1. Introduction
Cassava roots are a staple food that provides carbohydrates and energy for more than 2 billion people in the world, while representing the main source of carbohydrate and energy for the approximately 700 million people living in the tropical and sub-tropical areas [
1].
Vitamin A deficiency (VAD) is a widespread nutritional disorder in low-income countries, and is still a public health concern globally. VAD is the leading cause of preventable blindness in children. It leads to an increased risk of disease and death from diseases such as malaria, diarrhea and measles. [
2].
Yellow-fleshed cassava genotypes rich in provitamin A (pVA), are part of the outputs of an international biofortification effort by HarvestPlus, the International Institute of Tropical Agriculture (IITA), the International Center for Tropical Agriculture (CIAT) and other national agricultural research institutions, to reduce vitamin A and other micronutrient deficiencies through the development of staple food crops with enhanced micronutrient content. Provitamin A biofortified cassava is genetically improved for increased provitamin A content. In the case of this material, it was done through conventional breeding. Replacing the white-fleshed cassava varieties grown by most farmers with new high pVA (yellow) cassava varieties to address micronutrient and health needs of people, could benefit an estimated 20 million children under 6 years of age, who are currently at risk from diseases associated with VAD. Biofortified staple crops with higher micronutrient density, including yellow-fleshed cassava varieties biofortified with pVA carotenoids, have been developed to improve food and nutrition security reducing micronutrient deficiencies across the world [
3]. The United Nations have set 17 goals for Sustainable Development of which Goal 2 is the eradication of all forms of hunger, including hidden hunger, which refers to micronutrient deficiency [
4]. Biofortified crops contribute directly to this goal.
In 2013, 15 yellow-fleshed cassava genotypes with total carotenoid content (TCC) levels between 4–18 μg g
−1 were obtained from IITA-Nigeria. With the objective of releasing these genotypes, the Crops Research Institute (CRI) has been testing their agronomic performance across the various agro-ecologies in Ghana. Good cooking quality is an important parameter in selecting cassava for human consumption. Other factors important for selection are hydrocyanic acid (HCN) content, starch, fiber, cooking time, flavor, consistency and cooked pulp texture [
5].
The roots consist mostly of starchy flesh (80% to 90% by weight) with 60.3% to 87.1% consisting of water [
5,
6]. Moisture content is very important in the shelf life of cassava flour, since levels higher than 12% allows for microbial growth, which significantly reduces its shelf life [
5]. In cassava flour, the moisture is much lower than in roots and was reported to vary from 9.2% to 16.5% [
7,
8,
9,
10]. Cassava contains very low levels of protein of about 1–3% on a dry mass basis [
8] and between 0.4 and 1.5 g 100 g
−1 fresh weight [
11]. Cassava therefore has much less protein than cereals such as maize and sorghum, that have about 10 g protein per 100 g fresh weight [
12]. Cassava plants are very valuable, as they produce more weight of carbohydrate per unit area than other staple food crops under comparable agro-climatic conditions. Unfortunately, the low protein content and high starch content is the reason for the low nutritional value of the roots. About 50% of the crude protein in the roots consists of whole protein and the other 50% of free amino acids [
13].
The aim of the HarvestPlus program is the improvement of micronutrient content of crops to such an extent that it will impact on human nutritional and health status in a way that can be measured. Equally important is to ensure that the agronomic characteristics of the crop, such as yield and disease resistance, is not negatively affected. The process of developing biofortified crops include factors such as nutrient retention after harvesting, how much of the crop is consumed, and whether the biofortified crop is acceptable to the consumer. The bioconversion from pVA to retinol in the case of pVA rich foods (called bioavailability), is also an important factor. The mechanisms must also be in place for large-scale dissemination of the biofortified crop, which may differ in specific target countries [
14]. Carotenoids are very sensitive to light, heat and physical handling, which leads to losses during the processing of yellow-fleshed cassava roots into commonly consumed products [
15]. Total carotenoid retention is therefore largely dependent on specific genotypes and processing methods used to prepare products [
16].
The pVA content target level for cassava, set to reach 50% of the estimated average requirement for children and pregnant women in the DRC and Nigeria, assumes that up to 50% of pVA content in peeled roots is lost during processing, storage, and cooking [
17,
18]. Carotenoid retention higher than 50% in boiled cassava has been reported in different studies [
19,
20]. A study in Kenya demonstrated that feeding 2–4 years old children with boiled yellow-fleshed cassava improved their vitamin A status [
21]. Cassava in Ghana is mainly traded as either dry pieces of fermented cassava roots (
konkonte), that are milled into cassava flour to prepare
banku, or as fermented cassava paste (
bankye mole), used to prepare
koko. Cassava is also boiled and pounded with plantain to prepare
fufu. Generally,
fufu in Ghana is prepared by cooking peeled cassava in boiling water, whereas
chikwangue is prepared by precooking and steaming fermented cassava paste [
22,
23]. In Nigeria, a study found that apparent carotenoid retention in
fufu prepared with fermented cassava flour was 17–32%, but no information on true retention was presented [
24]. The same study also found that apparent retention of carotenoids was 86–90% when
fufu was prepared with a wet paste without a drying step. Another study in Nigeria reported true carotenoid retention between 12 and 36% when processing biofortified cassava roots into
fufu, using fermented cassava paste without a drying step [
15]. There is limited information on carotenoid retention in cassava in a country like Ghana.
