Étude du caractère cyanogénétique du manioc (Manihot esculenta Crantz)

The tuberous roots of cassava can release such quantities of hydrocyanic acid (HCN) that consumption without pretreatment may be very dangerous. Methods to eliminate the poison are known but cases of poisoning, of which some are fatal, still occur; moreover these methods often reduce the food value. For the preparation of cassava products for human and animal consumption it is very important to understand the poisonous character of the plant. Too little research has been performed in the past on the cassava toxicity, and the results obtained are often contradictory.

A review of cyanogenesis in plants is given (Chapter 2).

Cyanogenesis occurs in many families of higher plants but has also been observed in some lower organisms. Besides cassava some other cyanogenetic cultivated plants are: sorghum, flax, rubber, white clover, lima beans, almond and several other Prunus species.

HCN is liberated enzymically from cyanogenetic glucosides, this process starting as soon as the glucoside and the enzyme are brought together, e.g. after plant tissues are damaged. It is thought that the formation and degradation of cyanogenetic glucosides take place simultaneously in the living plant, whilst cyanogenesis is connected with protein metabolism. Recent studies show that amino acids can serve as precursors of cyanogenetic glucosides. There has proved to be a structural relation between the cyanogenetic glucoside and the precursor amino acid. Thus in cassava, valine and isoleucine serve as precursors of the aglycone moieties of linamarin and lotaustralin, cyanogenetic glucosides which occur in this plant (proportion 20:1).

The concentration of cyanogenetic glucosides (after this: glucosides) varies considerably according to species within the same family and also to individuals within the same species. The concentration depends on the stage of development of the tissue, or of the whole plant, and varies greatly according to environmental conditions.

The object of the research was at first to study the influence of some environmental factors on the toxicity of the tuberous roots. For a better insight in the cyanogenetic character we thought it necessary to also study other parts of the plant, notably the leaves.

Methods of determining the glucoside concentration in different parts of the cassava plant are described (Chapter 3). Some results of the relating studies are:- the amount of tuberous root to be grated may be reduced to a half or even a quarter by cutting the roots longitudinally;- samples of peeled and grated tuberous roots can be stored in closed bottles at 2°C for one week without important loss of HCN output;- storage at -15°C of various portions of plant material, whether whole or chopped, hardly reduces the HCN output for at least two months;- a favourable combination of maceration time and temperature for samples of peeled and grated tuberous roots is 16 - 20 hours at 35 - 40°C;- homogenization in a mixer of leaves, stem bark and bark of tuberous roots causes a rapid breakdown of all of the glucoside; prolonged maceration is not necessary and even reduces HCN output of leaf homogenates;- addition of diluted acid solution, buffer solution (pH 5.5-6.0) or extra enzyme during maceration of samples of peeled and grated tuberous roots does not increase HCN output;- glucoside concentration determined from liberated HCN is usually underestimated, due to partial blocking of the FICK.

Distribution of the glucoside in the plant, especially in the tuberous roots was studied (Chapter 4).

In the leaves glucoside content decreases with age, more so in leaf stalks than in leaf blades. In older leaves the concentration in leaf blades is higher, and in expanding leaves lower than in leaf stalks.

In the bark of the leafless part of the stem, glucoside concentration increases markedly from the top downward. In the bark of the original cutting, concentration is less than in the bark of the lower end of the stem. In the bark of tuberous roots, concentration equals or exceeds that in the bark of the lower end of the stem.

Glucoside concentration may vary greatly between the tuberous roots of one plant. Correlation between glucoside concentration and tuber size is not evident. Generally there is a variation in the pattern of concentration along the tuberous root; but in almost every case the highest concentration is found at the proximal end of the roots. In a transverse direction there is an increase from the centre outwards.

Concentration in the bark of tuberous roots is much higher than in the inner part and this difference is more obvious in less toxic clones than in very toxic ones. Concentration in leaves and bark of tuberous roots of less toxic clones is only a little lower than that in the same organs of very toxic clones. Thus as to toxicity, less toxic and very toxic clones differ mainly for the glucoside concentration of the inner part of tuberous roots. It is suggested that the ability to produce glucoside is, on average, about the same for less toxic as for very toxic clones, but that less toxic clones can easier metabolize the glucoside than the very toxic ones. This conversion is thought to take place mainly in the cambial zone of the tuberous roots.

Research on the effect of manuring with nitrogen, phosphate, potassium calcium, magnesium and farmyard manure (Chapter 5) showed that nitrogen has an increasing and potassium and farmyard manure a decreasing effect on the glucoside content of leaves and roots. The influence of potassium and farmyard manure was found to be more important in the roots than in the leaves. On the whole the effect of phosphate, calcium and magnesium was not important.

The supposition that glucoside concentration is positively correlated with the availability of valine and isoleucine in the plant may explain the influence of nitrogen and potassium, because manuring with nitrogen has been shown to increase, and with potassium to decrease, the amino acid concentration in the leaves of various plant species.

