplementation is frequently used during the pregnancy [33].
Manganese (Mn) is a trace element whose the main functions relate to carbohydrate (synthesis and secretion of insulin) and lipid metabolisms as well as the detoxification of free radicals from oxygen [34]. The results show that the fruits of Raphia and Saba were very rich in manganese with respectively 14.150 mg/100g DM and 6.855 mg/100g DM (Table 4). This potentiality could be exploited in the management of diabetes and other pathologies linked to oxidative stress.
The copper, manganese and zinc contents obtained from our wild fruits would be interesting in the repair of possible oxidative damage, since these trace elements are the cofactors of numerous enzymes which have antioxidant activities [35].
Despite its negative connotation as a pollutant and carcinogenic agent, the nickel (Ni) is an indispensable metal in the metabolism of methionine via homocysteine [34]. Our fruits contained the nickel (Ni) but the levels obtained (6.39 to 79.08 µg/100g) ( Table 4) would not be a danger for the consumption because they were all lower than the maximum dose set by AFSSA (2007) [33] which is 600 µg/day.
On the other hand, they were higher than the data registered by Leblanc et al. (2004) who revealed that the average nickel content of fruits alters between 1.68 and 5.56 µg/100g. Moreover, Daudet (2012) [36] had already drawn the attention to the micronutrient richness of wild products in these terms: "their contents in various micronutrients seem to be higher than the nutritional content of the agri-cultural products". As such, our wild fruits would constitute a potential local source of micronutrients and would be a glimmer of hope in the fight against the micronutrient deficiencies.

Conclusion
This work provided a comparative evaluation of physicochemical and nutritional parameters of four underexploited edible wild fruits. The results revealed that the evaluated parameters fluctuate from one fruit to another and sometimes from one zone to another. These picking fruits could be a potential source of macronutrients and calories. They could also offer many micronutrients such as minerals (Fe, Ca, Mn, etc.) and vitamins (A, C and E) which are essential for human nutrition. The fruits of R. sudanica due to their high level of calcium could be recommended to improve bone mineralization and growth, mostly in children. The richness in the iron of our fruits could make them potential candidates to fight against malnutrition and anemia, mainly in pregnant women. Thereby, in addition to their nutritional potential, these fruits would be promising candidates in the management of pathologies related to oxidative stress. So, to better valorize these edible wild fruits, it would be necessary to develop recipes incorporating these fruits to fortify the food basis in order to contribute to the fight against the malnutrition linked to certain micronutrient deficiencies.