Feedstuffs potential of harvest by-products from two oleaginous cucurbits

Among the generated crop residues and by-products in tropical agriculture, those of cucurbits represent great opportunities for animal nutrition. Nutritive profile of harvest by-products (dried leaves, fermented fruits, non fermented fruits and seeds shells) of two oleaginous cucurbits (Citrullus lanatus and Lagenaria siceraria) were investigated in order to explore their potential use as feedstuffs. The moisture, ash, and crude fibres contents were 4.81 to 12.87, 9.93 to 18.29, and 2.18 to 16.35%, respectively. Shells of L. siceraria seeds yielded the highest carbohydrate content (84.80 ± 2.78 %) while the highest calorific value (380.92 ± 11.40 kcal/100 g) was obtained in C. lanatus bebu. The contents of threonine (Thr), lysine (Lys) and methionine (Met) in dried leaves of C. lanatus bebu were 4.16, 6.86 and 6.89 g/100 g proteins, respectively. The content of methionine (Met) was 5.81 g/100 g proteins in fermented fruits of C. lanatus (wlêwlê). The harvest by-products analyzed in this study contained remarkably high amounts of potassium (671.78 – 4738.79 mg/100 g) and calcium (342.08 – 2963.95 mg/100 g) with highest value (4738.79 ± 230.10; 2963.95 ± 135.74 mg/100 g) for non-fermented fruits of L. siceraria and dried leaves of C. lanatus wlêwlê, respectively. The analyzed plants parts were also notable sources of magnesium, ranging from 221.45 ± 1.96 mg/100 g (non-fermented fruits of L. siceraria) to 872.10 ± 48.49 mg/100 g (dried leaves of C. lanatus wlêwlê). All these results suggest that the studied by-products could be used as valuable feedstuffs.


INTRODUCTION
Shortages of feed resources often impose major constraints on the development of animal production in tropical zones (Aregheore, 2000).In most developing countries, consi-derable quantities of by-products and crop residues generated every year and tending to accumulate can be utilized as feedstuffs (Ensminger et al., 1990).A by-product *Corresponding author.E-mail :banhiakalou@yahoo.fr .Tel: +225 07 39 02 31.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License feedstuff is a product that has value as an animal feed and is obtained during the harvesting or processing of a commodity in which human food or fibre is derived (Fadel, 1999).
Among the generated crop residues and by-products in tropical agriculture, those of cucurbits have a great economical importance (Dupriez and De Leener, 1987;Pitrat et al., 1999).Cucurbits belonging to the family Cucurbitaceae have common features: large leaves, creeping or climbing stems with simple or branched tendrils, and fleshy fruits containing numerous seeds (Ajuru and Okoli, 2013).Considering their consumption mode, two major groups are distinguished: those with edible flesh (watermelon, melon, squash, etc.) and those with oleaginous seeds (bitter cucumber, African melon, pumpkin, etc.) which are consumed as soup thickeners or cakes (Zoro Bi et al., 2003).Cucurbits cultivated for seed consumption are well adapted to extremely divergent agro-ecosystems and various cropping systems characterized by minimal inputs (Yaniv et al., 1999;Besognin, 2002).
Citrullus lanatus (Thunberg) Matsumura & Nakai, and Lagenaria siceraria (Molina) Standley are among the most widely cultivated oilseed cucurbits in tropical zones (Levi et al., 2001(Levi et al., , 2009;;Zoro Bi et al., 2006;Dane and Liu, 2007;Sarao et al., 2014).C. lanatus is a monoecious species, yellow flowered, and creeping annual vine, presenting leaves deeply divided into 5-7 more or less subdivided lobes.The fruits are round or oval, uniformly light green or mottled light and dark green and contain a white bitter flesh embedding about 200 seeds.Based on the morphology of seeds, two distinct cultigroups have been described (Zoro Bi et al., 2006).The first one, "wlêwlê", is characterized by glossy seeds with a tapered proximal extremity.Within the second cultigroup, "bebu", seeds are heavier and have a flat ovoid shape with rugged and thick ends (Zoro Bi et al., 2003).L. siceraria is also a monoecious species but white-flowered.Fruit and seeds shape and size are reported to be highly variable in the genus Lagenaria (Besognin, 2002).There are two varieties in L. siceraria, which are distinguishable by the hardness of their fruit exocarp and seed tegument.The variety with hard-shelled fruit and inedible seed is mainly cultivated for its dry fruit that is used mostly as containers, musical instruments, fishing floats, etc.The immature and tender fruits of some forms of this variety are also eaten as vegetable in some communities.The variety with soft-shelled fruit produces oleaginous edible seeds.The seeds of this variety are extracted after a 7day fermentation period of cut fruit containing a juicy pulp to which they are attached (Zoro Bi et al., 2003).This process is expected to facilitate the seed separation from the surrounding tissues, and increase their nutritive value and germination percentage (Achinewhu and Ryley, 1986).
Many studies on physicochemical and nutritive characteristics of cucurbit seeds have revealed that they are rich in minerals (calcium, potassium, phosphorus, iron, magnesium and zinc), proteins (36%) and fats (50%) (Achu et al., 2005;Loukou et al., 2011).In addition, oils extracted from cucurbit seeds contain relatively high content of essential fatty acid, particularly linoleic acid and this property made them recommended for healthy human nutrition (Achu et al., 2005;Anhwange et al., 2010).
Data from agro-ecological, cultural, genetic, and nutriational investigations (Pitrat et al., 1999;Yaniv et al., 1999;Besognin, 2002;Zoro Bi et al., 2006;Dane and Liu, 2007;Fuller et al., 2010;Loukou et al., 2011;Sarao et al., 2014) suggest that the oleaginous forms of C. lanatus and L. siceraria have a potential to play crussial roles in the improvement of food security in tropics, particularly though helping the poor for subsistence and income, reducing risks of over-reliance on major crops, increasing agriculture sustainability by mean of reduction in inputs, and contributing to food quality (Frison et al., 2011;Mayes et al., 2012).Despite these potentialities, these cucurbits are somehow neglected and classified as minor crops.Furthermore, there is a lack of scientific data concerning nutritive profile of some harvest by-products, derived from cucurbits, such as dried leaves, dried fruits, dried fermented fruits and seed shells.The study was undertaken to promote the harvest by-products from the oleaginous forms of C. lanatus and L. siceraria by exploring their nutritive profile for their potential use as feedstuffs.

