Nutritive Value and Inherent Anti-Nutritive Factors in Processed Peanut (Arachis hypogaea)

Processing conditions and even the form of the food being processed as they affect nutrient availability is critical to develop structured foods to meet the nutritional needs of end users. The effect of heat treatments (roasting, boiling and autoclaving) on the physical, nutritional and functional properties of in-shell and shelled peanut (Arachis hypogaea) was determined. Unprocessed shelled peanut served as the control. Nutrient and anti-nutrient compositions of peanut samples were determined by standard methods, while physical (colour) and functional properties were also carried out. 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 result of proximate composition revealed that raw peanut had protein (32.7%), ash (1.37%), fibre (5.15%), fat (42.9%) and carbohydrate (12.1%). However, processing methods significantly increased the fat and ash contents. Peanut is high in calcium, magnesium and potassium but low in iron and zinc; processing significantly increased the elemental concentration of peanut. Phytate, tannin, oxalate, alkaloid, trypsin inhibitor and flavonoid were determined in the peanut samples and all were significantly affected by the processing method employed. However, boiling of shelled peanut was more effective in reducing the anti-nutrients than roasting and autoclaving. The aflatoxin concentration was in a range of 2.06-8.05ppb. Shelled peanuts subjected to the processing methods had lower aflatoxin levels compared with in-shell processed peanuts. There exist variation in bulk densities (packed and loosed), water and oil absorption capacities as a result of processing method employed. The colour of peanut was significantly affected by the processing method. In general, processing of shelled peanut resulted in flour with better nutritional quality and functionality than in-shell peanut. The findings showed that boiling has proved to be an efficient method in processing of peanut.


Introduction
Peanut (Arachis hypogaea), also known as groundnut is a legume crop from the family Leguminosae or Fabaceae, native to Mexico, South and Central America [1]. The list of the five largest producing countries is headed by China with a production of 33,309,998 tonnes, followed by India with 6,857,000 tonnes, Nigeria with 3,028,571 tonnes, the United States with 2,578,500 tonnes and Sudan with 1,826,000 tonnes [2]. The leading producing state in Nigeria is Niger state and peanut has contributed extremely to Nigeria economy through the sales of seeds, cakes, oil and haulms [3]. Peanuts can be boiled, roasted with or without addition of sugar and salt and may be processed into other forms such as peanut butter, groundnut soup, groundnut oil peanut flour, etc. Large number of people eat processed peanut while a few eat it raw. Peanut is an excellent source of protein and also has high fat content which contributes to their use as oil seeds. Peanut is however low in carbohydrate though high in protein, fat and fibre which contribute to their very low glycemic index hereby making it suitable for diabetic patients [4]. On a 100 -point scale, the glycemic index (GI) of peanuts is 14, and the glycemic load (GL) of peanuts is one. Moreso, research has shown that when peanuts are added to a high glycemic load meal, they actually keep the blood sugar stabilized so that it does not rise too high too quickly [5]. The vitamins and minerals found in peanuts include: folate, niacin, thiamine, riboflavin, choline, magnesium, potassium, zinc, iron and selenium [6].
Asides, peanut contain phytochemicals such as flavonoids as reported by APC [6]. Peanuts also contain anti nutrients such as phytic acid, trypsin inhibitor, tannin, saponin, etc. and all these inhibit the absorption of some nutrient in the diet. Beside the anti-nutritional factors, peanuts are particularly vulnerable to contamination by fungi Aspergillus flavus and Aspergillus parasiticus. These fungi produce aflatoxins that are known to cause cancer in humans, lower immune response and impair growth in children [7]. As a result of this, significant risk is attached to eating raw peanuts [8]. Major process steps include processing peanuts for in-shell consumption and shelling peanuts for other uses. Peanuts are usually consumed after roasting or boiling in the shell or without the shell and are available in Nigeria throughout the year.
The knowledge of how processing conditions and even the form of the food being processed as they affect nutrient availability are critical to develop structured foods to meet the nutritional needs of end users. No comprehensive study has been reported simultaneously to show nutritional qualities of shelled and in-shell peanut growing in African tropics as affected by processing conditions. The processing of peanut in or out of shell will allow the products to be distinguished and their constituents to be checked. This study is aimed at evaluating the effect of processing conditions (roasting, boiling and autoclaving) on physical, nutritional and functional properties of shelled and inshell peanuts.

