AMERICAN JOURNAL OF FOOD AND NUTRITION Print: ISSN 2157-0167, Online: ISSN 2157-1317, doi:10.5251/ajfn.2014.4.1.1.10

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Proximate composition and carotenoid profile of yams (Dioscorea spp.)

and cocoyam (Xanthosoma maffa (Scoth)) root tubers from Nigeria Anthony N. Ukom1*, Philippa. C Ojimelukwe2, Chidi F. Ezeama2, Darwin O.Ortiz3, Ingrid. I. Aragon3,

Department of Food Science and Technology, Abia State University, Nigeria1 Department of Food Science and Technology, Michael Okpara University of Agriculture,

Umudike, Nigeria2 Nutrition Quality Laboratory, International Center for Tropical Agriculture, Cali, Colombia3

ABSTRACT

Major carotenoids of yams (D. cayenensis, D. dumetorum, and D. bulbifera) and cocoyam (Xanthosoma maffa (Scoth)) were identified and quantified using HPLC-DAD. The proximate composition of the samples was also determined. Carotenoid contents of these root and tuber crops varied. The carotenoid content of X.maffa (Scoth) was significantly (P

of epithelial cells, maintenance of epithelial barrier against attack by pathogenic organisms and plays important role in vision (Bovell-Benjamin, 2007). The all-trans-β-carotene exhibits the highest pro-vitamin A activity among carotenoids and is the usual configuration in nature. Some carotenoids are antioxidants which have been linked to enhancement of immune function and decreased risk of degenerative diseases like cancer, cardiovascular diseases and aging (Prasad et al., 2011; De Rosso and Mercadante, 2007; Gama and Sylos, 2005). The preventive role of carotenoids against diseases is attributed to its strong radical scavenging activity by capturing free radicals (atoms with unshared electron) owing to their conjugated double bonds or polyene chains (Rodriguez-Amaya, 1996). The role of food production is not just enough to feed

the people, but to feed the people well. Therefore

improvement of micro-nutrient dense staple food crops

(for minerals and vitamin) through biofortification for the

proper functioning of the body as well as prevention of

diseases should be a priority. Biofortification of staple

food crops with carotenoids and other micronutrients

like iron and zinc could play a key role in health

improvement programs. This breeding program utilizes

genetic manipulation that favors the genetic variability

for micronutrients in different staple crops (Chavez et

al., 2005; Champagne et al., 2010). Biofortification of

staple crops like cassava (Manihot esculenta Cranz)

clones with high provitamin A (β-carotene) in the root is

currently a program of

BioCassavaPlus{htt://biocassava.org/}. Although yams

and cocoyam have a narrow genetic base, it may be

that yams and cocoyam being the other very important

root and tuber crops in the tropical and sub-tropical

countries, especially West Africa, will receive attention

for genetic improvement with micronutrients. This study

is aimed at providing information on the proximate

composition and characterization of carotenoids in yams

(Dioscorea spp.) and cocoyam (X. maffa (Scoth)) grown

in Nigeria.

MATERIALS AND METHODS Roots and Tuber Samples: Three yams and one cocoyam variety samples planted in April, 2012 were taken after harvest in full physiological maturity in December, 2012 from Ututu, Abia State, Nigeria, and transported to International Center for Tropical Agriculture, Cali, Colombia The yams (Dioscorea spp.) were namely D.cayenensis (yellow yams), D.dumetorum (trifoliate yam), D.bulbifera (aerial yam) and cocoyam (X.Maffa (Scoth) (yellow cocoyam) were used.

Am. J. Food. Nutr, 2014, 4(1): 1-10

Sample Preparation: Sample preparation and analysis was carried out at Nutrition Quality Laboratory, International Centre for Tropical Agriculture, Cali, Colombia (CIAT). Tuber samples (aerial yam, trifoliate yam, yellow yam and yellow cocoyam) were washed with deionized water and immediately dried for morphological description. The tubers were chosen according to their colors varying from light to deep yellow. The whole and cross-section of the yams and cocoyam tubers were photographed. Then the tubers were peeled under deionized water. After peeling, the samples were diced into small cubes, pulverized (homogenized) using a blender (Retsch Grindomix, USA) at 1000rpm for 30 seconds. Each of these samples was packaged in cellophane bag filled with nitrogen gas

and stored at – 700C. After 24h, the frozen samples

were lyophilized (freeze-dried) at – 300C (Labconco

Corporation, Kansas City, MO). After freeze drying the samples were pulverized into powder, packaged

