Manihot esculenta Crantz, commonly known as cassava, is a plant belonging to the class Malpighiales, Family Euphorbiceae, and genus Manihot. It is a plant of great economic interest, mainly in developing countries, as it has an important role as human food. Brazil is the second greatest world producer among 100 countries that grow cassava, and is responsible for 10% of the world production. Cassava is grown in all Brazilian regions, and it is used as raw material for cassava products [1] .

From the 98 known species of the family Euphorbiceae, cassava is the only one that is grown as food [2] . Cassava roots are energetic foods, presenting high levels of carbohydrates, mainly polysaccharides. In general, cassava has an average composition of 68.2% moisture, 30.0% starch, 2.0% ash, 1.3% protein, 0.2% fat, and 0.3% fiber [3] .

Cassava species are classified in two groups, according to the difference in toxicity and palatability of the roots: sweet (slightly sweet; that is, plants that have less than 100 mg/kg cyanuric acid) or bitter (those that have a perceptible bitter taste due to the high concentrations of HCN, that is, values over 100 mg). There is no morphological characteristic that may tell these two groups apart [4] - [8] .

The most concrete difference between the sweet and bitter varieties is the type of use. Bitter cassava is used for flour, starch production, and other uses, but only after some type of detoxifying industrial process. Sweet varieties are more versatile, and may be processed similarly to bitter varieties, but they are also consumed after simpler preparations, such as boiling, frying, or roasting [9] .

Cassava belongs to the group of cyanogenic plants as it has cyanogenic glycosides and enzymes distributed in its roots and leaves in different concentrations. Cyanide (the chemical product that has a cyanide group-C≡N) leads to toxic acute and chronic effects on biological systems. When the cells of the cassava roots are ruptured, the enzyme linamarase degrades cyanide, releasing cyanuric acid (HCN), which is extremely toxic both to humans and animals [10] _.

The oral route is the main path of exposure to cyanuric acid, and occurs by means of unintentional ingestion. However, inhalation is also common, especially during cassava processing. According to the World Health Organization (WHO), the currently determined lowest lethal dose that kills 50% of the animals of a study (LD₅₀) is 10 mg/kg (by inhalation) [11] [12] . In another study, it was demonstrated that the oral dose of cassava extracted linamarin was 324.86 mg/kg by oral route [13] . Another study determined a concentration range with lethal doses for humans between 30 and 210 mg/kg of live weight [14] .

As cassava is a highly important food that may be easily grown in the most different parts of the country, and considering that Brazilian feeding habits involve cassava in different forms, such as flours; and that both chronic and acute ingestion may cause several diseases in men and animals, the objective of this study is to quantify cyanogenic glycosides found in different types of commercially available cassava flours.

Commercial samples of cassava products used in the study were purchased in different retail stores in the city of São Paulo, SP. Three packages of each of the following types of cassava flour were purchased: artisan toasted cassava flour; sweet cassava starch; artisanal dried cassava flour; “bijuzada” cassava flour; industrialized, toasted cassava flour; industrialized, raw cassava flour; and wet cassava flour. In the laboratory, these packages were opened, and 1 g was used in the chemical analyzes (Figures 1-3).

Variation coefficient observed between the analysts was 0.998%.

The calibration curve (Figure 4) was determined for the evaluation of the correlation coefficient (0.9982) and determination of the equation of the straight line (y = 0.0002 + 0.001).

The results obtained in the correlation coefficient were satisfactory for the method employed in the quantification of cyanide in the samples.

Results of cyanide quantification in the samples analyzed are shown in Table 1.

Among the different types of cassava flour analyzed, wet cassava flour showed the higher concentration of cyanide (225 mg/500g). This fact may be attributable to the moisture, once most of the glucosides able to generate cyanide, such as linamarin and lotaustralin, are soluble in water, and keeping the product in water prevents the elimination of the toxic products.

In this sense, the quantification of total cyanide in the different processing stages of wet and dry cassava flours showed that the wet flower showed lower levels of cyanide, and that the manufacturing process to produce these flours is effective in cassava cyanide detoxification.

On the other hand, artisan toasted cassava flour showed the lowest cyanide content (15 mg/500g) [10] . This finding evidences the importance of heat treatment to reduce cyanide content. The same finding was observed in industrialized, toasted cassava flour, which showed lower cyanide content (115 mg/500g) compared with industrialized, toasted cassava flour (140 mg/500g).

Results obtained in this study show the importance of quantifying cyanide content in cassava, as well as the need for official regulations that determine safe levels of exposure to cyanide in foods, in order to ensure the quality of the final product, preventing risks to consumer health.

Cassava consumption may be a risk factor for goiter, especially in regions where there is low iodine intake. Thiocyanate, a cyanide metabolite, changes thyroid metabolism, making iodine retention difficult. Therefore, goiter occurs mainly due to iodine deficiency, and may become more severe because of to the ingestion of glucosides [17] - [19] .

Protein deficiency associated with a diet rich in foods that contain cyanogenic glucosides may lead to neurological damage due to the deficiency in sulfureted amino acids, leading to the accumulation of cyanide in the body [17] [20] .

There are studies that correlate cassava intake with the onset of fibrocalculous pancreatic diabetes, which is considered to be a subclass of a new clinical type of diabetes that is related to inadequate nutrition [21] [22] . Besides, studies carried out in animals evidence that prolonged ingestion of cyanide is associated with hepatic and renal lesions in rabbits [18] [23] [24] .