Sample collection
In-shell peanuts were purchased from Bodija market, Ibadan, Oyo State, Nigeria and transported in polyethylene bags to the laboratory.

Treatment of samples
In-shell peanuts were sundried and processed as follows:

Raw peanut
In-shell peanuts (300g) were shelled and the nuts collected. The nuts were oven dried to constant weight and milled into flour. The peanut flour was then packed in plastic container and kept in freezer (-4°C) pending analysis. This served as the control sample.

Roasting of in-shell peanut
In-shell peanuts were roasted according to Ayoola and Adeyeye [9]. About 300 g of the dried pods were put into an iron pot and mixed with clean fine sand and stirred to prevent burning of the sample and to ensure uniform distribution of heat. The peanuts pods were roasted for 30 min at 120-130°C using Gallenkamp thermostat hot plate until a characteristic brownish nutty smell seeds were obtained which indicated complete roasting. The sand was then separated from the roasted pods using a sieve and allowed to cool. Thereafter, the pods were shelled and the peanuts collected. The nuts were then milled into flour, packed in plastic container and kept in freezer (-4°C) pending analysis.

Roasting of shelled peanuts
Shelled peanuts were roasted according to Ayoola and Adeyeye [9]. About 150 g of the shelled peanut were put into an iron pot and mixed with clean fine sand and stirred to prevent burning of the sample and to ensure uniform distribution of heat. The peanuts were roasted for 25 min at 120-130°C using Gallenkamp thermostat hot plate until a characteristic brownish nutty smell seeds were obtained which indicated complete roasting. The sand was then separated from the peanuts using a sieve. The nuts were cooled, milled into flour, packed in plastic container and kept in freezer (-4°C) pending analysis.

Boiling of in-shell peanuts
In-shell peanuts were boiled according to Ayoola and Adeyeye [9]. About 300 g of the dried peanut pods were put in aluminium pot, tap water added (peanut pods/water ratio 1:5 w/v), and cooked at 85-90°C on a Gallenkamp thermostat hot plate. The peanut pods got cooked after about 2½ hr. The nuts were considered cooked when they became soft to touch on pressing between the thumb and fingers. At the end of cooking time, the boiling water was drained and nuts were removed from pods and oven-dried to constant weight. The nuts were then milled into flour, packed in plastic bottles and kept in freezer (-4°C) pending analysis.

Boiling of shelled peanuts
Shelled peanuts were boiled according to Ayoola and Adeyeye [9]. About 150 g of the shelled peanuts were put in aluminium pot, tap water added (peanut/water ratio 1:5 w/v), and cooked at 85-90°C on a Gallenkamp thermostat hot plate. The shelled peanut got cooked after about 2 hr. The nuts were considered cooked when they became soft to touch on pressing between the thumb and fingers. At the end of cooking time, the boiling water was drained and the nuts were oven-dried to constant weight. The nuts were then milled into flour, packed in plastic container and kept in freezer (-4°C) pending analysis.

Autoclaving of in-shell peanuts
In-shell peanuts (300g) were autoclaved using vertical autoclave at 15 lb pressure (121°C) in tap water (1:2 w/v) for 1 hr. The nuts were considered cooked when they became soft to touch on pressing between the thumb and fingers. After autoclaving, the water was drained and nuts were removed from pods and oven-dried to constant weight. The nuts were then milled into flour, packed in plastic container and kept in freezer (-4°C) pending analysis.

Autoclaving of shelled peanuts
Shelled peanuts (150g) were autoclaved using vertical autoclave at 15 lb pressure (121°C) in tap water (1:2 w/v) for 45 min. The nuts were considered cooked when they became soft to touch on pressing between the thumb and fingers. At the end of autoclaving, the water was drained and the nuts were oven-dried to constant weight. The nuts were then milled into flour, packed in plastic container and kept in freezer (-4°C) pending analysis.

