Food Chemistry 136 (2013) 213217

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Food Chemistry
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Amino acid composition, antinutrients and allergens in the peanut protein fraction obtained by an aqueous enzymatic process
S. Latif a,, J. Pfannstiel b, H.P.S. Makkar a, K. Becker a
a b

Institute for Animal Production in the Tropics and Subtropics (480), University of Hohenheim, D-70593 Stuttgart, Germany Life Science Center Core Facility, University of Hohenheim, 70593 Stuttgart, Germany

a r t i c l e

i n f o

a b s t r a c t
Enzyme-assisted aqueous extraction (EAE) of peanut kernel was used to extract oil and protein. The aqueous fraction (AF) obtained by EAE had a better essential amino acid prole than the residues obtained by solvent extraction (Rs) and cold pressing (Rc). No major difference in the trypsin inhibitor activity among AF, Rs and Rc was observed; however, the trypsin inhibitor activity was drastically reduced in the residue obtained after EAE. AF was subjected to MALDI-TOF/MS, revealing it to be rich in peptides (107) with molecular masses from m/z 700 to 2369 Da. AF had an extremely low phytate content and was rich in peptides, which could be used as a food supplement. ESI-MS/MS data were used for the identication of major peanut allergens, viz., Ara h1, h3, h68. Their allergenic potential needs to be established. 2012 Elsevier Ltd. All rights reserved.

Article history: Received 29 May 2012 Received in revised form 19 July 2012 Accepted 30 July 2012 Available online 8 August 2012 Keywords: Enzyme-assisted aqueous extraction Peanut Allergens Peptides MALDI-TOF/MS Nano-LCESI-MS/MS

1. Introduction Peanut is ranked as the third largest source of vegetable oil in the world with a production of 31 million tonnes. Peanut is widely used in both western and oriental cooking and is added to a variety of foods such as pastries, sandwiches, egg rolls, chilli, syrups, ours, sauces and confections. Peanut seed contains 2529% protein, 2023% carbohydrate and 4050% oil. After oil extraction, the protein content in the residue may reach 50%. Peanut allergy appears to be on the rise in Western countries, affecting 11.5% of the population (Grundy, Matthews, Bateman, Dean, & Arshad, 2002). Curative treatment is not yet available and prevention is the best way to guard against manifestation of allergy. Traces of peanut can cause a severe allergic reaction in sensitive subjects and therefore strict elimination of peanut is required from the diets of such individuals (Hourihane et al., 1997). The nature of the allergenic proteins in peanut has been studied extensively and several allergens have been identied. Eleven allergenic proteins (Ara h1 through Ara h11) from peanut have been isolated and characterised. Ara h1 and Ara h2, the major peanut protein (allergens) account for 1216% and 5.99.3% of the total peanut protein, respectively (Jianmei, Ahmedna, Goktepe, Cheng, & Maleki, 2011). However, Ara h2 is a more potent allergen than Ara h1 (Koppelman, Wensing, Ertmann, Knulst, & Knol, 2004).
Corresponding author.
E-mail address: sajid.latif@yahoo.com (S. Latif). 0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.07.120

Various methods targeting proteins or DNA are used to determine the presence of allergenic ingredients. Protein-based methods utilise immunological techniques. A major drawback of immunological methods is that the epitopes detected are usually not known and cross-reactivity with matrix components can result in false positive results. DNA-based methods employ polymerase chain reaction (PCR). However, allergenicity of a food is caused by its proteins and not by its DNA, so therefore the presence of peanut DNA does not guarantee the presence of allergens. Consequently, a conrmatory method is needed to provide an unambiguous identication of (allergenic) proteins. A mass spectrometrybased approach combines the separation and identication of individual proteins and it might lead to identication of allergenic proteins. It is a direct method and hence is better than the DNA based method (Chassaigne, Norgaard, & Hengel, 2007). Enzyme-assisted aqueous extraction (EAE) is an environmentally friendly and safe alternative for simultaneous oil and protein extraction from oilseeds (Campbell et al., 2011; Hagenmaier, Carter, & Mattil, 1975). EAE has other advantages over the conventional hexane extraction. The oil obtained using EAE has low free fatty acid and phosphorous contents and hence can be used directly without further rening, and the protein-rich fraction obtained after oil extraction may have exceptionally low levels of anti-nutritional factors. The hydrolysed protein obtained through EAE may have a number of benecial human health implications. For example, enzymatically hydrolysed proteins are more rapidly digested and absorbed than whole proteins hence foodstuffs

