Effect of processing methods on the nutritional values and anti-nutritive factors of Adenanthera pavonina L. (Fabaceae) seeds

This research aimed at determining the effect of processing on the nutritional and anti -nutritional values of “food tree” [Adenanthera pavonina L. (Fabaceae)] seeds, a highly nutritional and underutilized legume. The seeds were separated into three groups namely, boiled, roasted and raw. Quantitative analysis was carried out to measure their proximate, minerals, vitamins and anti -nutrients compositions. Analysis of variance was used to analyze the treatment groups and Duncan’s multiple range tests to determine significant difference at P  0.05. The results show that processing significantly affected the nutritional and anti-nutritional constituents. The values for proximate composition in the raw, roasted and boiled seeds were: proteins (15.79±0.04, 18.86±0.02 and 23.25±0.02, respectively), carbohydrates (56.60±0.02, 54.89±0.02 and 52.05±0.02 respectively), fat (9.78±0.02, 11.70±0.02 and 11.40±0.02, respectively), crude fibre (9.80±0.04, 9.70±0.04 and 5.85±0.02, respectively), moisture (3.88±0.03, 0.10±0.02 and 3.20±0.02, respectively) and ash (4.03±0.01, 4.75±0.02 and 4.25±0.01, respectively). The values for mineral compositions in the raw, roasted and boiled se eds were: calcium (25.61±0.34, 30.34±0.02 and 80.88±0.02, respectively), magnesium (18.97±0.01, 22.76±0.01 and 60.68±0.02, respectively), phosphorus (7.00±0.06, 6.40±0.15 and 5.80±0.10, respectively), potassium (3.31±0.00, 2.43±0.02 and 4.23±0.02, respectively) and iron (0.41±0.02, 0.41±0.01 and 1.23±0.01, respectively). The values for vitamin compositions in the raw, roasted and boiled seeds were: β carotene (1458.33±0.01, 416.67±1.20 and 416.67±0.08, respectively) and vitamin E (22.50±0.02, 9.24±0.02 and 12.69±0.01, respectively). The mean values for anti-nutrient compositions in the raw, roasted and boiled seeds were: tannin (1.21±0.00, 0.049±0.00 and 0.15±0.00, respectively), phytate (5.16±0.02, 3.50±0.01 and 1.50±0.02, respectively), oxalate (0.34±0.00, 0.13±0.00 and 0.11±0.00, respectively), cyanide (1.17±0.00, 0.95±0.00 and 0.32±0.00, respectively) and trypsin inhibitor (0.92±0.01, 0.36±0.01 and 0.90±0.01, respectively). The results show that processing changed the nutritional constituents and reduced the anti-nutrients in the seeds of A. pavonina and boiling proven to be the best processing method.


