An Evaluation of the Protein Digestibility of Flours and Derived Protein Rich Product of Three Varieties of Mucuna Pruriens (L.) From Rats (Rattus Norvegicus) Males in Growth

The objective of the present study is to evaluate the proteinaceous digestibility of crude flours and derived protein rich product of three varieties of Mucuna pruriens of the male albino rats in growth. 48 rats have been taken randomly split up into 8 groups where 6 each according to their formulated feedings; those foods are: foods without protein (RSP), foods with casein (RC), food with the black macuna derived protein rich product (RMNc), food with the scratch macuna derived protein rich product (RMRc), food with the white macuna derived protein rich product (RMBc), food with the black macuna crude flours (RMNf), food with the scratch macuna crude flours (RMRf) et food with the white macuna crude flours (RMBf). All those foods content the same quantity of azote excepted foods without protein. Rats have been isolated in the semi metabolic cages which allow gathering the remaining of the foods supplied and the faeces to determine the parameter values of consumption. The experiment has taken 28 days. The apparent and reel protein digestibilities obtained in each food are respectively : RC (76,63±0,36% ; 95,86±0,06%), RMNc (71,20±0,77% ; 91,60±0,99%), RMRc (73,68±0,23% ; 93,49±0,85%), RMBc (71,16±0,59% ; 92,03±0,70%), RMNf (33,25±1,91% ; 53,881±1,70%), RMRf (31,3±2,40% ; 52,12±2,22%) and RMBf (31,12±4,00% ; 51,92±3,8%). The results show that derived protein rich product have ameliorated the protein digestibility and could be the alternative for the use of seeds of Mucuna pruriens to feed human beings and animals.


Introduction
In sub-Saharan Africa, the problem of protein malnutrition is recurrent because the diet of populations is characterized by protein deficiency, the consequences of which occur in  . Legume seeds, like those of Mucuna pruriens, are a rich source of quality proteins for humans and animals but have limited digestibility due to the antinutrients they contain (Gurumoorthi et al., 2003;Siddhuraju et al., 2005).
Indeed, Mucuna pruriens presents agronomic, medicinal and nutritional potential, but is very little used. The nutritional values determined for Mucuna seeds are comparable to those for conventional legumes (Pugalenthi et al., 2005). Protein values are generally between 22 and 35%. These values are higher than those found in conventional legumes such as Pisum sativum (22%), Phaseolus vulgaris (21%), Cicer arietinum (19%) and Lens culinaris (21%) (de Almeida Costa et al., 2006). However, it has been noted that when consumed without proper treatment, Mucuna seeds have adverse effects in humans and birds (Josephine and Janardhanan, 1992;Vijayakumari et al., 1996;Dossa et al., 1998Dossa et al., /1999Del Carmen et al., 1999). Among the toxic and anti-nutritional factors identified in these seeds are tannins, trypsin inhibitors, anticoagulants and L-dopa (Bell and Janzen, 1971;Daxenbichler et al., 1971;Ravindran and Ravindran, 1988). Traditional treatments such as cooking, fermentation or germination improve the protein digestibility of seeds by decreasing the levels of some anti-nutrients (Bishnoi et al., 1994;Chau and Cheung, 1997). In addition, methods for the production of isolates, concentrates and curds in seeds of certain legumes are an effective way to improve the nutritional quality of proteins as they reduce the toxic and anti-nutritional factors of proteins (Maoudombaye et al., 2012). The objective of this work is to evaluate the protein digestibility of raw meal and curds of three varieties of Mucuna pruriens (L.) in male rats (Rattus norvegicus).

Plant material
The plant material used consists of seeds of Mucuna pruriens, a legume little known and very rare on the Chadian market. Three varieties were used in this study: black, white and striped varieties (Fig. 1). The seeds were provided by the Chadian Institute of Agricultural Research for Development (ITRAD).

The animal material
For many years, nutritionists have adopted a particular rat strain, isolated by the Washington Wistar Institute as an experimental model (Adrian et al., 1991). The most obvious advantage of the rat is its omnivorous character and especially the sensitivity of its response to nutritional conditions. By simply monitoring its daily intake within a few days, it is possible to predict the nutritional quality of the component studied (Adrian et al., 1991;Giami et al., 2005). The animals are male albino rats of the Wistar breed, growing between 87 and 116 g.