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having hydrolysed proteins can be employed in the feeding of hospital patients with digestive diseases. Additionally, it has been reported that enzymatic processing is a useful approach to reduce/eliminate the allergenicity of foods and it has proved to be effective for producing hypoallergenic rice (Watanabe 1993). Recently, it has been observed that peanut allergenicity can be reduced by using an enzymatic treatment (Jianmei et al., 2011). Hence, we evaluated the presence of allergens in the aqueous fraction (AF) obtained through EAE by using a proteomic-based approach. Furthermore, the quality of AF and the residue obtained after oil extraction were analysed in terms of amino acid prole, phytate content and trypsin inhibitor activity, and the values obtained were compared with those of the residue obtained by solvent extraction and cold pressing. 2. Materials and methods Raw peanut seeds (with skin) were purchased from Nussdepot (Berlin Germany). The skin was removed by hand, the kernels were ground using a commercial grinder and passed through a 1 mm sieve. These ground peanut kernels were stored at 4 C and were used throughout the experiment. Protex 6L (protease) was kindly provided by Genencor Novozymes (Leiden, The Netherlands). 2.1. Enzyme-assisted aqueous extraction (EAE) Ground peanut seed was mixed with distilled water at a liquid: solid ratio of 6:1, v/w. The mixture was heated at 95 C in a water bath for 10 min and allowed to cool to room temperature. By using a pH-stat, the mixture was maintained at pH 8. Thereafter, enzyme Protex 6L (5:1000 enzyme/kernel; w/w) was added and the mixture was incubated at 50 C for 120 min with constant stirring at 500 rpm. These conditions were based on our previous study in which we had optimised operational parameters. The mixture was centrifuged (8545g, 30 C, 15 min), resulting in oil, creamy and aqueous phases (Latif & Anwar, 2011). Phases were separated using a micro-pipette, yielding the top, oil-rich phase along with the emulsion (Re: between oil and aqueous fraction) followed by the aqueous fraction (AF) and leaving the meal (R = Residue) at the bottom. The oil taken out by pipette was claried by centrifugation, leaving an emulsion. After removing oil, all the remaining fractions were lyophilised. All fractions were analysed for chemical composition, amino acid proles, phytate content and trypsin inhibitor activity (TIA). However, AF was also analysed for allergen peptide markers (APMs) by using MALDI-TOF and nano-LCESI-MS/MS. 2.2. Cold pressing and solvent extraction The cold pressed oil (CPO) was obtained by pressing the ground peanut kernels in a manual laboratory hydraulic press for one hour at a pressure of 10 ton per 78.54 cm2 (no heat was developed), the residue remaining after extraction of oil (Rc) was taken for the study. The same batch of ground peanut kernels was extracted with petroleum benzene (b.p. 4060 C) in a Soxhlet for 16 h (2 8 h each with the addition of fresh petroleum benzene each time) with solid to liquid ratio (1:10 w/w) to obtain solvent extracted oil. The residue after extraction of oil (Rs) was taken for the study. 2.3. Chemical composition and amino acids Samples of AF, Re and R were analysed for dry matter, ash, crude protein, lipid and amino acids. The moisture content was determined by oven drying at 105 C to constant weight. Lipid and ash