in a cellophane bag and stored at – 700C for

subsequent use. Analysis of proximate composition: Moisture

content, total ash (muffle furnace at 5500

C for 8h), crude protein (Kjeldahl method, %N x 6.25), crude fat (Soxhlet extraction system) and fiber were determined using AOAC (1995). Carbohydrate content was obtained by difference. Extraction of carotenoids from yams/cocoyam tuber Samples: Carotenoids were extracted in duplicate following the methods adopted in literature (Rodriguez-Amaya and Kimura, 2004), except that the separation of the solid and liquid phases was carried out by centrifugation and not by filtration (Chavez et al., 2007). Approximately, 5g of the each powdered tissue was added to a vial (50ml BD Falcon tube) with (10:10ml) acetone: petroleum ether and mixed using an Ultra-Turax Vortex Mixer (IKA Janke and Kunkel, Staufen, Germany) for 1 minute. Samples were then centrifuged (Eppendorf 5804R, Hamburg, Germany), at 6000rpm, for 6 minutes at 40

C. The organic phase was collected and extraction repeated on the residue with 5 ml acetone and 5 ml petroleum ether, followed by centrifugation at

6000rpm, for 6 minutes at 40C. Extractions were

continued until residues turned colorless, usually three times. The organic phases were combined with 10 ml of 0.1M NaCl solution and centrifuged (6000

rpm, 6min., at 40C). This washing process was

repeated two additional times. The aqueous phase was extracted with Eppendorf micro- pipitte and the carotenoids extract was made up to 15 ml when the

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Am. J. Food. Nutr, 2014, 4(1): 1-10 volume of extract was less. Precautionary steps were taken to protect extracted carotenoids from UV light and high temperatures. Special care was equally taken to avoid direct exposure to sunlight. The lights in the laboratory were protected with UV filters. Samples were covered with aluminum foils when not under processing. Carotenoid Quantification: Total carotenoids concentration (TCC) was determined by UV/Visible absorption spectrophotometry (uQuant, Biotech Instruments, USA), at absorbance of 450nm and using the absorption coefficient of β-carotene in petroleum ether (2592) (Rodriguez-Amaya and Kimura, 2004; Sanchez et al., 2013). For the HPLC-DAD determinations, 15ml of the organic phase (used for the spectrophotometric quantification of Total Carotenoid Concentration (TCC)) were transferred to a glass tube, and dried by nitrogen evaporation (N-Eva 112, Organomation Associates, Berlin, MA, USA). The nitrogen dried extract was dissolved in 2ml (1:1v/v) methanol and methyl tert-butyl ether (MeOH:MTBE) HPLC-grade and agitated in a VWR multi-tube Vortexer (2400 rpm, 60 seconds). This was immediately followed by filtering the carotenoids through a 0.22nm polytetra-fluorothylene (PTFE) filter into a sample vial and injected into the HPLC system. Separation and quantification of carotenoids were achieved using a YMC carotenoids S-5 (30 reversed-phase column (4.6mm x 150mm: particle size, 5nm), with a YMC carotenoid S-5 guard column (4.0 x 23mm) in an HPLC system (Aligent Technologies 1200 series, Waldbronn, Germany), with a cooling auto-sampler

unit at 40

C to avoid a evaporation of the injected

solvent and carotenoid degradation. DAD detector was set at three wavelengths: (286nm for phytoene; 348nm for phytofluene and 450nm for colored carotenoids). Peaks were identified by comparing retention time and spectral characteristics against a pure standard from CaroteNature (GmbH, Lupsingen, Switzerland: Lutein – No. 0133 HPLC 97%; α-carotene – No. 0007 HPLC 97%; β-carotene – No. 0003 HPLC 96%; (E/Z) – phytoene – No. 0044 HPLC 98%; Zeaxanthin – No. 0119 HPLC 97%; Lycopene – No. 0031 HPLC 95% and Xanthophyll – No. x 6250 minimum 70% from Sigma-Aldrich. Quantification of each carotenoid was carried out by integration of their peak area against respective standard curves. Two independent calibration curves were used to quantify β-carotene and phytoene. For all-trans-β-carotene, the linearity (correlation coefficient) was 0.99925 at 2, 6, 10, 14, 18, 25 and 30 ppm levels.For phytoene, the linearity was 0.99959 at 1.25, 2.5, 5,

3

10, 15 and 20 ppm levels. 9 –cis and 13 – cis isomers of β-carotene were identified according to the following combined information: elution order, UV-visible spectrum (גmax, spectral fine structure, peak cis intensity). Their calibration was made using the All-trans-β-carotene curve. For phytofluene, a similar approach was followed using the phytoene calibration curve (Ceballos et al., 2012).