Production of Mucuna Pruriens Flours and Derived Protein Rich Product
The seeds of the three varieties (white, striped and black) of Mucuna pruriens were soaked separately in distilled water in a 1/10 (w / v) ratio for 24 h at 35 °C. They were squeezed and then dried for 48 h in an oven (Towson & Mercer, England) ventilated at 40 ± 5 °C. The dried seeds were husked using a cereal mill (SAMAP brand) and manually wound to remove the seed coats. They were then crushed in the same mill and then sieved using a sieve of mesh 0.5 mm in diameter. The flours thus obtained were stored at 4 °C. in plastic bags for the production of curds, the nutrition of the rats during the experiment and the determination of the physico-chemical characteristics.
The derived protein rich product are produced according to the procedure described in (Ngatchic et al., 2005). Proteins coagulate in the presence of acids and heat. The principle of this process is to produce Mucuna milk from the flour and to curdle it in the presence of hot citric acid. 500 g of mucuna flour were dissolved in 2500 ml of distilled water (1/5 weight / volume ratio). The solution obtained was stirred with an electric stirrer (TECHNICON stirrer motor, England) equipped with an autotransformer (DIDALAB, France) set at 130 rpm for 3h and then centrifuged using a Centrifuge (DL-600OB) for 10 min at 4000 rpm to collect the supernatant. The pellet was recycled once under the same extraction conditions. The supernatant obtained is mucuna milk. This milk was poured into a pot and then acidified with citric acid at a rate of 0.325 g / 100 ml. The whole was boiled on a hot plate at 97.5 °C. for 10 min. The solution was then poured into a pressing cloth and then pressed for 10 min. The compact mass obtained is the mucuna Thederived protein rich product.
After pressing, the mucuna curd was removed and dried in a ventilated electric dryer at 40 ± 2 °C for 48h. It was finally ground and sieved to a particle size of 0.5 mm and then the powder conserved as in the case of flours at 4 ° C. in plastic bags for the physicochemical characterization and nutrition of the animals during the experiment.

Determination of the Protein Content of Flour and Faeces
The protein content was determined using the Kjeldahl method (AFNOR, 1982) for mineralization and the method of (Devani et al., 1989) for the determination of nitrogen. The crude protein content was obtained by multiplying the nitrogen content by the conventional factor 6.25 (Séraphin et al., 2015).

Formulation of Diets for In Vivo Digestibility
The in vivo digestibility of proteins evaluates the nutritional quality of a protein from the analysis of the results of the measurement of the nitrogen retained and of the growth of the rats. The quality of dietary proteins was assessed by the balance sheet method, which examined the measurement of ingested nitrogen and that lost in faeces (Mezajoug et al., 2010). The digestibility tests are carried out on animals placed individually in cages specially adapted to collect faeces, which are analyzed physico-chemically at the end of experimentation (Mezajoug et al., 2010).
The basic rule is that all rations are iso-nitrogenated (ie, they have the same nitrogen content) and the only variable between the positive control (containing casein), the negative control (containing protein ), And experimental rations, is the nature of proteins. The protein content of food is maintained at 10% (Giami et al., 2005). The rations were formulated according to (Bonafou et al. , 2007)[26]. In total, eight diets were prepared as follows:  A protein-free regimen representing the negative control (RSP);  A positive control protein diet having casein (RC) as a protein source;  Three protein diets from raw meal of the three varieties of Mucuna which are raw mucuna flour (RMNf), raw mucuna flour (RMRf) and raw mucuna flour (RMBf);  Three protein diets from the curd flours of the three varieties of mucuna which are black derived protein rich product mucuna (CWM), curdled mucuna (RMRc) and white curd mucuna (RMBc).