were determined using the standard methods of the Association of Ofcial Analytical Chemists (AOAC, 1990) while crude protein (CP) was determined using a C/N analyser (C/N varioMAX, Elementar Analysensysteme, Hanau, Germany) and a conversion factor of CP = N 6.25. The amino acid composition was determined using an automated amino acid analyser after hydrolysing the samples with 6 M HCl at 110 C for 24 h (Bassler & Buchholz, 1993). The sulphur-containing amino acids were oxidised using performic acid before acid hydrolysis. The tryptophan contents of the above-mentioned samples were determined spectrophotometrically by the method of Pinter-Szakacs and Molnar-Perl (1990). 2.4. Determination of phytate content and trypsin inhibitor activity (TIA) TIA was determined according to Smith, VanMegen, Twaalfhoven, and Hitchcook (1980) except that the enzyme was added last, as suggested by Liu and Markakis (1989). In brief, about 0.25 g defatted sample was Ultra-turraxed for 30 s in 12.5 ml of 0.01 M NaOH. The pH of this mixture was adjusted in the range of 9.4 9.6 with 1 M NaOH or 1 M HCl and was centrifuged at 3000g for 15 min. The supernatant was taken for the assay using benzoylDL-arginine-p-nitroanilide (BAPA) as the substrate. Phytate content of the sample was determined by a spectrophotometric procedure described by Vaintraub and Lapteva (1988). The results are expressed as g/100 g phytic acid, using standard phytic acid (sodium salt; Sigma, St. Louis, MO, USA). 2.5. MALDI-TOF MS-spectra of AF MALDI-TOF MS-spectrum of AF was acquired on an Autoex III MALDI-TOF-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). The instrument was operated in the positive ion mode and calibrated externally using peptide calibration standards (Bruker Daltonics, Bremen, Germany). AF was desalted using C18 ZipTips (Millipore, Schwalbach, Germany) according to the manufacturers protocols. Proteins were eluted from the ZipTips directly onto a stainless steel target using a-cyano-4-hydroxy-cinnamic acid (HCCA) matrix solution (5 mg/ml in 50% ACN/50% 0.1% TFA, v/v). Peptide mass ngerprint data were recorded in reector mode using an accelerating voltage of 21 kV. Typically, 1000 laser shots were accumulated for each peptide mass ngerprint spectrum. FlexAnalysis 3.0 and BioTools 3.0 software (Bruker Daltonics, Bremen, Germany) were used for data processing. 2.6. Nano-LCESI-MS/MS and database searches Nano-LCESI-MS/MS experiments were performed on an ACQUITY nano-UPLC system (Waters, Milford, USA) directly coupled to a LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Fisher, Bremen, Germany). Peptides extracted from AF were concentrated and desalted on a pre-column (2 cm 180 lm, Symmetry C18, 5 lm particle size, Waters, Milford, USA). Separation of peptides was performed on a 20 cm 75 lm BEH 130 C18 reversed phase column (1.7 lm particle size, Waters, Milford, USA) using a linear gradient of 150% ACN in 0.1% FA within 1 h. The LTQ-Orbitrap was operated under the control of XCalibur 2.0.7 software. Survey spectra (m/z = 2501800) at a resolution of 60.000 at m/z = 400 were detected in the Orbitrap using lock-mass ions from ambient air for internal calibration (Olsen et al., 2005). Data-dependent tandem mass spectra were generated for the ve most abundant peptide precursors in the linear ion trap. Mascot 2.3 software (Matrix Science, London, UK) was used for protein identication. Spectra were searched against the Green Plant subset of NCBI protein sequence database downloaded as FASTA-formatted sequences from ftp://ftp.ncbi.nih.gov/blast/db/

S. Latif et al. / Food Chemistry 136 (2013) 213217 Table 1 Chemical constituents in fractions obtained by an enzymatic extraction process of Peanut seeds (%, w/w). Component Peanut seed Enzymatic aqueous extraction process Dry matter Aqueous fraction (AF) Emulsion (Re) Residue (R) 94.1 92.2 90.6 94.4 Crude lipid 51.8 4.3 81.1 22.2 Protein 26.6 63.1 18.6 23.9 Crude ash 2.1 6.1 1.9 7.8
a

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Table 3 Anti-nutritional components of peanut fractions obtained by different extraction processes. Components EAAEa Aqueous fraction Phytates (% dry matter) Trypsin inhibitor (mg trypsin inhibited/g sample dry matter basis) 0.62 1.92 Residue 6.64 0.59 Soxhlet extracted Residue 1.98 1.92 Cold pressed Residue 2.13 2.23

EAAE: Enzyme assisted aqueous extraction.

