The study focuses on the untreated seeds of the four Mucuna varieties and the seeds are treated according to four protocols. The species studied are the seeds of Mucuna deerigiana, Mucuna nagaland, Mucuna cochinchinensis and Mucuna rajada harvested in Burkina—Faso. To better identify the part of the seed containing more tannins, the different parts, namely the epicarp, the starch and the crushed seed set, of each of the four untreated species were studied. The tannin determination for the treated species was done on the whole seed set.

The different seed processing methods [7] for four Mucuna species are as follows:

§ Mucuna seeds are boiled directly for 2 hours;

§ Mucuna seeds are soaked for 12 hours in water followed by boiling for 2 hours;

§ Mucuna seeds are boiled in the presence of KOH (0.5%) for 2 hours;

§ Mucuna seeds were soaked in water for 12 hours followed by scalding in the presence of KOH (0.5%) for 2 hours.

Different species of the seed were finely ground in a mortar. Then, 5 g of powder of each species were weighed and put into 30 mL of acetone-water system (80: 20, v/v) and kept in the refrigerator at 4˚C for three days [8] . The extracts were filtered and the filtrates kept in the refrigerator at 4˚C for subsequent tannin determination.

The method adopted to determine the tannins of the different Mucuna species is the reference method used to quantify the tannins of sorghum [9] .

Ferric ammonium citrate (III) and ammonia were added to part of the acetone phase and the absorbance of the resulting solution was measured spectrophotometrically at 525 nm.

The determination of the tannin content is done using a calibration curve obtained from gallic acid as reference.

2.5 g of powder of the plant material were added into 50 mL of boiling water and left to infuse for 15 min before filtering. A few drops of FeCl₃ are added to the infused, precipitation with green, brown or blue coloration shows:

§ The presence of gallic tannins if the color is dark blue;

§ The presence of catechic tannins if the color is green brown [10] .

After extraction of phenolic compounds by chloroform, then comes the processing of the total tannin determination.

1 mL of the acetone phase was measured with a pipette and introduced into a test tube N˚ 1. 5 mL of distilled water and 1 mL of ammonia were added and the whole was stirred for a few seconds with a vortex mixer.

A second sample of 1 mL of the acetone phase is taken in a test tube N˚ 2 by adding 5 mL of distilled water and 1 mL of the iron (III) ammonium citrate solution while stirring for a few seconds with a vortex mixer.

The two solutions from tubes N˚ 1 and N˚ 2 are transferred to the measuring vessels (1 cm) for measurement of absorbances with a spectrophotometer (UV-1700: UV-Visible spectrophotometer—SHIMADZU) at 525 nm against water, 10 min after operations 1 and 2. The result is based on differing absorbances.

The establishment of the calibration curve was done as follows:

Ÿ The gallic acid solution was pipetted into 6 test tubes 0, 1, 2, 3, 4, and 5 mL, respectively, and then each test tube was supplemented with acetone such that 10 mL of solution was obtained.

Ÿ 1 mL of each of these 6 solutions was pipetted into 6 other test tubes and successively supplemented with 5 mL of distilled water, 1 mL of the iron (III) ammonium citrate solution. The whole was shaken for a few seconds with a vortex mixer.

Ÿ The curve was plotted with the absorbance values on the ordinates and the corresponding concentrations on the abscissa.

The resulting solutions are transferred to the measuring cessels and the absorbances are measured 10 minutes later at 525 nm against water.

The statistical study was carried out by Excel software at the 5% probability threshold. All experiments were performed in triplicate. The comparison of means was performed by analysis of variance (ANOVA). Results are expressed as mean ± standard deviation. Values of p < 0.05 are considered statistically significant [11] .

The results of the phytochemical screening on the tannins of the Mucuna different species seeds are shown in Table 1.

A calibration curve was plotted with gallic acid at different concentrations. The tannin content was determined from Equation (1):

y = 0.0234 x + 0.0911 (1)

With the correlation coefficient R² = 0.9985.

The results of tannin determination in different parts of untreated seeds of the four Mucuna species are shown in Table 2.

++: intense coloring and +: less intense coloring.

Superscripted values with the same letters in the lines were not significantly different (p < 0.05) according to Duncan’s multiple comparison test.

The tannin content values in the treated seeds of the different Mucuna species are recorded in Table 3.

The percentages of tannin reduction in the treated seeds of the four Mucuna varieties are recorded in Table 4.

