1. Introduction
Vitamin A deficiency (VAD) is still a prevailing public health challenge in many sub-Saharan countries [
1]. While several interventions have attempted to reduce this burden, few have provided the promise of sustainable impact on a large scale when compared with biofortification of crops [
2,
3]. The main advantage of biofortification rests on the selection of crops which are usually staples of selected populations, thus increasing their adaptability [
4]. This is true of a crop like cassava in Africa where it is a widely used and consumed staple especially in underdeveloped populations [
5,
6,
7,
8,
9]. Another advantage of biofortification is that unlike other interventions that may seek to deliver a high instant dosage of micronutrient through food supplementation/fortification, biofortifying staples will consistently contribute to daily micronutrient intake, so far, the crop is consumed [
10].
Thus cassava, which is a chief source of dietary carbohydrate in local diets, when biofortified with increased levels of carotenoids, can now offer other nutritional benefits, such as contributing to improved functioning of visual and immune systems [
11], and possible inhibition of carcinogenic pathways [
12]. In Nigeria, where a strong breeding effort exists, there have been releases of high pro-vitamin A content cassava varieties since 2011 [
13,
14]. These varieties also popularly referred to as “yellow cassava” which are being promoted in various communities and are gaining momentum across the country [
15]. These successes have diffused into neighboring countries within sub-Saharan Africa (SSA) with a release of similar varieties to combat VAD in burdened populations.
However, retention of carotenoids is still a challenge during the processing of fresh yellow cassava roots into commonly consumed products mainly due to the sensitive nature of carotenoids to light, heat and physical handling [
16,
17]. Thus, the retention of total carotenoids is usually dependent on the prevalent processing method and the variety being used. The former being difficult to control especially in large scale processing which is common in SSA
While previous studies have highlighted the retention of total carotenoids in cassava products [
13,
17,
18] few studies have specifically examined the effects of processing on β-carotene—the principal carotenoid in biofortified cassava [
19,
20,
21]. Also, studies examining β-carotene retention at each step of processing of cassava into commonly consumed local products are scarce [
17,
20,
21]. Another justification for this probe is that, even though there is a report on retention in fermented dough made from biofortified Cassava [
20], no study has evaluated β-carotene retention in high carotenoid content varieties, which were most recently released in 2014, especially when processed into commonly consumed products (gari and its dough). Gari is a roasted granule obtained through processing (fermenting, grating, dewatering, and frying) of fresh cassava roots, and its dough “eba” is obtained by cooking gari in hot water to the constant dough. These products constitute a major part of the dietary intake of cassava products in Nigeria and SSA [
22,
23].
In this study, β-carotene concentrations and their retention, in gari and its dough “eba” were studied under two fermentation periods. The study also evaluated the possible contribution of these products to Vitamin A intake by comparing β-carotene concentrations in yellow varieties with dietary data of analogous products from the white cassava variety.
2. Materials and Methods
2.1. Experimental Design
A laboratory experimental design was used to evaluate the concentrations and retention of total β-carotene in four varieties of biofortified cassava and their products. Comparison of laboratory results with dietary data of cassava products (white variety) was used to estimate the contributions of yellow cassava products to the Recommended Dietary Allowance (RDA) of vitamin A in selected respondents.
2.2. Harvesting and Processing
Matured roots (aged between 11 and 12 months) of four recently released yellow-fleshed cassava varieties—TMS 0593, TMS 0539, NR 0220, and TMS 1371—were harvested from the research farm of International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. Large and small roots were selected in a proportional manner across all varieties. The total weight ranged between 5 kg and 7 kg. Damaged roots were sorted out. The roots were then peeled, washed, and processed into commonly consumed cassava products—gari and its dough. Two processing batches were carried out by fermenting the grated mash for one day and three days as explained in
Figure 1. The same experimental conditions were applied uniformly across both processing steps. Gari frying was carried out at 165 °C for 12 min. The dough was prepared by introducing the gari into hot (boiled) water and stirred until a smooth textured dough was achieved.
2.3. β-Carotene Extraction and HPLC Analysis
The extraction and instrumentation were carried out using HarvestPlus methods [
28] with slight modifications to the sample weight, which varied across each step of processing. Waters HPLC system (Water Corporation, Milford, MA, USA) consisting of a guard-column, C30 YMC Carotenoid column (4.6 × 250 mm, 3 μm) supplied by YMC Korea Co., Ltd., Sungnam-si, Korea, Waters 626 binary HPLC pump, 717 autosampler, and a 2996 photodiode array detector (PDA) was used for β-carotene quantification. Chromatograms were generated at 450 nm (
Appendix C) and subsequent identification of cis and trans isomers of β-carotene was done. The modifications to the extraction and the full description of the instrumentation applied were adapted from literature where they have been fully described [
29,
30].
2.4. Dry Matter Content
An oven-drying method was used to determine dry matter content. Samples (fresh cassava roots, intermediate, or final products) were oven-dried for 20 to 24 h at 105 °C until a constant weight was achieved. Weight before and after drying was taken and used to calculate the lost weight and the dry matter content [
31].
2.5. True Retention Calculation
After adjustments were made for weight and moisture content changes, percentage true retention was calculated as reported [
20,
32,
33]. Refer to
Appendix A for sample calculation.
2.6. Dietary Intake Assessment
Retrospective dietary intake data from 100 primary school children, 102 female and 100 male in-school adolescents, and 108 adult women were used for the study. The data was obtained from dietary intake assessments which used a multipass 24-h dietary recall method to elicit information from selected respondents. These assessments are periodically carried out by the Department of Human Nutrition of the University of Ibadan, Nigeria, and are always collected under the full guidance and approval of the University’s ethics review committee. The mean portion sizes (in grams) of commonly consumed cassava products (gari and its dough) were extracted from the full dietary survey data and averaged using a spreadsheet.
2.7. Estimation of Possible Contribution to Vitamin A Intake
Mean portion size (in grams) of the commonly consumed cassava products from white variety gari and its dough was compared to β-carotene concentrations in the similar products from yellow cassava varieties and was used to calculate possible contribution to Estimated Average Requirement (EAR) for vitamin A intake. The age range of the children whose dietary intake data was considered was 4–8 years. The adolescents ranged from 14 to18 years old and the women were aged between 20 and 50 years. The EAR values were extrapolated from the Dietary Reference Intake Tables [
34]. EAR values were 275 µg for children, 630 µg for adolescent males, 485 µg for adolescent females, and 500 µg for women. The bioconversion factor of 12 µg to 1 Retinol Activity Equivalent (RAE) was applied [
34]. Refer to
Appendix B for sample calculation.
2.8. Statistical Analysis
Data of analytical values were expressed as Mean ± Standard deviation (SD). The Statistical interaction between varieties and different processing methods on β-carotene concentrations and corresponding retention of intermediate and final products were evaluated using a linear regression analysis while means separation was analyzed using Duncan’s Multiple range test. The level of significance was set at p < 0.05. IBM SPSS Statistics for Windows, version 20 (IBM Corp., Armonk, NY, USA) was used for the statistical analyses.