FASTA/nr.gz. Search parameters specied no cleaving enzyme, a 3 ppm mass tolerance for peptide precursors and 0.6 Da tolerance for fragment ions. Cysteine carbamidomethylation was selected as a xed modication, methionine oxidation was selected as variable modication. 3. Results and discussion 3.1. Oil and protein content The peanut seed used in this study had 94.1% dry matter comprising 51.8% lipid, 26.0% protein and 2.1% crude ash (Table 1). We used previously optimised conditions for EAE and were able to extract 92% oil and 83% protein (Table 1). Among all the three fractions (AF, Re and R), AF had highest protein (63.1%) and lowest lipid content (4.3%). However, Re had highest lipid content (81.1%) because it was present between free oil and protein. After simultaneous extraction of oil and protein from peanut kernel, the remaining fraction (R) had a signicant portion of lipid (22.2%) and protein (23.9%) which can also be used as a valuable byproduct. 3.2. Amino acid composition and antinutrients In order to evaluate the quality of AF and R obtained through EAE, the amino acid composition and anti-nutritional factors were determined and compared with those in the residues obtained by solvent extraction (Rs) and cold pressing (Rc) (Table 2). Higher amount of essential and non essential amino acids were observed in the AF and R as compared to Rs. This decrease of amino acids
Table 2 Amino acid compositions of peanut fractions obtained by different extraction processes. EAAEa Aqueous fraction Protein (%) 63.1 Essential amino acids composition (g/100 g crude protein) Methionine 1.13 Cystine 1.35 Valine 3.68 Iso leucine 2.92 Leucine 5.94 Phenylalanine 5.09 Tyrosine 3.66 Histidine 2.33 Lysine 3.20 Threonine 2.61 Tryptophan 0.81 Non-essential amino acid composition (g/100 g crude protein) Serine 4.98 Arginine 11.57 Glutamic acid 19.78 Aspartic acid 11.73 Proline 4.18 Glycine 5.13 Alanine 3.88
a

in Rs may be due to the severe and prolonged heat treatment during solvent extraction. On the other hand, the amino acid prole of AF and R were close to untreated ground peanut seed which may be attributed to the gentle operational conditions of EAE. Exceptionally low contents of phytate (% in DM: dry matter) were observed in the AF (0.62) as compared to those in Rs (1.98), Rc (2.13) and R (6.64) (Table 3). The exceptionally higher amount of phytate in R may be due to the extraction of the major portion of protein from the residue by the enzymatic action and their release in the AF, resulting in concentration of the phytate in the R. There are a number of studies that have proved phytate to be an antinutrient component having a negative impact on the bioavailability of divalent and trivalent mineral ions and forming complexes with proteins at low and high pH (Kumar, Sinha, Makkar, & Becker, 2010). Therefore, efforts are made to degrade phytate during food processing. Exceptional phytate reduction in the AF will be an additional benet for the EAE. On the other hand, phytate has been reported to be anti-carcinogenic, an antioxidant, to regulate insulin secretion, to prevent heart disease and to be used for the removal of traces of heavy metal ions (Kumar et al., 2010). The R fraction having enriched phytate can be explored for various medicinal and industrial applications. The trypsin inhibitor activity (TIA; mg trypsin inhibited/g dry sample) was remarkably reduced in the R fraction (0.59 mg) when compared to that in Rs (1.92) and Rc (2.23 mg) fractions (Table 3). On the other hand, TIA in the AF was also 1.92 mg trypsin inhibited/g dry sample. The trypsin inhibitor is heat labile and can be removed by hydrothermal treatment and roasting (Alonso, Aguirre, & Marzo, 2000). The lower TIA in the R fraction compared with those

EAAEa Residue 23.9 0.92 1.00 4.02 3.26 6.61 4.60 3.56 2.22 3.72 2.80 0.96 4.85 10.04 12.30 8.37 4.23 7.70 4.02