Conduct of the Experiment
The rats are weighed and randomized into 8 groups of 6 rats each, a total of 48 rats. They are each kept in semi metabolic cages (Giami et al., 2005).
During the experimental period, the foods were weighed and given to the rats in the form of cossettes once every two days in the morning in a fixed time interval (8 to 10h). The water is served in baby bottles at will and replaced every other day. Before distributing new foods, the remains of the foods offered to the animals were picked up and weighed. The difference between the quantities of feed served and the leftovers determined the amount consumed per animal (Adrian et al., 1998). The animals were weighed every other day at the same time interval. The last weighing took place at the end of the experiment. The feces of the last two days of the experiment were collected and their nitrogen content was determined for the evaluation of protein digestibility (Adrian et al., 1991). The variation in body mass and the amount of food consumed during the experimental period were used to estimate the protein efficiency coefficient (PEC). The loss of mass presented by the group of rats subjected to the protein-free diet was used to calculate the net protein efficiency coefficient (FNEC) (Mezajoug et al., 2010).

Evaluation of Nutritional Value Parameters of Diets
The weight gain (GP), expressed in g, is defined as the difference between the final weight and the initial weight of the animal. The weight gain per day was obtained by dividing the total weight gain by the number of days of the experiment.
GP (g) = Final weight -initial weight GP/j (g) = Final weight − initial weight Number of days The total quantities of food consumed (QTAC) represent the total quantities of food ingested during the experimental period. Its expression in g / d is obtained by dividing the amount of QTAC (g) by the number of days of the experiment.
QTAC (g) =Total quantity of food ingested during the experimental period. The protein efficiency coefficient (PEC) is obtained by comparing the weight gain (g) to the PTI (g).

CEP =
Growth of the test group (g) Total protein ingested (g) .

Protein Digestibility In-Vivo
Protein digestibility in vivo is determined by apparent digestibility (DA) and actual digestibility (DR).

Data Analysis
The data obtained were expressed as mean ± SD. The Statgraphics 5.0 software (Manugistics, Rockville, Maryland, USA, 1997) was used for ANOVA and the DUNCAN multiple comparison test for significant differences. Statistical significance was defined for p <0.05.

Results and Discussions
The amounts of food and nitrogen ingested as well as nitrogen excreted were measured in male albino rats subjected to a 28-day experiment. Rat weight measurements were also performed. These results are reflected in Fig. 2 (Fig. 2). On the other hand, the values of the regimes NMR, RMBC, NMR, RMRf and RMBf differ significantly from CPR at the threshold of p <0.05. The high level of consumption observed in the batch of rats subjected to the casein diet is due to the palatability of the presence of this protein in the food. Maga (1981) reported that basic amino acids and glutamic acid help to improve the flavor and palatability of foods rich in it. On the other hand, the low consumption rate of the batch of rats subjected to the protein-free diet would be due to the total absence of protein which would decrease the palatability. The results obtained are much greater than those of [31], which obtained the mean consumption of 17.8 ± 1.7 g and 8.8 ± 2.1 g respectively for the casein diet and the protein-free diet. This paper can be downloaded online at http://ijasbt.org&http://nepjol.info/index.php/IJASBT