Chronic cyanide intoxication may lead to several consequences to humans, such as Tropical Ataxic Neuropathy (TAN), a syndrome involving myelopathy, bilateral optic atrophy, bilateral deafness, and polyneuropathy, which is observed in some areas of Nigeria and Tanzania. Other severe health problems that may be found together with TAN are Parkinson’s disease, cerebellar degeneration, psychoses, and dementia [17] [20] .

Cyanogenic glucosides are organic compounds made up of sugar and anaglycone (no sugar). They are formed, in general, by a hemiacetal bind between reduced sugar groups and the aglyconephenolic hydroxyl. The aglyconeportion of these cyanogenic compounds may be made up by an alquil, aril/alquil, or aril group which, generally, defines the characteristics of the glucoside. Therefore, when this group is represented by hydroxynitriles (cyanohydrin), it may be classified as cyanogenic [25] [26] .

There are factors that influence the concentration of cyanogenic glucosides in plants, such as age, and seasonal, environmental, nutritional, and genetic factors [27] .

Cassava accumulates two glucosides in its roots and leaves: linamarin and lotaustralin (93:7 ratios). These glucosides are able to produce cyanuric acid by hydrolysis. The enzyme responsible for the hydrolysis is linamarase; when the plant tissue is hydrolyzed/degraded, linamarin gets in contact with this enzyme, and when it is broken down, it yields glycose and α-hydroxynitriles, which, when catalyzed by a hydroxynitrillyase, yields cyanuric acid and ketones [28] . As linamarindoes not accumulate, LD₅₀ is not reached. Therefore, animals recover. There is no mention to this compound in Brazilian official regulations [12] .

Glucosides are easily hydrolyzed and degradation products are sugar and cyanohydrin, which, in its turn, yields the toxic compound cyanuric acid. Several factors are associated with hydrolysis, such as acid pH, endogenous enzymes, or even the action of beta-glucosidases found in foods that are eaten at the same time [27] [29] .

Plant that have more than 20 mg of cyanide in 100 g are considered of high risk for intoxication, even when ingested in small amounts. Cyanide is found in larger concentrations in the leaves of these plants. However, it may also be found, in considerable amounts, in roots, seeds, and other parts of the plant [30] [31] .

As cassava is a product used as human food and animal feed, cassava products are subjected to a series of health regulations, such as:

Brazilian Ministry of Agriculture (MAPA) regulation no. 554, from August 30^th, 1995, which approves the standard for identity, quality, storage, and transport of cassava flour to be commercialized, determining new specifications for cassava flour classification [32] .

Collegiate Directory Resolution (RDC) no. 359, from December 23^rd, 2003, which approves the technical regulation national labels in portions of packaged food [33] .

Collegiate Directory Resolution (RDC) no. 263, from September 22^nd, 2005, which approves the technical regulation for cereals, starches, flours and meals [34] .

As cassava flour is an industrialized product, it is inferred that it undergoes processes that would eliminate most cyanide found in the original product. However, residues may be found in amounts that are sufficient to lead to signs of intoxication [28] .

According Naves et al. consumption of significant cyanide doses arising from foods rich in cyanogenic glyco-sides and poorly processed may result in acute and chronic poisoning, causing abnormalities such as Konzo dis-ease (disease found in Africa that causes paralysis of the legs. This disease arises especially in poor rural areas and affects mainly young women and children and causes paralysis of the legs). Cyanide also features remarkable toxic power because it is a potent inhibitor of cytochrome oxidase, which results in blocking the electron transport chain during cellular respiration process. It is estimated that the consumption of food containing hy-drocyanic acid (HCN) in a concentration between 0.5 to 3.5 mg of HCN per kg body weight, may cause the in-dividual to death within minutes. However, the risk of intoxication can be minimized from the use of preparation processes, such as baking, frying and drying to reduce the content of such a compound in food. This beneficial effect results from the removal of cyanogenic glycosides, the inactivation of β-glucosidase, or both [35].

Cassava and its products are widely used in Brazil (and also, in other countries), being part of daily feeding habits in different Brazilian regions. Although cassava products are industrially processed, there is no official standard for the maximum amount of CN⁻ residues in these products, or established limits for safe human and animal intake.

Besides, there is an inherent occupational risk for operators working directly with the raw material, and, therefore, exposed to the toxic compounds discussed here. There also is an environmental risk posed by residues generated in the industrial process, with negative effects in animal health.

Industrial development has to be followed by the determination of standards for the consumer, environment, and workers to be safe when in contact with and when using these products. It should be emphasized that this food production segment is related with regional habits, which make it difficult, but not impossible, to update regulations related to this product, considering the impact these products may cause, as its effects are not clearly defined.

André Rinaldi Fukushima,Maria Aparecida Nicoletti,Almir Junior Rodrigues,Caroline Pressutti,Jeandro Almeida,Tamires Brandão,Rosilene Kinue Ito,Luis Antônio Bafille Leoni,Helenice De Souza Spinosa, (2016) Cassava Flour: Quantification of Cyanide Content. Food and Nutrition Sciences,07,592-599. doi: 10.4236/fns.2016.77060