Despite its nutritional and commercial benefits, cassava contains toxic substances that limit its utility, the most important being cyanogenic glucosides, which are responsible for the bitter taste of some cassava cultivars. [
25]. Glucosides such as linamarin and the linamarase enzyme react when cassava cells are mechanically damaged during harvesting. They then release acetone cyanohydrin, and this then decomposes to release cyanide [
26], either by hydroxyl nitrile lyase or spontaneously when the pH is higher than 5 [
27]. Cassava cultivars are therefore classified into two major types: bitter and sweet [
28] based on the cyanogenic content. “Sweet” cassava variety roots contain less than 50 µg g
−1 HCN on a fresh weight basis, whereas those classified as “bitter” varieties may contain up to 400 µg g
−1 HCN [
29]. However, the level of cyanide in the cassava roots can be effectively reduced with different processing and fermentation methods [
30].
Cyanide is stored in vacuoles of cassava cells, and is known to be more concentrated in leaves and the root cortex compared to root parenchyma [
31]. Several neurological diseases, including ataxic neuropathy, cretinism, and xerophthalmia are seen in areas where cassava is the staple food, and this has been attributed to cyanide poisoning [
32,
33]. Cyanide can also cause thyroid disorders, goiter and stunting in children [
34]. Cassava toxicity levels vary depending on altitude, geographic location, the period of harvesting, crop variety and seasonal conditions [
35]. Several cases of cassava poisoning have been recorded in Nigeria, all resulting from improper fermentation and processing of cassava. Cyanide exposure of more than 50 µg g
−1 caused symptoms such as headache, weakness, changes in taste and smell, irritation of the throat, vomiting, lacrimation, abdominal colic, pericardial pain and nervous instability [
36].
Cyanide content of cassava is higher during drought periods due to water stress in the plant [
37]. The response of cassava plants to water stress is a function of both the duration and severity of water deficit and the cultivar. Cyanogen is the most important toxic substance in cassava, which is formed because of enzymatic hydrolysis of linamarin and lostaustralin. Cyanogen increases during drought because of the “concentration effect” from reduced yields (which increases cyanide per mass), due to water stress. The naturally high cyanogenic glucoside content of bitter cassava varieties is further increased by water stress. Dry season (inter-seasonal dry spells) water stress, is similarly known to result in increased cyanogenic glucoside levels in cassava [
37]. During the dry season, cassava cyanogen levels can increase by 9–10 times their normal levels [
38]. In Mozambique, more than 55% of fresh sweet roots became extremely toxic during drought periods, a trend which was also observed in other countries in Africa [
31]. Cassava must, therefore, be processed to make it safe for consumption. Numerous processing techniques are used in cassava consuming countries. These techniques often improve palatability, extend shelf life, but also decrease the cyanogenic potential of cassava [
39].
The aim of this study was to determine the TCC, proximate values and HCN in yellow-fleshed cassava genotypes and to measure the retention of carotenoids during the processing of biofortified cassava into boiled cassava. This will help breeders to identify genotypes with the best nutritional quality across the tested locations for planting and promotion.
4. Discussion
The results of the proximate analysis of the different cassava genotypes samples from three locations revealed wide variation for all traits with ranges of 50.48–90.4% for moisture content, 6.85–45.79% for carbohydrate, 0.01–1.26% for protein, 0.07–1.24% for fat, 0.47–2.62% for fiber and 0.37–2.34% for ash. Significant differences (
p < 0.001) were found amongst the genotypes for each of the proximate analysis parameters. In general, the observed ranges were below values reported previously [
45,
46]. The maximum limit for the crude fiber and fat content observed agreed with values reported previously [
47]. However, the values observed for fat content was higher than the values reported previously [
36].
The carbohydrate values obtained in this study were lower than values reported previously, which had a range of 62.0–72.4% [
48], 87–89% [
49] and 85–89% [
45]. The results in this study generally indicated that yellow-fleshed cassava tends to have less carbohydrate than white-fleshed varieties.
Crude ash content is usually indicative of inorganic constituents (minerals such as K, Zn and Ca) and for cassava, and generally ranges from 1% to 2%. Ash contents represents the total mineral content in food after it has been burned at a very high temperature. The ash and protein contents were lower than values reported by other studies [
45,
50], but were similar to those reported by others [
32,
45] from six yellow and white cassava varieties cultivated in Umudike, Nigeria.