Processing methods
Fruits and leaves of the studied cucurbits were processed as depicted in Figure 1.The leaves were sun-dried on clean papers for 10 days with constant turning over to avert fungal growth.The fruits containing seeds were entirely cut up using a stainless steel laboratory knife.The pieces obtained were divided into two lots.The first lot was packaged in sterile airtight polythene bags and allowed to ferment for 7 days in the darkness.Afterwards, the fermented pieces of fruits were oven-dried (Memmert, Germany) at 50°C for 7 days.The seeds of fermented fruits were removed, sundried for 3 days, sorted to remove bad ones and manually shelled.The second lot was immediately oven-dried at 50°C for 7 days.
The dried materials (leaves, fermented fruits, non-fermented fruits and shells) obtained were ground with a laboratory crusher (Culatti, France) equipped with a 10 µm mesh sieve.The powdered samples obtained were stored in airtight polythene bags at 4 °C until further analyses.Three replicates were performed for each selected by-product.

Proximate analysis
Moisture, ash, proteins and lipids were determined using AOAC (1990) official methods.For crude fibres, 2 g of dried powdered sample were digested with 0.25 M sulphuric acid and 0.3 M sodium hydroxide solution.The insoluble residue obtained was washed with hot water and dried in an oven (Memmert, Germany) at 100°C until constant weight.The dried residue was then incinerated, and weighed for the determination of crude fibres content.Carbohydrates and calorific value were calculated using the following formulas (WHO/FAO, 1985): Carbohydrates = 100 -(% moisture + % proteins + % lipids + % ash + % fibres) Calorific value = (% proteins x 4) + (% carbohydrates x 4) + (% lipids x 9) Polyphenols content was determined using the method reported by Singleton et al. (1999).A dried powdered sample (1 g) was soaked in 10 mL of methanol 70% (w/v) and centrifuged at 1000 rpm for 10 min.An aliquot (1 mL) of supernatant was oxidized with 1 mL of Folin-Ciocalteu's reagent and neutralized by 1 mL of 20% (w/v) sodium carbonate.The reaction mixture was incubated for 30 min at ambient temperature and absorbance was measured at 745 nm using a spectrophotometer (PG Instruments, England).The polyphenols content was obtained using a calibration curve of gallic acid (1 mg/mL) as standard.

Amino-acids analysis
Free amino acids were extracted from 5 g dry matter with 20 mL sodium citrate 0.2 N, pH 2.3 and protein hydrolysis was performed with hydrochloric acid 6 N at 110°C for 24 h.After evaporation, the aliquots were re-suspended in sodium borate buffer 0.4 N, pH 9.5.The homogenized mixture was filtered through a 0.45 mm Millipore membrane (Sartorius AG, Goettingen, UK).The extracts obtained were stored at -20°C until HPLC analysis.An aliquot of 20 µL of filtered sample was injected for HPLC analysis after derivatisation of the amino acids on a pre-column with orthophtaldialdehyde.The HPLC equipment was composed of a pump (Shimadzu LC-6A Liquid Chromatograph), an UV detector (Shimadzu SPD-6A UV spectrophotometry detector) and an integrator (Shimadzu CR 6A Chromatopac).Chromatographic separation of amino acids was performed with an ion exclusion column Hypersil C8 (25 cm x 4.6 mm x 5 µm, Interchom, France) maintained at 35°C.The mobile phase was a buffer solution of phosphate/methanol (88:12, v/v) with a flow rate of 0.6 mL/min and the detector was set at 280 nm.The standards were filtered and injected separately and each analysis was performed in triplicate.Amino acids were identified and quantified by comparison of their retention time and peak area with those of standard solutions.

Mineral analysis
The mineral content was estimated by dry ashing of dried powdered sample (5 g) in a muffle furnace (Pyrolabo, France).The ash obtained was dissolved in 5 mL of HCl/HNO3 and analyzed using the atomic absorption spectrophotometer (AAS model, SP9).