Determination of nutritional constituents
The proximate composition of peanut samples was determined using standard procedures [10]. Carbohydrate content was determined by difference. Potassium content was determined using flame photometry (Corning, UK Model 405). The other elemental contents (Ca, Mg, Fe and Zn) were determined, after wet digestion of sample ash with a mixture of concentrated nitric acid, sulphuric acid and perchloric acid (10:0.5:2, v/v) using Atomic Absorption Spectrophotometer (AAS, Hitachi Z6100, Tokyo, Japan). All the determinations were carried out in triplicates.

Determination of secondary metabolite constituents
Tannin content was determined by the modified method described by Trease and Evans [11]. The chemical method described by Maga [12] was used to determine the phytate content. The titration method was used to determine the oxalate content according to Day and Underwood [13]. Trypsin inhibitor activity (TIA) was determined according to the procedure described by Prokopet and Unlenbruck [14]. Alkaloid content was determined gravimetrically [15]. Total flavonoid content was determined by Boham and Kocipal-Abyazan [16].
Total aflatoxin (B1, B2, G1 and G2), were determined according to the method described by CSAN [17] using an ELISA (Enzyme Linked Immuno-Sorbent Assay) test kit. A portion of the sample (50g) was extracted with a sachet of the extraction powder and 150 mL of distilled water was added. The mixture was stirred properly for 30 sec to extract and centrifuged at 4000 rpm for Sustainable Food Production Vol. 8 90sec. 300 µL of the supernatant was pipetted using a micropipette into a clean tube containing 600 µL of aflatoxin dilution buffer. 300 µL of the mixture was then pipetted and plated on the test strip in the reader. The reader incubates the sample in the test strip at 45±1ºC for 5 min and displays the result in parts per billion (ppb).

Determination of functional properties
The loose and packed densities were determined using the method of Ikpeme et al. [18]. Water and oil absorption properties of the peanut flour were determined following methods of Adebayo et al. [19] with slight modifications. Flour sample (1g) was mixed with 10mL distilled water for water absorption and 10mL of oil for oil absorption in a blender at high speed for 30 sec. Samples were allowed to stand for 30min at room temperature then centrifuged (Uniscope, England) at 2000rpm for 30 min. The volume of supernatant in a graduated cylinder was noted. Density of water was taken to be 1g/mL and that of oil was determined to be 0.993g/mL.

Colour value measurement
The colour of different samples of peanut flour was measured using the Konica Minolta Spectrophotometer (CR-410, Japan). The flour sample (30g) was placed in the sample holder and the surface colour was measured at three different positions. The colour readings were displayed as L* a* b* values where L (100 = white; 0 = black) is an indication of lightness; a* measures chromaticity, with positive values indicating redness and negative values indicating greenness; while b* also measures chromaticity, with positive values indicating yellowness and negative values indicating blueness.

Statistical analysis
Determinations were carried out in triplicates and the error reported as standard deviation from the mean. Analysis of Variance (ANOVA) was performed and the least significant differences were calculated with the SPSS software for window release 16.00; SPSS Inc., Chicago IL, USA. Significance was accepted at p ≤ 0.05 levels.