Total carotenoids concentration (TCC) (μg-1

) was

calculated on fresh weight basis. Before determination, the method was validated according to the requirements of Thompson et al. (2002) and Eurachem-citac (2002). The relative standard deviation in reproducibility conditions is 2.1%. Statistical Analysis All tests were carried out in triplicate for proximate composition and in duplicate for carotenoids analysis. Means were separated using LSD at P

Am. J. Food. Nutr, 2014, 4(1): 1-10

Fig. 1A: Whole and Cross-section of D. cayenensis (yellow yam)

Fig. 1B: Whole and Cross-section of D. dumetorum (trifoliate yam)

Fig.1C: Whole and Cross-section of D. bulbifera(aerial yam) Fig.1C: Whole and Cross-section of D. bulbifera(aerial yam)

The result showed low dry matter content with high moisture content ranging from 622.1gkg-1 ±0.08 (D.cayenensis) to 694.9gkg-1 ±0.06 (X. maffa

(Scoth). This moisture level is in close agreement

with the data reported in literature (Osagie, 1992).

The protein content of the yams and cocoyam 4

Am. J. Food. Nutr, 2014, 4(1): 1-10

varieties in this study were between 31.95gkg-1

±1.59

(D. cayenensis) to 71.3gkg-1

±1.32 (X. maffa (Scoth),

and compared with the values reported for D. alata (Udensi et al., 2008). D. dumetorum (trifoliate yam) and X.maffa (Scoth) (yellow cocoyam) protein content were twice that of D. cayenensis (yellow yam) and D. bulblfera (aerial yam) and may be regarded as food of good nutritional quality to help prevent protein malnutrition. Yams with relatively high protein content are thought to be important food in many African

countries (Omonigho and Ikenebomeh, 2000). The yams and cocoyam varieties contained 27.3gkg- 1±1.65 to 71.0gkg-

1±7.41 crude fiber. The result

indicates high fiber content when compared to the value of yams (1.6±0.4) reported by Hsu et al.(2003).Foods with high dietary fiber is reported to promote beneficial physiological effects, including laxation, blood cholesterol and glucose attenuation (Llobera and Canellas, 2007).

Table 1: Proximate composition of the three yams (Dioscorea spp.) and cocoyam (X.maffa Scoth)(gkg-1)

Sample Moisture Dry matter Protein Crude fiber Crude fat Ash Carbohydrate

D.cayenesis 622.1c±0.08 37.90

a±0.08 31.95

b±1.59 27.30

b±1.65 4.4

a±1.91 19.64

c±0.67 294.5

a±0.24

(Yellow Yam) 690.5

a±0.52 31.28

c±0.09 69.15

a±4.49 56.50

a±4.10 3.67

a±1.60 28.38

b±0.56 151.8

c±1.18

D. dumetorum

(Trifoliate Yam) 653.1

b±0.17 34.69

b±0.17 37.14

b±1.54 71.00

a±7.41 3.47

a±1.40 30.66

b±2.79 204.6

b±1.03

D.bulbifera

(Aerial Yam)

X. maffa 694.9

a±0.06 30.51

d±0.06 71.3

a±1.32 55.3

a±1.32 2.20

a±0.35 47.29

a±1.28 129.2

d±0.91

((Scoth)(Yellow

Cocoyam)

Values are means of triplicate determinations. Mean values having the same letters within a column are not significantly

different (p

Am. J. Food. Nutr, 2014, 4(1): 1-10 Figure 1.The concentrations of colorless carotenoids, phytoene and phytofluene are also presented in Table 2. X. maffa (Scoth) contained the highest concentration of these colorless carotenoids, 1581µg/100g for phytoene and 1574µg/100g for phytofluene. The yam (Dioscorea) species maintained low values.

Phytoene and phytofluene are significant in the biosynthesis of colored carotenoids (Fanciullo et al., 2007).The concentration of these carotenoid precursors may be important when considering increasing the content of provitamin A carotenoids in X. maffa (Scoth) through biofortification.

Table 2: Carotenoids content (μg/g) of Yams (Dioscorea spp.) and Cocoyam (Xanthosoma

maffa (Scoth)) Tubers

Yam Lutei Zeax 13-cis- Alpha All- 9-cis- Total Vit. Phytoe Phytoflu Variety n anthi β-C carotene trans- βC Carotenoi A(RE/1 Ne ene