Statistical analysis
All the analyses were performed in triplicate and data were analyzed using SPSS 11.5.Values were expressed as mean ± standard deviation (SD).Treatments (species and processing methods) were compared using two-way analysis of variance (ANOVA2).Differences between means were evaluated by Duncan's test.Statistical significant difference was stated at p < 0.05.

Physicochemical properties
The proximate composition of harvest by-products (leaves, fruits and shells) from C. lanatus and L. siceraria is presented in

Amino acid composition
The amino acids contents of leaves, fruits, and shells from C. lanatus and L. siceraria are presented in Table 2. Seven major amino acids, namely threonine (Thr), proline (Pro), serine (Ser), Tyrosine (Tyr), methionine (Met), lysine (Lys), and cystine (Cyst) were determined.The contents of these amino acids differed significantly (p < 0.05) and ranged from 0.045 to 6.89 g/100 g proteins.The contents of Thr, Lys, and Met in dried leaves of C. lanatus bebu were 4.16, 6.86, and 6.89 g/100 g proteins, respectively.The content of Met was 5.81 g/100 g proteins in fermented fruits of C. lanatus (wlêwlê).

Mineral composition
Mean values for mineral content of analyzed samples are presented in

DISCUSSION
The relatively lowest values of moisture obtained in this study indicated that the analyzed plant parts were in storage condition avoiding microbial spoilage (Fennema and Tannenbaum, 1996).The contents of all samples in ash suggested that they might be good sources of minerals for feedstuffs (NRC, 1994).The relatively high contents in fibres observed referred to structural carbohydrates that are composed of cellulose, hemicelluloses (pentosans, hexosans) and indigestible materials as lignin ( ottg ter, ).With respect to their contents in crude fibres, dried leaves, fermented and non-fermented fruits of the selected cucurbits could be used as feeds for ruminants which need fibres in their daily ration for stable and healthy digestion ( ottg ter, ).The dietary carbohydrate contents of seed shells of C. lanatus bebu and L. siceraria were in the range of that of cereals (80-90%), which constitute important sources of energy for poultry (Moran Jr, 1985).However, it is worth noting that the energy content of by-product feedstuffs is usually determined by the level of residual fat and percentage of fibres in the meal (Aherne and Kennelly, 1982;Schingoethe, 1991).
Polyphenols are the main dietary antioxidants and possess higher in vitro antioxidant capacity than vitamins and carotenoids (Gardner et al., 2000).Plant phenolics include phenolic acids, coumarins, flavonoids, stilbenes, hydrolysable and condensed tannins, lignans, and lignins (Naczk and Shahidi, 2004).Tannins present in many feedstuffs do not only affect the feed quality but can also be toxic to animals because they are often considered as anti-nutritional compounds (Lowry et al., 1996).With regards to their low contents in polyphenols, seed shells of C. lanatus and L. siceraria could be more exploited as feedstuffs for monogastrics.
Dietary requirements for protein in feedstuffs are linked to the amino acids contents.Indeed, amino acids obtained from dietary protein are used by animals to fulfill diverse functions, as primary constituents of structural and protective tissues and precursors of many important non-protein body constituents (NRC, 2001).Threonine (Thr), methionine (Met), and lysine (Lys) are essential amino acids which cannot be synthesized by animals and need to be supplied by feedstuffs (Mendonca and Jensen, 1989).The content of dried leaves of C. lanatus bebu in these essential amino acids was higher than those of standard protein (WHO/FAO, 1985): 2.80, 2.20, and 4.20 g/100 g protein, respectively.Therefore, the consumption of dried leaves of C. lanatus bebu by animals could increase their adequate growth and productivity (NRC, 1994).
The macro mineral (Ca, P, Mg, K) concentrations of the analyzed by-products were lower than the maximum tolerable levels (10-40 g/kg) for poultry (McDowell, 1992).In addition, the contents of these macro minerals could meet the requirements (200-750 mg/100g) of poly-and monogastric animals such as beef cattles, sheep and pigs (NRC, 2001).The copper (Cu) content of the shells from seeds of C. lanatus and L. siceraria was lower than the maximum tolerable level (25-30 mg/100 g) for pigs and poultry (McDowell, 1992).These plants could therefore be exploited as non-toxic feedstuffs for these monogastric animals.Indeed, copper is known to affect entero-hepatic function in small ruminants, especially sheep and goats by decreasing the ability of liver to metabolize this trace mineral (Church and Pond, 1988).

Conclusion
The data obtained in this study showed that the harvest by-products from C. lanatus and L. siceraria contained appreciable amount of ash, fibres, carbohydrate, essential amino acids and mineral elements.Consequently, the plant parts analyzed could satisfy the nutrient requirements of animals and should be used as valuable feedstuffs.However, it is necessary to consider other aspects such as the in vivo bioavailability of these nutrients after various feed formulations.

Figure 1 .
Figure 1.Processing diagram of fruits and leaves from C. lanatus and L. siceraria.

Figure 1 .
Figure 1.Processing diagram of fruits and leaves from C. lanatus and L. siceraria.