INTRODUCTION
Food legumes constitute a major source of nutrients such as proteins, lipids, carbohydrates, and other important substances such as fibre, minerals and vitamins (Deshpande, 1992) which are necessary for human and animal health.Similarly, they contain anti-nutritional components such as saponins, tannins, phytates, lectin/haemagglutanin, oxalates, polyphenol, among others, which hinder the body from digesting the nutrients in pulses.These toxins cause food poisoning to human beings and animals (Osifo, 1974).According to Olusanya (2008) and Geil and Anderson (1994), legumes contain some toxic components such as anti-trypsin factors which impair the digestion of proteins and hence prevent its efficient utilization.Phytates, oxalates and cyanides cause various physiological disorders like increase in relative weight of pancreas and liver, and also diarrhea (Arija et al., 2006).Fortunately, many of these toxic components are destroyed by heat provided by different food processing methods (Olusanya, 2008).
Boiling and roasting are important household food processing methods.Boiling is a method of cooking food in water such that it bubbles vigorously, while roasting is achieved in an uncovered pan without water to produce a well-browned exterior and a moister cooked interior.These processing treatments increase the nutritional quality of food plants and are also effective in eliminating the anti-nutritional factors in them and thus the need for their proper processing to levels where they are safe for human and animal consumption (Hotz and Gibson, 2007;Nzewi and Egbuonu, 2011).
Adenanthera Pavonina L. (Fabaceae) is a woody Southeast Asian species of legume mostly known for its edible seeds (Arzumand et al., 2010).It is endemic to India and Southeast China, where it is considered as an alternative nutrients source for animals and humans, but has been introduced into tropical and sub-tropical areas of the world including Malaysia, Polynesia and eastern and West Africa.The plant is known as "food tree" because its seeds and leaves are valued for food, and the seeds, which when roasted are said to taste like soy bean, possess high percentage of proteins, fatty acids, minerals and other nourishing properties (Olajide et al., 2004;Senga et al., 2013).
It has been used in traditional medicine practices to treat many diseases such as asthma, boil, diarrhoea, gout, inflammations, rheumatism, tumor and ulcers, and as a tonic (Ghani, 2003;Arzumand et al., 2010).Several parts of the plant have been verified for its medicinal importance hence, the bark and leaves are used in the treatment of gonorrhea, ulcers and rheumatism.The powdered seeds are applied as a poultice to abscess and to promote suppuration (Hussain et al., 2010;Sujit et al., 2010).Physicochemical characterization of the seed oil showed appreciable amounts of neutral lipids and unsaturated fatty acids including linoleic, oleic and lignocerotic acids (Robert et al., 2004).However, report on its antinutritive constituents is totally lacking.
In Nigeria and other parts of West African where it has been introduced, the tree has been economically utilized as source of timber and wood fuel.However, there is no report on utilizing any part of it, including the seeds as food.Therefore, the present research was to analyze the nutritional potentials of the seeds in combating malnutrition and food insecurity.

Source of plant m aterial
Dried pods of A. pavonina L. w ere obtained from the Botanic Garden, Department of Plant Science and Biotechnology, University of Nigeria, Nsukka, Enugu State in May, 2016.The seeds w ere separated from the dried pods, cleaned and freed from foreign matters and air dried.The dried seeds w ere stored in airtight bottles for further studies.

Preparation of sam ples
The preparation of the various sample groups w as done follow ing the methods of Ajeigbe et al. (2012).

Boiling
Whole seeds (100 g) w ere w eighed using digital w eighing balance and soaked in distilled w ater for overnight.The soaked seeds w ere boiled for 1 h and then rinsed w ith distilled w ater.Further boiling w as done for another 2 h before the seeds w ere dried using Gallenkamp hot air oven at 40°C for 15 min.

Roasting
With the aid of a digital w eighing balance, 100 g w hole seeds w ere w eighed and roasted in Gallenkamp hot air oven at 120°C for 1 h.The roasted seeds w ere allow ed to cool.

Raw sample
Raw processing w as done by drying 100 g w hole seeds in hot air oven at 40°C.The prepared samples w ere separately ground w ith Thomas hammer mill blender to obtain pow dered particle size of 1 mm.The pow dered samples w ere stored in air-tight bottles at room temperature for further analysis.

Determination of proximate composition of seed samples
The proximate composition of the samples w as done follow ing the standard methods as recommended by Pearson (1976) and the Association of Official Analytical Chemists (AOAC, 1990).*Corresponding author.E-mail: felix.nwafor@unn.edu.ng.Tel: +2348036062242.
Author(s) agree that this article remains permanently open access under the terms of the Creativ e Commons Attribution License 4.0 International License

Crude protein
The crude protein content of foods or plant sample was determined by using the Micro Kjedahl Nitrogen Method (Pearson, 1976).The method involves digestion of samples, distillation of digests and titration of distillate.

