Nutritional value of a dietary supplement of Moringa oleifera and Pleurotus ostreatus

1 Department of Biochemistry, Faculty of Science, University of Yaoundé I, Cameroon. 2 Laboratoire de Biotechnologies des champignons, Département de Biologie et Physiologie Végétale, Faculté des Sciences et Techniques, Université Cheikh Anta Diop de Dakar, Sénégal. 3 Laboratories for Food Safety Research, Biotechnology Center, University of Yaoundé I, Cameroon. 4 Laboratory of the Institute of Livestock Research for Development of N’Djamena, N’Djamena, Chad. 5 Laboratore de Biotechnologie, Institut de Technologie Alimentaire, Dakar, Sénégal. 6 Department of Biochemistry, Physiology and Pharmacology, Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, Yaoundé, Cameroon.


INTRODUCTION
Many people living in developing countries all over the world suffer of hunger -12.9% of the population is underfed. Sub-Saharan Africa is the region with the highest prevalence of hunger. About 25% of the populations suffering from chronic malnutrition live in sub-Saharan Africa (FAO, 2015). Every one child on six in developing countries, so like 100 millions of children, is suffering of insufficient weight (WHO, 2010). In the world, 66 millions of children go to school with an empty belly and of which 23 million live in Africa (WFP, 2012). According to World Food Program (WFP, 2012), reported that annually 3.2 billions of dollars for feeding 66 millions of children who go to school. However, according to currents previsions, by 2020, Africa will have the most increasing percentage of death because of noncommunicable diseases. Among the risk's factors, bad feeding and a poor consumption of fruits and vegetables could be the cause of 1.7 million of death (WHO, 2010). In order to fight hunger and diseases, many organisms and governments, encourage the use of vegetal species as minerals and proteins sources (FAO, 2015). In some developing countries like Senegal and Cameroon, governments encourage the use of naturals and bioproducts against under-feeding which cause many infantile mortality and morbidity (UNICEF, 2007;Shetty, 2010). Many studies have been shown the health and nutritional interest of edible mushrooms (Zhang et al., 2016;Alam et al., 2008;Pornariya and Kanok, 2009). Mushrooms and some plants provide proteins, carbohydrates, minerals, fibers, vitamins, minerals and fatty acids (Khatun et al., 2012;Okwulehie et al., 2014;Barros et al., 2007). They have therapeutic properties and many of them have been used in medicine all over the world (Badalyan, 2014). Researchers have shown that P. ostreatus has antitumor effects, antioxidant properties, antihyperlipidemic effects, antidiabetic effects, (Zhang et al., 2016;Abrams et al., 2011;Alam et al., 2008;Elmastas et al., 2007;Jayakumar et al., 2007;Jayakumar et al., 2006). Also, the plant M. oleifera has multiple therapeutic effects (Farooq et al., 2007;Ferreira et al., 2008;Sholapur and Patil, 2013). High malnutrition rates are common in the pastoral regions in different parts of the world. In order to solve this complex problem, simple alternative solutions that contribute considerably to immediate food self-sufficiency are required. The objective of the present study was to determine the nutritional value of Moringa oleifera and Pleurotus ostreatus mixture in specific proportions.

Plant material
The mushroom P. ostreatus was cultivated and harvested at the Biotechnology Laboratory of mushrooms at the University Cheikh Anta Diop de Dakar (UCAD), Senegal. They have obtained on a substrate from straw peanut and rice bran; the mycelium was inoculated on corn seeds. Fresh leaves of M. oleifera were collected from the Botanical garden of UCAD, identified and authenticated by a taxonomist from the Laboratory of Botany and Plants systematic, UCAD.

M. oleifera and P. ostreatus powder mixture preparation
Carpophores of P. ostreatus were washed with distilled water and then heated at 50°C for 10 min (Manzi et al., 2004). They were drained and dried at 40°C till a dry texture was obtained which was then molded in a sterilized at 170°C for 30 min local molding machine in order to obtain a fine powder. The leaves of M. oleifera were washed thoroughly two times with distilled water, and put inside the bag containing 1% of sodium hypochlorite for 5 min as describe by Bénissan et al. (2012). They were washed again with distilled water and dry on tissue paper in a clean room sheltered from the sun during 7 days. The dry leaves were then reduced to get powder for further exploration by an artisanal machine after sterilization at 170°C during 30 min (Rutala et al., 2008). According to the recommendation of FAO (2015), WHO (2007) and Goyens (2009), one mixture of two powders (M. oleifera and P. ostreatus) was prepared in function of the recommended daily intake under the aseptic conditions. These studies notified that when people take 30 g of M. oleifera and 15 g of P. ostreatus, more than 50% of daily recommended intakes of total proteins, calcium, potassium, magnesium, phosphore, iron, manganese, copper, zinc are covered by these proportions. For that reason, a mixture with these two species according to these recommendations was made.
All metallic serving utensils were sterilized with local autoclave at 170°C for 30 min, plates in autoclave at 121°C for 20 min, the others that didn't support high temperature were sterilized at UV for 30 min.