N βC ds 00gFW) D.Cayenesis 0.18

c 0.01

d 0.34

b 1.37

b 2.83

b 1.93

a 11.99

b 77.5 0.22

b 0.41

b

D. dumetorum 0.18 0.07 0.00 0.02 0.59 0.00 3.79 10 0.52 0.91

D.bulbifera 2.07a 0.03

b 0.02 0.20

c 0.27 1.14

b 10.11 15.8 2.29

b 0.41

b

X. Maffa 1.67 0.01 0.91 3.36 3.88 1.13 14.87 109.7 15.84 15.7 (Scoth) 4

a

Mean 1.03 0.03 0.32 1.24 1.89 1.05 10.19 53.25 4.21 4.36

LSD 0.05 0.27 0.01 0.25 0.29 0.67 0.32 2.71 _ 0.61 0.74

Values are means of Duplicate determinations. Mean values having the same letters within a column are not significantly different (P

hereby highlighted. The profiles of the yams and cocoyam tuber samples monitored in three wavelengths is presented in Figure 2A, B, C and Fig. 3. The chromatographic profiles of figures 2A (D. cayenensis), 2B (D. dumetorum) and 2C (D. bulbifera) may not have been well resolved. Champagne et al. (2010) observed similar trend on fresh yam and cocoyam tuber species from Vanuatu. Figure 3(X. maffa (Scoth)) showed better resolution and was the chromatographic profiles chosen to interpret the carotenoids of each yam species monitored in three wavelengths. Each species of the yam and cocoyam possessed notable differing numbers of carotenoids peaks, and retention time in line with the chromatographic standards (Figures 2 and 3). D. cayenensis possessed five identified carotenoids peaks, lutein (peak 1,), 13-cis-β-carotene (peak 4,), α-carotene (peak 5), all-trans-β-carotene (peak 6)

Am. J. Food. Nutr, 2014, 4(1): 1-10 and 9-cis-β-carotene (peak 7). Two major carotenoids, lutein (peak 1) and all-trans-β-carotene (peak 6) were identified in D.dumetorum. Lutein

(peak 1) and 9-cis-β-carotene (peak 7) were major carotenoids identified in D. bulbifera. In X. maffa (Scoth), lutein (peak 1), 13-cis-β-carotene (peak 4),α-carotene (peak 5), all-trans-β-carotene (peak 6), and 9-cis-β-carotene (peak 7) were the major carotenoids profiled. Several unknown peaks were observed in the yam and cocoyam tuber species. Recent study on the chromatographic profiles of yams and cocoyam attributed many unidentified carotenoid peaks to be xanthophylls (Champagne et al., 2010). It is believed

that yams possess low carotenoid content, but report on the carotenoids of yams/cocoyam (Ferede et al., 2010; Champagne et al., 2010) and the present study

show that yams/cocoyam have appreciable amount of carotenoids to benefit human nutrition and health.

6 6

A B

5 7

1 4

1

1

C

7

Fig 2: Chromatograms and peaks of yam Dioscorea spp. A (D.cayensis), B ( D.dumetorum), and

C (D.bulbifera) in three wavelengths (286nm (blue), 348nm (green) and 450nm (red))

Am. J. Food. Nutr, 2014, 4(1): 1-10

α-carotene: 5

Phytoene: 2 All-trans β-carotene: 6

Phytofluene: 3

13 cis-β-carotene: 4

Lutein: 1

9 cis-β-carotene: 7

Fig. 3; Chromatograms and peaks of (X.maffa (Scoth)) in three wavelengths (286nm (blue), 348nm (green) and

450nm (red)), typical of the carotenoid species identified in the yams and cocoyam tuber extracts. Conclusion The data generated in some of the yam and cocoyam tuber varieties have shown evidence of micronutrient staple root crops. Provitamin A and antioxidant carotenoids as characterized in this study can play important physiological role in human health and immune function. However, many of these traditional tuber crops are underutilized despite their potential as sources of micronutrients in addition to their major roles of providing energy in the diets. There is an urgent need to further investigate the carotenoids and micronutrient contents of many traditional root and tuber crops with modern methods in order to harness their potentials for the supply of micronutrients whose adverse deficiency affect human health especially in the tropical regions. It was observed that the differing visual yellow color shade in each Dioscorea and Xanthosoma species correlated with their carotenoids composition and concentration. This raises the hope (from the data generated) that D. cayenensis and X.maffa (Scoth) can provide some amount of provitamin A carotenoids (α and β- carotene) which in supplementation with other β-carotene rich food sources can meet the Recommended Daily Intake of vitamin A (retinol) among the groups of yams and cocoyam consumers Therefore, considering the spread and consumption of these tuber crops in West Africa and other Tropical regions of the world, biofortification strategy can be employed to further enhance the concentration of provitamin A and other micronutrients to combat hidden hunger and other related health challenges.

ACKNOWLEDGEMENT This work was financially supported by Tertiary Education Fund, Nigeria, and Abia State University, office of the Vice Chancellor for Research and Development, Nigeria. We appreciate the International Centre for Tropical Agriculture (CIAT), Cali, Colombia for providing the equipment and facilities used for the research work and Godwin Kalu Nwankwo for supplying the yam and cocoyam tubers. REFERENCES Aguaya, V.M., Kaln, S., Ismael, C and Meershock, S

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