Crude fat (using Soxhlet apparatus)
Tw o grammes aliquot of the processed sample w as w eighed into a 250 ml clean flask and put into the thimble.The boiling flask w as filled w ith 250 ml n-hexane.The Soxhlet apparatus w as set and refluxed for about 3 h.The thimble w as removed w ith care and the hexane w as collected in the top container of the set up and drained into a container for re-use.When the flask w as almost free of hexane, it w as removed and dried at 105°C to a constant w eight.It w as transferred from the oven into a desiccator and allow ed to cool, and then w eighed.

Fibre
Tw o grammes aliquot of the sample w as w eighed and 150 ml of heated H2SO4 w as added and heated to boiling for 30 min and filtered.The residue w as w ashed three times w ith hot w ater.Preheated KOH (150 ml) w as added and the residue w as heated to boiling.Few drops of anti-foaming agent w ere added and boiled slow ly for 30 min.The residue w as filtered and w ashed three times w ith hot w ater, then w ashed w ith acetone, dried at 130°C for 1 h and w eighed.

M oisture (using oven method)
A crucible w as thoroughly w ashed and dried in the oven, then cooled in a dessicator and w eighed.Tw o grammes of the sample w as w eighed into the crucible.The crucible and the content w as transferred into a hot air oven and dried at 105°C to a constant w eight.The sample w as then cooled in dessicator and the w eight of the crucible and the content w as taken, recorded and calculated.

Ash (using muffle furnace)
Tw o grammes aliquot of the sample w as put into a w eighed crucible and pre-ashed to drive off most of the smoke.The pre-ashed sample w as transferred into a furnace at 550°C and allow ed to ash until w hite ash w as obtained.The desiccator w as cooled and rew eighed.Ash content w as calculated.

Carbohydrate
Carbohydrate content w as determined by the difference in the percentage composition of protein, crude fat, ash, moisture and crude fibre (AOAC, 1990).

Calcium
Calcium w as determined using Pearson (1976) method.25 ml of the sample w as pipetted into a conical flask, a pinch of EBT w as added, 2 ml of the NaOH solution w as also added and the mixture w as titrated w ith standard EDTA solution.
Where, T = Titre value; M= molarity of EDTA; E = equivalent w eight of calcium.

M agnesium
Magnesium w as determined using Pearson (1976) method.Aqueous extract of the sample (25 ml) w as pipetted into a conical flask and a pinch of EBT w as added and then shaken.This w as follow ed by the addition of 2 ml buffer.The mixture w as then titrated using 0.01 M EDTA.
Where T = Titre value; M= molarity of the standardized EDTA; E = equivalent w eight of magnesium.

Phosphorus
Phosphorus w as determined using Pearson (1976) method.Aqueous extract of the sample (5 ml) w as pipetted into a test tube and 5 ml of the molybdate solution w as added and the absorbance read at 420 nm.The concentration w as calculated using the standard curve.

Potassium (using flame photometer)
Potassium w as determined using Pearson (1976) method.The instrument w as sw itched on and allow ed for about 20 min to stabilize.The gas w as then turned on, distilled w ater w as aspirated through the siphon in order to zero the instrument and the samples w ere aspirated and the emission recorded.The concentrations w ere calculated using sodium and potassium calibration curve for sodium and potassium readings, respectively.

Iron
Iron w as determined using Pearson (1976) method.The sample (10 ml) w as added into a 100 ml flask and made up to 50 ml w ith deionized w ater.Concentrated HCI (20 ml) w as added follow ed by the addition of 1 ml of hydroxylamine solution.About 0.5 g glass beads w as added and heated to boiling point till the volume reduced to 2 ml.
Ammonium acetate buffer solution (10 ml) and 2 ml of phenanthroline w ere added and the content made up to 100 ml mark w ith de ionized w ater.

Vitamin A (β-carotene)
One gram of the sample w as w eighed.Then, the proteins w ere first precipitated w ith 3 ml of absolute ethanol before the extraction of vitamin A w ith 5 ml of heptane.The test tube containing this w as shaken vigorously for 5 min.On standing, 3 ml from the heptane layer w as taken up in a curvette and read at 450 nm against a blank of heptane.The standard w as prepared and read at 450 nm w avelength using UV/Vis spectrophotometer (Model: CE 2041), and vitamin A calculated from the standard (Pearson, 1976).