Effect of processing on nutritional constituents of peanut flour
The proximate composition of peanut samples is as presented on Table 1. Processing methods were observed to significantly (p≤ 0.05) affect the nutrient composition when compared with the raw seeds. Results showed that, the moisture content of raw peanut (control) was 5.68%. The moisture levels in processed samples were: boiled in-shell (5.94%), boiled shelled (6.53%), roasted in-shell (3.15%), roasted shelled (1.49%); autoclaved in-shell (6.32%), and autoclaved shelled (8.40%). The moisture of in-shell nuts decreased during roasting though the most substantial water losses were observed in shelled nuts. The lower values reported for roasted peanut compared to raw sample could be attributed to the fact that the nuts were in direct contact with dry heat at very high temperature. The significantly higher moisture level of boiled and autoclaved peanut flour respectively compared to raw sample could be attributed to absorption of moisture from processing water medium. Moreso, the moisture content value increased in boiled shelled sample compared to in-shell peanut possibly due to the direct absorption of water into the nuts during moist cooking. Increased moisture content could also be due to water absorption by fibre and other natural chemical component [20]. A similar relationship exists between autoclaved shelled peanut and autoclaved in-shell peanut (8.40% and 6.32%, respectively).
The ash content of peanut was significantly (p≤0.05) increased by all treatments. This may be attributed to the heat employed during processing which led to greater reduction in anti-nutrients binding to the mineral elements. Moreso, the ash content of the in-shell processed peanut samples was significantly lower than shelled samples. This could be explained by the fact that there are anti-nutritional factors such as phytic acid in peanut shell [21]. Similar observation was reported by Nwafor et al. [22] on food tree (Adenanthera pavonina) seeds. The fat content in control sample 20 SFP Volume 8 (42.87%) was the least, followed in ascending order by boiled in-shell (45.79%), roasted in-shell (47.43%), autoclaved in-shell (47.53%), boiled shelled (47.60%), autoclaved shelled (47.76) and roasted shelled (49.37%), respectively. This shows that processing enhances the fat content of peanuts. The effect of direct heat on shelled peanut samples resulted in better release of fat from the nuts due to higher disintegration of inherent complex organic compounds at high temperatures to release more free fat molecules [23] compared with processed in-shell peanuts. Raw sample had the lowest protein content (32.72%). Generally, the protein value significantly increased after boiling, autoclaving and roasting, respectively. The increase in protein content of processed peanut is attributed to a positive effect of heat treatment on proteins due to the reduction in the level of anti-nutrients such as phytate which bind to them and subsequently reduce their availability and digestibility through the formation of phytic-protein complexes. However, processed shelled peanut samples had higher protein contents than processed in-shell samples. This could be attributed to the direct contact of the nuts with the heat employed in the processing leading to the disruption or breakdown of inherent organic compounds binding to the proteins and hence, further release of protein molecules. The protein content of peanut is generally high and hence suitable for therapeutic aids [6]. The fibre content of peanut was significantly (p≤0.05) affected by all treatments. Moreso, processed in-shell peanuts had significantly higher fibre than processed shelled peanuts. The observed higher fibre content could be attributed to high level of crude fibre in peanut shells [21] which permeate into the nuts during processing.
Moist heating (boiling or autoclaving) significantly decrease the carbohydrate content of peanut. This is due to the flow of free sugar (leaching) to the water during processing. Considering dry heat treatment (roasting), the reduction in carbohydrate content could be due to Maillard reaction in which the simple sugar forms complexes with protein molecules. In addition, the observed the lower carbohydrate content in processed shelled peanut samples compared to its inshell counter-part could be attributed to direct contact with heat which results in higher decrease in simple sugars as reported by Nwafor et al. [22].  Table 2 depicts the effect of boiling, autoclaving and roasting on mineral content of peanut flours. Flour prepared from raw nuts had significantly (p≤0.05) lower Ca, Mg, K, Fe and Zn contents compared with flour from boiled, autoclaved and roasted nuts, respectively. This may be attributed to the fact that the phytic acid in the raw sample is higher and it binds to inherent minerals in the nuts. Generally, boiled and autoclaved samples had the higher mineral concentration than roasted peanut. This implies that moist heat is more effective than dry heat in the release of mineral from the nuts. In all the samples, processed shelled peanuts had greater mineral concentrations than in-shell peanuts counterpart due to better release of minerals in shelled Sustainable Food Production Vol. 8 peanuts than encapsulated peanuts. The report from this study supports the claim of Mustapha et al. [24]. In essence, peanut is a viable option for improving the nutritional status of malnourished, developing and growing population in developing countries.