Cyanide concentrations vary in different cassava genotypes according to the altitude, geographical location and seasonal and production conditions [
51]. Reports have shown that age, variety and environmental conditions influence the occurrence and concentration of cyanide in various parts of the cassava plant and at different stages of development [
7], hence the genotypes need to be tested at different ages of maturity for further inferences. Cassava is classified as sweet if cyanide content is less than 50 µg g
−1 or bitter if the total cyanide is more than the 50 µg g
−1. In drought conditions, there is an increased total cyanide content due to water stress [
29]. Thus, a variety is considered to be “sweet” under one set of conditions may be “bitter” in a different geographical location or climatic conditions [
43]. Values from 15–400 µg g
−1 fresh weight of total cyanide in cassava roots have been reported in different studies, and there were reports of even higher levels, depending on where the crop was grown [
29,
51]. However, the rates can be reduced in cassava with different processing and fermentation methods. The observed levels of cyanide obtained in the present study showed that all the genotypes sampled could be classified as sweet varieties. The values were lower than those reported previously [
25] but it is not advisable to eat it raw, since the range is above the acceptable limit (10 µg g
−1).
A loss of micronutrients during processing and cooking is undesirable, as it reduces the nutritional value of biofortified foods. It is therefore particularly important that biofortified crops should be able to retain sufficient levels of micronutrients after typical processing, storage and cooking practices for successful biofortification.
All eight tested yellow-fleshed genotypes had TCC higher than the farmer preferred variety (Husivi) and the improved check (Cape Vars) in both fresh and boiled states. TCC of freshly peeled cassava of the evaluated genotypes was 1.18 to 18.81 µg g
−1 on fresh weight basis, whereas in boiled cassava it was lower, with a range of 1.01 to 13.36 µg g
−1 across the three locations. Large genetic variation for TCC in 12 yellow cassava genotypes in Brazil was also reported [
52]. Genetics was also reported to be a large contributor to variation in yellow and white cassava and their products [
53]. In general, there was a decrease in TCC level during boiling. The average TCC loss was 27.26% for Cape Coast, 15.03% for Fumesua and 16.59% for Ohawu, but there was large variation between genotypes for TCC loss, for example, I090151 lost only 7.96% TCC across locations, while I070557 lost 31.23% (
Table 6). This is in contrast with previous findings [
54] that carotenoid retention was better when sweet potatoes were boiled for the shortest possible time compared to methods like drying, frying and roasting that caused reduced retention. Another study [
13] also reported that boiling lead to higher TCC retention compared to other processing techniques in sweet potatoes. A study on yellow cassava in Colombia [
55] showed a mean retention of 87% of carotenoids after 30 min of boiling, which was higher than the 80.4% average in the current study. Their study also showed that dry matter content after boiling influenced TCC, and should be considered when measuring TCC retention. Another study [
56] showed a much lower TCC retention of 47.87–83.79% in three yellow cassava genotypes after 10 min of boiling. Both these studies indicated that initial TCC influenced retention after boiling, therefore genotypes that had high TCC before boiling also had the highest TCC after boiling. This was generally also the case in the current study, where genotypes that had high TCC before boiling, ranked high for TCC after boiling. Contrary to these studies, almost no influence of boiling for 30 min on β-carotene content in yellow cassava (on a dry weight basis) for both fufu and boiled cassava was reported in another study [
57].
Different factors separately or combined, such as heat, light, oxygen and enzymes, can lead to major or minor losses of carotenoids in yellow cassava during processing into consumable products [
18,
58]. The losses observed in the study for boiled roots could also be due to carotenoid isomerization and oxidation, which is the breakdown of trans-carotenoid to their cis-isomers due to increased content with moisture and heat treatment during boiling [
59]. Gari is also one of the most popular products of cassava processing in Ghana and sub-Saharan Africa, and it has been reported that extended roasting during its processing results in higher carotene content [
19]. Gari may therefore, be a useful way of efficiently utilizing biofortified cassava in VA deficient population. Further studies on more varieties commonly used for cassava dough, fufu, konkonte and gari may be needed to ascertain how yellow-fleshed cassava varieties may respond for TCC during processing into Ghanaian food forms.
Previous findings [
60,
61,
62] confirm TCC loss patterns in cassava products consumed in sub-Saharan Africa. The result further suggests that the current yellow-fleshed cassava genotypes being evaluated could provide more VA in diets and contribute to the reduction of health challenges associated with VAD, which is widespread in Ghana and sub-Saharan Africa. Following the agricultural transformation agenda in Ghana (Modernization of Agriculture in Ghana), which has resulted in the availability of improved varieties (including biofortified cassava), there is a great need to scale up micronutrients in staple foods produced in the country [
63]. Even though the impact of consuming yellow flesh cassava products on VA serum concentration is not yet fully established in VAD populations in Ghana, the results give an indication that yellow-fleshed cassava varieties are better than white-fleshed ones in terms of carotenoid and protein contents, and have the potential of reducing VAD in Ghanaian populations, where it is still endemic.