Vitamin E
One gram of each sample w as macerated w ith 20 ml of petroleum ether for 10 min.The macerated samples w ere allow ed to stand for 1 h w ith intermittent shaking at every 10 min and thereafter, centrifuged for 5 min.Three millilitres of supernatant w as transferred into triplicate test tubes, evaporated to dryness and then re-dissolved w ith 2 ml of ethanol and shaken.One millilitres of 0.2% ferric chloride in ethanol, 1 ml of 0.5% and dipyridyl in ethanol and 1 ml of ethanol w ere added and the resultant solution w as made up to 5 ml.The solution w as mixed thoroughly by shaking and absorbance w as taken at w avelength of 520 nm using UV/Vis spectrophotometer (Model: CE 2041), against the corresponding blank.

Tannins
This w as determined as described by Pearson (1976).Distilled w ater (10 ml) w as added to 1 g of the test sample and shaken at 5 min interval for 30 min.The solution w as centrifuged to get the extract.Tw o and half millilitre of the supernatant w as transferred into a test tube and 2.5 ml of standard tannic acid solution w as also transferred into a 50 ml flask.One millilitre Folin-Denis reagent w as added into the flask, follow ed by 2.5 ml of saturated Na2CO3 solution and the solution w as made up to the mark.Absorbance w as read after 90 min incubation at room temperature using UV/Vis spectrophotometer (Model: CE 2041).

Phytate
The sample (0.5 g) w as extracted w ith 100 ml of 2.4% HCl for 1 h at room temperature.The extract (5 ml) w as pipetted into a test tube and diluted w ith 25 ml of distilled w ater.0.7 M sodium chloride (15 ml) w as added and the absorbance w as read at 520 nm using UV/Vis spectrophotometer (Model: CE 2041).The value w as calculated from a prepared standard curve and blank (Pearson, 1976).

Oxalate
One gram of the pow dered sample w as w eighed and put into a test tube and 47.5 ml of w ater and 2.5 ml of 6 N hydrogen chloride w ere added to the pow dered sample.It w as boiled for 1 h and made up to 62.5 ml w ith w ater.The solution w as cooled at room temperature and filtered.Some filtrate (12.5 ml) w as taken and the pH w as adjusted to the range of 4.0 to 4.5 w ith dilute ammonia (NH3).The solution w as heated up to 90°C, filtered and heated up again to 90°C.Then, 5 ml of calcium chloride w as added to the solution w ith constant stirring.The solution w as allow ed to stand overnight.The solution w as centrifuged for 5 min and the supernatants w ere decanted off.The precipitate w as dissolved w ith 5 ml of 20% sulphuric acid.It w as heated until about to boil.The solution w as then titrated w ith 0.5 N standard KMNO4 until a pale pink colour that persisted for 30 s w as attained and the percentage oxalate w as calculated (Pearson, 1976).

Cyanide
Five grams of the sample w as prepared into a paste and the paste w as dissolved in 50 ml of distilled w ater and allow ed for the cyanide extraction to stay overnight, then filtered and the filtrate w as used for the cyanide determination.To 1 ml of the sample filtrate in a test tube, 4 ml alkaline picrated w as added and allow ed to stand for 5 min.The absorbance w as read at 490 nm after colour development (redish brow n colour).The absorbances of the blank and standard w ere also read and the cyanide content of the test sample w as extrapolated from cyanide standard (Pearson, 1976).

Protease inhibitor
Tw o grammes of the finely ground sample w as extracted w ith 10 ml of 0.01 N NaOH for 1 h. 5 ml of benzoyl-DL arginine-p-nitro anilide hydrochloride (BAPNA) solution w as hydrolyzed w ith 2 ml of 0.2 mg/ml trypsin (Sigma Type 11) in 0.0001 M HCl.P-nitro anilide w as released as a coloured product and absorbance w as read at 410 nm (Pearson, 1976).