Intense staining was observed in the tubes containing extracts of Mucuna deerigiana and Mucuna nagaland seeds. This implies a strong presence of tannins in the seeds of these Mucuna species. The tubes containing the extracts of Mucuna rajada and Mucuna cochinchinensis seeds are less colored indicating a light presence of tannins in the seeds of these two species.

The action of iron (III) chloride on the aqueous extract showed that all four seed varieties contain tannins (Table 1). This corroborates to the work of Agbafor and Nwachukwu [12] and later Murthy et al. [13] .

Superscripted values with the same letters in the columns were not significantly different (p < 0.05) according to Duncan’s multiple comparison test. ^†A: seeds scalded directly for 2 hours; ^†B: seeds soaked for 12 hours in water followed by scalding for 2 hours; ^†C: seeds boiled in the presence of 0.5% KOH for 2 hours; ^†D: seeds soaked in water for 12 hours followed by scalding in the presence of KOH (0.5%) for 2 hours.

The test results of the different parts of the untreated seeds (Table 2) revealed, on the one hand, that the extract of the starch contains more tannins than the extract of the epicarp. On the other hand, for the crushed seed set, the results show that the Mucuna deerigiana seed extract is richer in tannins (0.370 g GAE/100g DM). It is followed by the seed extracts of Mucuna nagaland, Mucuna cochinchinensis and Mucuna rajada with the respective values of 0.336 g GAE/100g DM, 0.266 g GAE/100g DM, and 0.203 g GAE/100g DM thus confirming the results of the identification test. These values are similar to those found by Murthy et al. [13] which was 0.306 g GAE/100g DM for Mucuna cochinchinensis (white seeds) but lower than the value of 0.577 g GAE/100g DM found for Mucuna cochinchinensis from black seeds [13] .

Tannins are much more concentrated in starches than in epicarps. If they were more concentrated in the epicarp, the epicarp would be removed and the starch left for feeding. Since the starch constitutes the largest part of the seed, hence the need for the treatment processing.

Determination results of extracts from treated seeds of the four Mucuna varieties showed an effective decrease in tannins (Table 3).

All seed treatment methods resulted in a decrease of tannin content. However, the method of soaking the seeds in water for 12 hours and boiling in the presence of KOH (0.5%) for 2 hours gave the best result.

The percentages of tannin content reduction in the treated seeds of different Mucuna species are recorded in Table 4. The results revealed that the method of direct boiling of seeds for 2 hours decreased the tannin content by 24.86% in Mucuna deerigiana seed extract, 42.85% in Mucuna nagaland seed extract, 18.42% in Mucuna cochinchinensis seed extract and 3.94% in Mucuna rajada seed extract. This decrease in tannins from the different seeds of four species of Mucuna can be explained by the increase in the temperature of the water which broke some tannin-protein bonds allowing the polar solvent to extract the tannins from these seeds.

In addition, the method of soaking the seeds for 12 hours in water followed by boiling for 2 hours reduced the tannin content in the extracts of Mucuna deerigiana, Mucuna nagaland, Mucuna cochinchinensis and Mucuna rajada by 27.02%, 45.53%, 32.27% and 5.91% respectively. It is noted that this method reduced more tannins in the seeds of the different Mucuna species than the previous one. The high rate of tannin reduction can be explained by the duration of the soaking of the seeds.

On the other hand, the method of treating seeds by boiling in the presence of KOH (0.5%) for 2 hours resulted in low tannin reduction rates. It resulted in 8.37% tannin reduction in Mucuna deerigiana seed extract, 31.25% in Mucuna nagaland seed extract, 1.50% in Mucuna cochinchinensis seed extract and 3.94% in Mucuna rajada seed extract. This low reduction rate can be explained by the formation of potassium salts with some tannins that are trapped.

Finally, the method of soaking seeds in water for 12 hours, then boiling in the presence of KOH (0.5%) for 2 hours gave for the extracts of the seeds of Mucuna deerigiana, Mucuna nagaland, Mucuna cochinchinensis and Mucuna rajada, the percentages of reduction of tannins respectively 47.29% , 48.21% , 66.16% and 15.76%. These results, compared to those obtained by the first treatment method which seems to be the best, indicate a clear increase in the rate of tannin reduction for the seeds of these different Mucuna. However, this method of soaking seeds in water for 12 hours and then boiling in the presence of KOH (0.5%) for 2 hours resulted in a high reduction of tannins in the seeds of all four species of Mucuna. This high reduction rate can be explained by the soaking time, which has already extracted some seeds that can form salts.