Fertilizer trials in the field with mature plants and in plastic pots or bags with young plants gave similar results and therefore indicate that experiments may be simplified by using young plants.

There was no indication that glucoside content of tuberous roots changed because of the dry season in Adiopodoumé, Bouaké and Man; there may be an increase in Ferkéssédougou.

However, experiments with young plants in bags showed clearly an increasing effect of drought on glucoside in leaves and roots (Fig. 6).

It is concluded that drought increases glucoside content, but in the field only after a very long dry period, because plants can adapt to short droughts by abscission of some leaves.

Glucoside concentration of peeled tuberous roots was studied during growth for three years in a field recently cleared of forest (Chapter 7). In the first year there was a considerable increase in glucoside concentration; during the second and the third year no further increase was observed. The increase in the first year is thought to be due to a decrease in soil fertility, especially for organic matter.

Experiments in four regions of the Ivory Coast indicated that glucoside content of peeled tuberous roots can rise remarkably in the beginning of the rainy season (figs 7-9). This increase may be mainly due to increased demand of the plant for nutrient elements, especially potassium and nitrogen, and to a change in the availability of the elements (increased nitrification after first rains; afterwards leaching due to heavy rains).

The toxicity of the roots proved to vary considerably between the fields in the four regions. The way clones reacted to the environmental conditions was far from identical.

No indications were found that the glucoside concentration of the tuberous roots is directly related to plant age, contrary to assertions of other authors. It is supposed that fluctuations in glucoside content during growth are mainly due to changes in ecological conditions.

Shading young plants was found to increase glucoside concentration of leaves and to decrease that of roots (Fig. 11).

Glucoside concentration in the leaves, on a dry matter basis, increases during the night and decreases during the day (figs 12-15), probably corresponding with the daily fluctuation of dry weight itself. Regular diurnal fluctuation of glucoside concentration expressed on basis of fresh weight of leaves is less important; quite often there is a slight increase in the morning, probably due to a rapid decrease of moisture content. Thus, a diurnal fluctuation of the absolute amount of glucoside seems unlikely.

Planting cuttings upside down did not influence glucoside concentration of the tuberous roots of the resulting plants. Sometimes glucoside concentration of young plants was influenced by this type of planting (tables 36 and 37).

A search for correlations between glucoside concentration and other characteristics of plants within clones often revealed a correlation with average root weight (r = + 0.35; 48 degrees of freedom).

A comparison of characteristics of 67 clones gave more results but often the coefficients were low (Table 39). Glucoside concentration of leaves was found to correlate with: glucoside of peeled tuberous roots (r = +0.55), dry matter content of roots (r = -0.34), number of roots per plant (r = +0.22). Glucoside concentration of peeled tuberous roots was found to correlate with: glucoside of leaves, dry matter content of leaves (r = -0.33) and of tuberous roots (r = -0.40), average root weight (r = +0.26), and amount of leaf (r = +0.20), stem (r = +0.24) and tuberous root (r = +0.20) per plant.

Ringing of stems (Chapter 10) caused a considerable increase of glucoside in the bark above the incision, especially during the first days. Such an increase was not observed when leaves were eliminated. Stem ringing, leaf elimination and stem cutting caused a decrease of toxicity of tuberous roots.

The ringing experiments indicate that the glucoside or products that cause its formation (amino acids) are synthesized in the leaves and transported, at least partially, to the tuberous roots.

A method to determine the activity of the enzyme linamarase in different parts of the plant is described and the distribution of this enzyme in the plants has been examined (Chapter 11). The activity was found to be highest in the very young expanding leaves, and lowest in the inner part of tuberous roots. In very young leaves enzyme activity may be as much as 100 times as high as that in peeled roots.

In the bark of tuberous roots activity is about 20 times as high as in the inner part. In the leaves activity decreases with age, more so in the leaf blades than in the leaf stalks. In the bark of the leafless part of the stem, activity is much lower than in the leaves and there is a marked decrease from the top downwards.

Enzyme activity in the tuberous roots of very toxic and less toxic clones is hardly different, either in the bark or in the inner part.

The danger of poisoning due to consumption of cassava products is discussed (Chapter 12). For various methods of preparation of cassava roots and leaves, the chapter indicates how far they result in a decrease in toxicity. Results of our research are added.

After boiling poisonous tuberous roots, 90 % of the glucoside was found to be still present but the enzyme was destroyed. Supposedly the danger of poisoning due to eating them is reduced but not eliminated by boiling.

Addition of glucose before maceration of homogenates of peeled tuberous roots hardly reduces HCN output.

Boiling is a very suitable method of eliminating glucoside from leaves, if these are previously crushed or chopped.

Addition of juice prepared from leaves or bark of tuberous roots, caused a considerable acceleration of the hydrolysis of the glucoside present in grated peeled tuberous roots, due to the high enzyme activity of these juices.

After crushing unpeeled tuberous roots, all glucoside present is hydrolysed within one hour, after which HCN can be driven off.