Data analysis
The data obtained for the nutritive (proximate, mineral and vitamins) and anti-nutritive composition w ere statistically analyzed using one w ay analysis of variance (ANOVA) and reported as mean ± standard error of triplicate data.Duncan's multiple range test w as used for mean separation.

RESULTS
Proximate compositions of the raw and processed (roasted and boiled) seeds of A. pavonina are presented in Table 1.The processed seeds had the highest values of protein, crude fat and ash while the raw seeds had more carbohydrate, crude fibre and moisture.The proximate composition also varied significantly (P ˂ 0.05) between the boiled and roasted seeds.Analysis of variance (ANOVA) showed that there is significant difference (P < 0.05) in the mineral composition of the processed (roasted and boiled) seeds of A. pavonina when compared with the raw seeds.Processing methods affected the composition of mineral  nutrients in the seeds (Table 2).Processing significantly increased the percentage compositions of calcium, magnesium, iron and potassium.Analysis of variance (ANOVA) showed significant difference (P<0.05) in the vitamin composition of the processed (roasted and boiled) seeds of A. pavonina when compared with the raw (control) seeds.Processing methods affected the composition of vitamin nutrients in the seeds.Processing significantly reduced the vitamins A and E constituents (Table 3).
There was significant difference (P<0.05) in the antinutrients composition of the processed (roastesd and boiled) seeds when compared with the raw seeds.Processing methods affected the composition of antinutrients in the seeds.The anti-nutrients were generally reduced in the processed seeds and the boiling gave the most significant effect (Table 4).