Effect of processing on secondary metabolite constituents
Data on anti-nutrient concentrations in raw and processed peanut samples is presented in Table 3.
The level of phytate in raw peanut was 0.006g/kg and a significant reduction (p≤0.05) was observed after all heat treatments. Boiling, autoclaving and roasting of the nuts led to significant (p≤0.05) reductions in oxalate, phytate, tannin, alkaloid, flavonoid and trypsin inhibitors, respectively in the flour when compared with their values in the flour from the raw nuts. However, boiling of the nuts was more effective in reducing the anti-nutrients than autoclaving and roasting of the nuts. The apparent decrease in phytate content during thermal processing may be partly due either to the formation of insoluble complexes between phytate and other components, such as phytate-protein and phytate-protein-mineral complexes or to the inositol hexaphosphate hydrolyzed to penta-and tetraphosphate [25]. On the other hand, some authors reported that phytic acid contents were unaffected or increased after heat treatments [26,27]. Similarly, processing significantly (p≤0.05) reduced the oxalate content of the samples. Boiled peanut samples had the lowest oxalate content and hence, it can be said that it is the most effective processing method in reducing oxalate content in peanuts. In peanut, most tannin is located in seed coats (skin) while fruit (meat nut) are practically tannin free [28]. In the present study, tannins were determined in the raw and processed peanuts without removal the skin. Edible parts of the peanut consist of the kernel and the protective skin/testa. This could explain the reported higher levels of tannins (20.49-56.03g/kg) in peanut samples. There was significant reduction in tannin concentration in processed peanut compared to raw peanut. The loss of tannin may be due to the degradation or interaction with other components of seeds, such as proteins, to form insoluble complexes [27]. The tannin content of the boiled shelled sample was the lowest and this implies that boiling has greater diminishing effect on this compound compared to other processing methods. This is sequel to the fact that tannins are water soluble in nature resulting in their leaching out during boiling into the cooking medium especially when the water is discarded after boiling. The result indicated higher trypsin inhibitor activity in raw (5.6 mg/g) compared to processed peanut samples. Significant reduction of trypsin inhibitor activity was noted in all heat treated peanut samples. Boiling of the nuts brought about highest reduction in trypsin inhibitor activity and this could be attributed to the faster and more efficient heat transfer (into the core of the nuts) than other treatments. Similar inactivation of trypsin inhibitor activity had been reported in cooked, autoclaved and microwave cooked legumes [27]. Processing significantly (p≤0.05) reduced the alkaloid content of the samples. Boiling of peanut resulted in the most significant reduction of alkaloids. This is due to leaching out of this toxic substance into the processing water. Also, apart from the raw sample,

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SFP Volume 8 the alkaloid content of all the samples are less than 10% and this implies better taste or improved palatability [29]. Processing also significantly (p≤0.05) reduced the flavonoid content of the samples. Aside the significant reduction in anti-nutrient concentration in peanut subjected to boiling, autoclaving or roasting, it was noted that in-shell processed samples had significantly higher concentration of anti-nutrients compared to the shelled samples. Lindsey and Turner [30] found out that there are various anti-nutritional factors present in peanut shells, cotyledons and skin such as phenols, tannins and related pigment. Similarly, Sim et al. [21] reported that anti-nutrients are present in peanut shell.