Chemical composition analysis
Chemical composition of sample (M. oleifera and P. ostreatus mixture powder in 2:1 proportions) was determined for moisture, crude protein, total fat, minerals (sodium, calcium, magnesium, phosphorus, potassium, iron, copper, zinc, manganese). Chemical analysis of samples was conducted at the "Laboratoire National d'Analyses et de Contrôle" in Dakar (Senegal), at Institute of Medical Research and Medicinal Plants studies MINRESI and "Centre Pasteur du Cameroun". The determination of crude proteins was made by mineralization process in the flasks, the distillation by machine VELP® Scientifica UDK 127 device, and titration relatively with the Kjeldahl method according to the AOAC (2002). Total lipids were determined by the method of Soxhlet through the solubilization of lipids in an organic solvent, hexane. The samples weighed in Whatman paper and placed in extraction inners of the balloons ramp heating Bistabil BRAND 6 positions and heated for 12 h and then weighed (AOAC, 2002). Total ash was determined by the weight difference by incinerate the samples in the oven electronic Heraeus (T 5042, Germany) at 550°C for 24 h carbohydrates by the difference between dry matter and (crude proteins+ total ash + fats+ total fibers). Energy was calculated by *Corresponding author. E-mail: wfmbacham@yahoo.com Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License using carbohydrates, fats and crude proteins contents (Italian law, 1993). Minerals as calcium, magnesium, potassium, sodium, copper, iron, manganese, zinc, phosphorus are determined by flame atomic absorption spectrometry after mineralization of parallel samples in microwave mineralization (Milestone model START-D, Italy) with power of 800 W. The dry matter was determined by the method of drying according to the AOAC directions (AOAC, 2002). Samples of known weight were placed at 105°C for 48 h. The water content was calculated by the difference of dry matter obtained before and after drying.

Microbiological analysis
Sanitary quality of samples (M. oleifera powder, P. ostreatus raw powder, and mixture powders of M. oleifera and P. ostreatus cooked) were analyzed by identifying microorganisms including thermoresistants coliforms, Escherichia coli, Salmonella and Fungi commonly used as indicators of sanitary quality of water and foods (NF V08-060, 2009;NF ISO 16649-2, 2001;NF V08-061, 2009;NF ISO 21527-2, 2008). The detection of Salmonella was investigated according to the standard method of NF EN ISO 6579 (2002). A portion of each 25 g of mixture product was placed in 225 ml of buffered peptone water for pre-enrichment. The homogenized solution was incubated at 37°C for 24 h. Then, 0.1 ml of the solution was added in a tube containing 10 ml of Rappaport Vassiliadis broth (Biolife, Italy) incubated at 42°C for 24 h. The selective isolation was realized on medium Hektoen agar (Biolife, Italy). After researching thermoresistants coliforms, Escherichia coli were searched at 44°C for 24 h. For each tube of 10 ml of EPSI (Eau Peptonée Sans Indole), one colony was plated (NF ISO 16649-2, 2001). According to the standard method, Sulfito-reducer Anaerobe in mixture product was carried out by conventional methods NF V08-061(2009) based on the use of Tryptone Sulfite Néomycine medium (Biolife, Italy).

Data analysis
All experimental results are the averages of two parallel measurements (means ± SD). Quantitative data were expressed as means and standard deviation (SD) of at least 2 measurements. Each experimental set was compared with one way analysis of variance (ANOVA) procedure using Statistical Package for Social Sciences (SPSS) version 20 (SPSS Inc., Chicago, IL, USA). Duncan's new multiple range test was used to determine the differences of means. P values <0.05 were regard as significant.

RESULTS
The macronutrients levels are presented in Table 1. The highest content of carbohydrates was 25.13 ± 0.20 mg for P. ostreatus, 36.81±0.21 mg was the highest value of crude proteins in M. oleifera powder. Raw P. ostreatus had the highest content in fibers. The most abundant macronutrients in the mixture were crude proteins followed by fats, fibers and carbohydrates.
As can be seen in the Table 2, M. oleifera had the highest content of calcium, magnesium, manganese and sodium. Others minerals (iron, potassium, zinc, copper and phosphorus) were abundant in raw P. ostreatus. The most abundant mineral in the mixture was potassium followed by phosphorus, calcium and magnesium.
As can be concluded from data present in Table 3, almost elements contents except sodium could cover minimum 20% of DRI for children (1 to 8 years). For some nutrients, the levels were more than 100% of DRI.
Some minerals covered a minimum of 20% DRI while others contained more than 100% of children's DRI within the age group 9-18 years (Table 4). This is valid for adults who had more than 100% of crude proteins, iron and manganese (Table 5).
As can be concluded from data present in tables, almost elements contents could cover more than 20% of DRI of all groups. For some nutrients (proteins, iron, manganese), we have noticed more than 100% of DRI.
According to referenced standards, we didn't find any pathogens in the product (Table 6).