Amount of Nitrogen Ingested Per Diet
The results of the analyzes yield values of 0.46 ± 0.01 g of nitrogen ingested for the batch of rats subjected to the casein (RC) regimen and 0.00 ± 0,00 g for the group of rats consuming the food Without protein. Nitrogen intake increases with the amount of food consumed for each diet (Mezajoug et al., 2010). After ingestion of a meal, nitrogen of exogenous origin represents between 50 and 70% of the nitrogen flux at the jejunal level (Baglieri et al., 1994;Mahé et al., 1996;Gaudichon et al., 1996;Gausseres et al., 1996). The nonprotein fraction of exogenous nitrogen is slightly higher than that of endogenous nitrogen, as it varies between 40 and 80% depending on the type of meal and the time (Baglieri et al. 1994). The levels of ingested nitrogen obtained from the different curd diets were significantly different: 0.43 ± 0.00 g for cMMR, 0.44 ± 0.00 g for CMA and 0.42 ± 0.00 g for RMBc. At the level of flours, the levels of NMR (0.43 ± 0.00 g) and FMRR (0.43 ± 0.00 g) were significantly different from each other and different from that of RMBf (0.42 ± 0.00 g) (Fig. 3). The results of casein diet and protein-free diet confirm their respective intake. In general, the quantities of nitrogen ingested are appreciable for all experimental diets except for the protein-free diet.
Fecal nitrogen test results yielded a maximum amount of excreted nitrogen for FMRR (0.29 ± 0.01 g) and a minimum value for the batch of rats subjected to the protein-free diet (0.089 ± 0.00 G). The fecal nitrogen values of the clotted groups are significantly identical with each other, as well as for the crude flour diets (Fig. 4). The values of the curds are close to those of the casein, which attests that the proteins of the curds have been well digested. On the other hand, the faecal nitrogen levels of raw meals are very high: 0.29 ± 0.00 g for (RMBf) and for (RMRf) and 0.28 ± 0.00 g (NMR). The maximum fecal nitrogen excreted by the groups of rats subjected to raw meal was due to the high content of raw fiber foods and the antinutrients which would have complexed the proteins thus preventing their digestion. The fibers of the different regimes were increased by the incorporation of 5% cellulose.Increased dietary fiber content increases fecal nitrogen excretion following an increase in microbial nitrogen (Zhu et al., 1990). The protein fraction of endogenous nitrogen mainly consists of salivary amylases, pepsin, pancreatic proteases (trypsin, chymotrypsin, elastase, carboxypeptidases) and lipases (Baglieri et al., 1994;Mahé et al., 1996;Gaudichon et al., 1996;Gausseres et al., 1996). Moreover, because of their high water retention capacity, the fibers exert a "barrier" effect which would impede the digestion and reabsorption of digestive secretions and explain the higher endogenous losses observed in their presence (Mahé et al., 1997). All this explains the rate of 0.089 ± 0.00 g for the diet without protein and that of fecal nitrogen excreted (0.10 ± 0.00 g) by the casein-fed animals because they lack fiber (Mezajoug et al., 2010).  Théopihile et al. (2017) Int. J. Appl. Sci. Biotechnol. Vol 5(3): 284-292 This paper can be downloaded online at http://ijasbt.org&http://nepjol.info/index.php/IJASBT

Weight Gain of Animals
Evaluation of animal weights yielded results ranging from 11.55 ± 0.30 g for casein-fed rats to -8.6 ± 0.84 g for NMRfed rats. The weight gains of NMR (10.78 ± 0.10 g) and C-RMR (11.28 ± 0.52 g) were identical to each other and significantly differed from RMBc (10.13 ± 0.45 g) at the threshold of P <0.05. There was no significant difference between cMRc and casein group (Fig. 5). These results show that the proteins of the curds are assimilated to the same title as the reference protein which is casein. In the case of crude flours, the weight losses of NMR (-8.6 ± 0.84 g) with RMRf (-7.94 ± 0.13 g) and RMBf (-8.01 ± 0.25 g) Between them. The weight losses of RMRf, RMBf and protein-free diet are identical to the threshold of p <0.05. The weight loss of the animals would be attributed to the poor protein digestibility of the raw meal on the one hand and to the total absence of it in the ration on the other hand. The results obtained are lower than those of (Bonafou et al., 2007) which reported a gain of 24.22 ± 9.580 g for the casein diet and a loss of -12.26 ± 5.200 g for the proteinfree diet.

Consumption Index (ICONS)
The Consumption Index (ICONS) is the ratio of the amount of food ingested to the weight gain of the animal. It reflects overall food efficiency and figures the yield of the ration. The lowest result reflects better production (Mezajoug et al., 2010). Under normal feeding conditions, the value of the consumption index is between 1.9 and 2.1; Or an average value of 2. The value 2 means that the animal consumed 2 kg of food to produce 1 kg of live weight (www.avicultureaumaroc.com). A negative result is a sign of misuse of the food (Maoudombaye et al., 2012). Table 1 shows the consumption indices ranging from 2.64 ± 0.09 for RMBc to -3.38 ± 0.07 for RMRf. From this table, it appears that the best production returns to the CMA RMR (2.46 ± 0.11) which has the lowest index. The cMNA consumption indices (2.55 ± 0.05) and cMRc (2.46 ± 0.11) were statistically identical to each other and that of casein and differed from that of RMBc (2.64 ± 0, 09). In general, the indexes of consumption of curds reflect a good protein efficiency because they have allowed weight gain comparable to that of casein. In contrast, the RMRf (-3.38 ± 0.07) and RMBf (-3.35 ± 0.10) consumption indices for the raw meals were significantly different from each other and different from that for NMR (-3.17 ± 0.24 g) at the threshold of p <0.05. These negative cues indicate poor digestibility of protein diets that resulted in weight loss of rats. However, ICONS alone can not predict the effectiveness of a protein as the amount consumed does not always correspond to the assimilated quantity (Bonafou et al., 2007).