Effect of processing on aflatoxin composition of peanut flour
The total aflatoxin content (B1, B2, G1 and G2) of peanut samples were as follows, in increasing levels: boiled shelled (2.06 ppb), boiled in-shell (2.27 ppb), autoclaved shelled (3.10 ppb), roasted shelled (4.12 ppb), autoclaved in-shell (4.14 ppb), roasted in-shell (5.19 ppb) and raw (8.05 ppb) as indicated in Figure 1. Aflatoxins are secondary metabolites produced by fungi in the genus Aspergillus, including A. Flavus and A. parasiticus [31]. The four major naturally produced aflatoxins are known as aflatoxins B1, B2, G1 and G2. 'B' and 'G' refer to the blue and green fluorescent colours produced by these compounds under ultraviolet light on thin layer chromatography plates, while the subscript numbers 1 and 2 indicate major and minor compounds, respectively [32]. Processing significantly (p≤0.05) reduced the aflatoxin content of the samples. This may be due to the removal of mould-damaged kernels, seeds or nuts physically from food commodities before processing which as stated by Park [33] reduces aflatoxin by 40-80%. However, the efficacy and extent of reduction depends on several factors, including aflatoxin concentration, extent of binding between aflatoxin and food constituents, heat penetration, moisture content, pH, ionic strength, processing conditions and source of contamination (naturally or artificially) as reported by Hussain et al. [34]. Increases in moisture content noted in boiled and autoclaved peanut samples justify the significant reduction compared to dry heat (roasting) treatment. The relationship between moisture content of foods and reduction of aflatoxin has been demonstrated [35]. According to this report, by increased moisture content, the reduction of aflatoxin is increased during processing. Moreso, it has been suggested that the presence of water helps opening the lactone ring in aflatoxin B1 (by the addition of water molecule to the ring) to form a terminal carboxylic acid that subsequently undergoes heat-induced decarboxylation [36].
The level of aflatoxin contamination differs in the shelled and in-shell samples respectively. The aflatoxin concentration was lower in shelled nuts after processing compared to in-shell nuts. This can be explained by the fact that shelled batches are composed of nuts from which Sustainable Food Production Vol. 8 unacceptable parts were withdrawn (e.g. fungi, stains) and thus the probability of presence of aflatoxin would be lower compared to encapsulated (in-shell) nuts. In fact, the contamination seems to be more associated with the shell and not in the nut, and after its removal the aflatoxin concentration can be reduced to levels below the lowest LOD value [37]. However, in this study, the aflatoxin content of the shelled and in -shell processed peanuts ranged from 2.06 to 8.05 ppb and these values are below the European standard (10 ppb) for permissible limit of aflatoxin in food [38].

Effect of processing on functional properties of peanut flour
The functional properties of the samples of peanut flour are as presented in Table 4. The loosed bulk density (LBD), which is the lowest attainable density without compression, was higher in processed peanut than raw sample. The increase in loosed bulk density of boiled and autoclaved samples may be due to the fact that the nuts were conditioned by water used in processing and later dried back and this affected the size of the particles during milling as they could not be milled enough to achieve fine texture due to the absorbed water components [39]. Loosed bulk density value in roasted shelled peanut and in-shell roasted peanut was 0.39 g/ML and 0.40 g/ML, respectively. It is revealed from the results that roasting significantly increased the loose bulk density. In contrast, packed bulk density was lower in processed peanut than raw sample. The reduction in packed bulk density in processed peanut samples makes the products suitable as functional ingredient in weaning diet formulation.
Water absorption capacity was higher in processed peanut than raw sample. Higher water absorption capacity noted in processed peanut may be due to the high polar amino acid residues of their proteins which have strong affinity for water molecules. Similarly, oil absorption capacity was higher in processed peanuts than raw sample. Boiling, autoclaving and roasting increased the oil absorption capacity of the peanut samples and this implies that there will be high interactions among the hydrophobic/ lipophilic lipoprotein and oil and hence they could be a good thickener in food systems. Generally, bulk densities, water and oil absorption capacities of processed shelled peanut were found significantly different from processed in-shell samples. The variation in functionality of raw, processed shelled and in-shell peanuts could be due to the existing differences in the conformational characteristics of their proteins.

Effect of processing on colour attributes of peanut flour
The measured values of the colour dimensions, L*, a* and b*for raw and processed peanut flour are presented in Figure 2. The lightness (L*), of processed peanut samples decreased significantly compared with raw peanut. The lower the L* value of a sample is, the darker the product becomes. The decrease in lightness of processed samples may be due to Maillard reactions and other chemical reactions that affect the colour of food during heat processing. Similarly, parameter a* significantly decreased in boiled, autoclaved and roasted peanut samples compared with raw peanut. In terms of b* values, there are variations depending on the processing condition. Also it is worth noting that processed shelled peanuts presented higher L* values compared to processed in-shell peanut. In view of this it is possible to suggest that various processing methods accelerated the decrease in L* value noted in-shell samples. However, the colour of autoclaved in-shell sample was the most affected as indicated by lowest L* value. The lower L* values in processed in-shell samples reported in the present work suggested that peanut shells could have played a role in protecting the kernel, however the shells were not efficient in preventing the changes to the colour of the skins.