DISCUSSION
The results from the nutritional analysis showed that the values for the major nutrients tested are within the reported values for other legumes (Aremu et al., 2006).
Protein composition of processed A. pavonina seeds is comparable to that found in the seeds of soybean, Canavalia ensiformis and cowpea (El-Adaway and Taha, 2001), and much higher than that of bambara groundnut (Akaninwor and Ogechukwu, 2004).Carbohydrate level is favorably compared with the acceptable range mean values for legumes (20 to 60%) (Aykroyed and Dought, 1964), and higher as compared to that of C. ensiformis, soybean and Mucuna utilis (Balogun and Olatidoye, 2012).The carbohydrate content gave an indication that the seeds of A. pavonina studied here can be considered as a rich source of energy and is able to supply the daily energy requirements of the body in children and adults (Aranda et al., 2001;Balogun and Olatidoye, 2012).The seeds of A. pavonina contained higher crude fats than most other legumes.Legumes generally have low fat content in the range of 1 to 2% with the exception of Cicer arietinum, Glycine max and pea nut (Costa et al., 2006).The same appreciable result was recorded for crude fibre, moisture content and ash.
Furthermore, processing methods were observed to significantly (P˂0.05)affect the nutrient composition when compared with the raw seeds.Boiled seeds had higher amount of protein than roasted seeds.In addition, regarding the recommended daily allowance for proteins for children, which ranges from 23.0 to 36.0 g, and for adult (44 to 56 g), it can be considered that the boiled seeds of A. pavonina can supplement the recommended daily intake of this nutrient, particularly for children.Therefore, this appreciable proteins content in the processed seeds suggests their usefulness as alternative source of protein nutrients.The fat composition obtained from the processed seeds of A. pavonina studied showed that processing significantly (P ˂ 0.05) affected the fat composition of the seeds.Roasted seeds had the highest amount of fat than the boiled seeds.
The crude fibre content of the roasted seeds was significantly (P ˂ 0.05) higher than the boiled seeds and the overall fibre content of A. pavonina was higher when compared with those of other legumes, for example, Dolichos tribalus, Vigna radiata and Vigna unguiculata (Aremu et al., 2006).Processing methods therefore, affected the crude fibre composition, with the roasted seeds having more amount than the boiled seeds, and this implied that more of the crude fibres were probably leached into water during boiling (Aremu et al., 2006).
The moisture composition of the roasted seeds of A. pavonina was significantly (P˂0.05)lower in comparison with boiled seeds.This was expected as seeds are subjected to higher temperatures during roasting.The result indicate that roasting may favor keeping quality and acceptability of A. pavonina seeds as texture, taste, appearance and stability of foods depends on the amount of water they contain (Isengard, 2001).
The ash composition of the processed seeds of A. pavonina was reflective of the high level of some mineral elements presented in Table 2.The roasted seeds had significantly (P ˂ 0.05) higher amount of ash than the boiled seeds.The low value of ash in the raw seeds may be as a result of the effects of anti-nutrients on the mineral contents of the food sample.The ash content reported here is higher as compared to the recommended values and suggested that these seeds are rich source of ash (Kala and Mohan, 2008).
Table 2 shows that processing significantly (P<0.05)increased some mineral contents of A. pavonina seeds with boiling having the most significant increased effect.This is probably because minerals are not destroyed by heat.The reduction in some cases may be as a result of leaching of minerals into the boiling water and through roasting process (Amarowicz et al., 2009).This study revealed that seeds of A. pavonina are rich in mineral elements including calcium, phosphorus, potassium, magnesium and iron.These minerals are necessary for cell formation, transmission of nerve impulse, fluid balance and bone formation (Ezeagu and Ologhobo, 1995).
In this study, vitamins A (β carotene) and E compositions of the seeds of A. pavonina were significantly (P<0.05)reduced by processing methods when compared with the raw seeds.This agrees with an earlier report that processing of legumes by heating lead to reduction of vitamin content (Asogwa and Onweluzo, 2010).This could be explained by the fact that vitamins are lost during processing because of their high sensitivity to oxidation, and leaching into water soluble media during storage (Davy et al., 2010).
The result of the anti-nutrient values of the processed seeds of A. pavonina showed that processing methods significantly (P<0.05)reduced the anti-nutrient composition of the seeds when compared with the raw seeds (Table 4).It has been reported that some antinutrients are heat labile and therefore will be reduced to a great extent by the application of heat to the food (Apata and Olegbobe, 1994), and this statement has been justified in this result as boiling most significantly reduced all the anti-nutrients to very low levels in seeds of A. pavonina.Roasted seeds showed higher increase in phytate content because of the increase in phosphorus concentration since phytate is the major store of phosphorus in mature seeds, while the boiled seeds showed reduced amount of phytic acid which is attributed to leaching in water.

Conclusion
Processing methods adopted in this study have been proven to have significant effects on the nutritional and anti-nutritional compositions of the seeds of A. pavonina, a highly nutritious and neglected legume, in agreement with earlier reports that thermal processes of legumes enhance tenderization of the cotyledons, thereby increasing palatability and nutritional value by inactivating endogenous toxic factors.In addition, it was observed that boiling gave higher significant effect than roasting and proved as more efficient method of processing the seeds.Furthermore, this study revealed that the seeds, when properly processed, have high nutritional values that can be exploited and considered as an alternative source of nutrients to reduce malnutrition among economically weaker categories of people in the developing countries.
ith different superscript alphabets in each column are significantly different from each other by DMRT (P < 0.05).
ith different superscript alphabets in each column are significantly different from each other by DMRT (P<0.05).

Table 1 .
Mean proximate constituents observed in raw and processed (roasted and boiled) seeds of A. pavonina.

Table 2 .
Mean constituents of the mineral nutrients observed in raw and processed (boiled and roasted) seeds of A. pavonina.

Table 3 .
Mean constituents of the vitamin nutrients observed in raw and processed (boiled and roasted) seeds of A. pavonina.

Table 4 .
Mean constituents of the anti-nutrients observed in raw and processed (roasted and boiled) seeds of A. pavonina. c1.17