Coefficients of Protein Efficiency (CEP) and Net (FNEC)
The protein efficiency coefficient (PEC) is a parameter for measuring the growth of animals. CEP is the ratio of animal weight gain on the amount of protein ingested. According to Friedman (Friedman et al., 1996), proteins with a PEC less than 1.5 are of low protein quality, those with a PEC between 1.5 and 2 are of intermediate quality, when greater than 2, the proteins are good quality. Based on this classification, protein of curds; 3.93 ± 0.06% (C NMR), 4.06 ± 0.18% (CPR) and 3.78 ± 0.13% (RMBc); Are of good nutritional quality such as casein (4.08 ± 0.11%). However, a low CEP value does not always indicate a low ability of the protein to grow. The calculation of the FNEC is more appropriate and more accurate (Friedman et al., 1996).
The efficiency of a protein for growth can be estimated by calculating the net protein efficiency coefficient (FNEC) which takes into account the weight loss of the animal fed to the staple food (Cheftel et al., 1985). This is the ratio of animal weight gain added to the weight loss of the proteinfree group divided by the amount of protein ingested by the animal. The CEPN values of the curds are significantly equal to each other. There was no significant difference between the value of casein CEPN (6.73 ± 0.03) and that of cMNA (6.67 ± 0.05) and MRC (6.66 ± 0.04). FNEC levels of raw meals, all negative, are statistically identical at the p <0.05 threshold. The values of the curds obtained are higher than those of rapeseed (4.59) and soybean (2.74) reported by Friedman (Friedman et al., 1996). This demonstrates the good nutritional quality of mucuna curds. Mezajoug (Mezajoug et al., 2010) reported a value of 5.89 ± 0.50 for casein.
The digestibility of a food is defined as the ability of a food to be transformed into chemicals capable of passing through the doorway, while the digestibility of a protein is the capacity of the digestive tract to effectively absorb the food, Nitrogen intake [38]. The apparent digestibilities obtained ranged from 76.63 ± 0.36% for the batch of rats subjected to the casein diet to 31.12 ± 4.00% for rats consuming foods based on crude mucuna flours black. The apparent digestibility values of rats fed with curds did not differ significantly from each other but were slightly lower than that of casein ( Table 2). The apparent digestibility rates of the crude flour diets, which are very low compared with those of the curds, are significantly identical to the p <0.05 threshold.
On the other hand, the actual digestibility rates range from 95.86 ± 0.06% for RC to 51.92 ± 3.8% for RMBf. The actual digestibility value of cMRc (93.49 ± 0.85%) was significantly identical to that of RC (95.86 ± 0.06%) but differed from that of cMR (91.60 ± 0.99%) In addition, RMBc (92.03 ± 0.70%) which are equal to each other. The actual digestibilities of crude flours, NMR (53.881 ± 1.70%), RMRf (52.12 ± 2.22%) and RMBf (51.92 ± 3.8%) are all identical to one another at the threshold of p <0.05. The digestibilities of the curds are included in the animal protein digestibility ranges of 95 to 98% and vegetable proteins of 75 to 95% reported. On the other hand, the low digestibility rates of the raw meal diets relativize their low nutritional quality (maoudombaye et al., 2012).

Conclusion
From this work, the curd production process not only concentrated proteins but reduced the levels of mucuna to antinutrients that complexed the proteins preventing their proper use. Nutrition tests carried out on the young growing male rats showed that the curds favored good growth of the rats compared to those fed with casein. The digestibility rates in these rats showed that the curds were not toxic and showed good nutritional quality of proteins. On the other hand, animals fed raw meal during the same tests lost weight. Mucuna raw meals showed a poor nutritional quality of proteins which resulted in substantial loss of animal weight. In view of these results, the curds of Mucuna