DISCUSSION
The dried leaves of M. oleifera and P. ostreatus are rich in macronutrients and minerals. According to Breene (1990), Çokuner and Özdemir (2000), Bénissan et al. (2012), proteins content in 100 g dried matter of P. ostreatus ranged from 19 to 39 g; for M. oleifera average amounts 35 g. In this study, the protein content is in conformity with Shin et al. (2007). Watanabe et al. (1994) found that the carbohydrates value of P. ostreatus as 47.9 g in 100 g dry matter which is higher than this study, however, Alam et al. (2008) found that a value as 37.8 g which is also not compatible with result 16.70 g in 100 g dry matter. This difference might be due to the difference of environment factors. The results are similar those of Moyo et al. (2011) who worked on nutritional characterization of M. oleifera leaves. The most abundant mineral in both species is potassium 1566.8 mg followed by phosphorus 318.5 mg and calcium 284.3 mg and the least content was copper with 0.53 mg.
According to Daily Reference Intakes in the Essential Guide of Nutrients Requirements (EGNR) (2006), sodium contents is low and the great amount of potassium suggest the utilization of this mixture in anti-hypertensive diet because of the fact, potassium from fruits and vegetables can lower blood pressure.These results and observations are similar those of Manzi et al. (1999). Calcium was observed to be higher compared with other plant sources (Nkafamiya et al., 2010). It plays a key role in bone health. In fact, more than 99% of total body calcium found in the teeth and bones. Calcium deficiency can result from inadequate intake or poor intestinal absorption and can cause osteopenia, osteoporosis, and an increased risk of fractures (EGNR, 2006). Interestingly too, iron, commonly deficient in many plant-based diets, which is a necessary component of hemoglobin and myoglobin for oxygen transport and cellular processes of growth and division (EGNR, 2006), an essential trace element for normal functioning of the central nervous     system and in the oxidation of carbohydrates, proteins and fats (Umar et al., 2007), was found in abundance in these two species. These results are comparable to data published by Moyo et al. (2011). Results from this study had higher levels of zinc 4.36 mg/100 g. These results corroborate studies by Barminas et al. (1998), who reported 25.5 mg/kg in dried Moringa leaves. Zinc has been shown to boost the immune system, is also required for cell reproduction andgrowth especially sperm cells (Brisibe et al., 2009). Raw P. ostreatus and M. oleifera leaves were analyzed separately, then the mixture was done by mixing both,but in this case, P. ostreatus were cooked. According to Manzi et al. (2004), the effect of the cooking process is explained as a decrease of the nutrients contents of raw sample in the water, and consequently their concentration due to thermal degradation. The cooking process might affect the nutritional value by decreasing minimum 70% of his content. However, this decrease does not affect fibers which stay constant. Also, according to Barminas et al. (1998) and Broin (2006), the mode of conservation and time between collection and analysis might influence the nutritional composition; it is worth mentioning that these analyses were completed 6 months after collection and transformation.
According to the daily recommendations in the EGNR (2006), by the Institute of Medicine of the National Academies, vitamins and minerals requirements (FAO, 2004), 100 g of this dietary supplement composed by M, oleifera and P, ostreatus might cover many recommended daily reference intakes of minerals and macronutrients. Percentages covered by each element are represented in Tables 3 to 5; showed that the dietary supplement is a good source of minerals and macronutrients more than many others plants which are used like food supplement (Bénissan et al., 2012;Moyo et al., 2011;Maiga et al., 2005).

Conclusion
In conclusion, mushrooms such as P. ostreatus could be excellent food that can be used in alimentation for malnutrition problem in sub-Saharan Africa for their high contents in macronutrients and minerals. In this study confirmed the high nutritional quality of M. oleifera leaves which can be used to improve health and reduce malnutrition in the world. The data derived from nutritional value of this mixture of M. oleifera and P. ostreatus are clear indications that these species are rich in nutrients and had potential to be used as a food supplement and a promising dietary supplement that may overcome proteinenergy malnutrition problem in the third word. For that reason, our dietary supplement composed of these two species could be a very good source of nutrients for reduce malnutrition rate.