Cold pressed Residue 48.6 1.01 1.17 3.44 2.65 5.76 4.69 3.42 2.20 3.13 2.51 0.88 4.75 10.45 17.55 10.49 3.91 5.23 3.74

Solvent extracted Residue 59.4 0.89 1.13 3.18 2.66 5.54 4.51 3.20 2.10 3.01 2.41 0.42 4.48 9.90 17.10 9.95 3.74 5.02 3.55

Untreated kernal Residue 26.6 1.09 1.20 3.83 3.12 6.24 5.04 3.65 2.33 3.35 2.67 0.86 4.92 11.35 18.72 11.17 4.32 5.49 3.98

EAAE: Enzyme assisted aqueous extraction.

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Fig. 1. MALDI-TOF/MS of peanut protein extracted by enzyme-assisted aqueous extraction.

Table 4 Identied allergen peptide markers in the protein extracted through aqueous enzymatic process. Protein description Ara Ara Ara Ara Ara Ara h1 h3 h6 h7 h8 h8 isoform pep_exp_mz 745.3793 634.8632 564.8124 435.2453 494.2535 686.8682 pep_exp_mr 1488.744 1267.7119 1127.6102 868.476 986.4924 1371.7218 pep_calc_mr 1488.746 1267.714 1127.612 868.4766 986.492 1371.725 pep_score 39.59 60.98 28.75 27.3 24.39 45.43 Peptide sequence DLAFPGSGEQVEKL RSVNELDLPIL KRELMNLPQ ELRNLPQ KPDEEELK KATVVDGDELTPK

Peptide experimental m/z value (pep_exp_mz); peptide experimental molecular weight (pep_exp_mr); peptide calculated molecular weight (pep_calc_mr); peptide score according to MASCOT (pep_score).

in Rs and Rc suggests that, along with other proteins, the enzyme treatment also releases TIA (a protein) from the R fraction and releases it into the AF. It may be noted that the enzyme used for oil extraction is protease based and this enzyme was not able to completely degrade TIA. 3.3. MALDI-TOF/MS spectra of AF Fig. 1 presents the MALDI-TOF/MS spectra of the protein extracted as AF during EAE. One hundred and seven peptides with molecular masses ranging from m/z 700 to 2370 Da were identied. Most of the peptides were in the range of 320 amino acids per peptide and peptides within this mass range have the potential to be bioactive (Pownall, Udenigwe, & Aluko, 2010). As a rich source of peptides, this protein fraction can be a potential source of bioactive peptides (e.g. antioxidant and antihypertensive peptides) and may be used for therapeutic applications. By using EAE, we can extract both a good quality oil (unpublished data) and a protein fraction with a rich source of peptides as a byproduct. This will denitely improve the economics of this environmentally friendly process as compared to conventional extraction methods. 3.4. Nano-LCESI-MS/MS identication of allergen peptide markers (APMs) The aqueous fraction (AF) was analysed further by nano-LCESIMS/MS and the data obtained were used to perform a database search using the Mascot algorithm to identify peptide sequences. This program nds the sequence that best ts the MS/MS data and assigns a probability score to the peptides (peptide score). Based on the peptide score we were able to identify the APMs

listed in Table 4. For Ara h 1, a peptide marker (DLAFPGSGEQVEKL, m/z 745.3793, charge 2+) was found which was similar to that previously reported by Chassaigne et al. (2007) (DLAFPGSGEQVEK, m/z 688.85, charge 2+) using trypsin. The presence of leucine at the C terminal of the identied peptide may be attributed to the different enzyme used. Chassaigne et al. (2007) reported another allergen peptide marker (APM), namely, VLEENAGGEQEER (m/z 786.88, charge 2+) which may not be detected in our trial because of possible hydrolysis during the enzymatic treatment. We did not nd any APM for Ara h 2 having an appreciable score. Although based on a previously reported APM (RQQWELQGDR, m/z 439.23, charge 3+) for Ara h 2, we found a peptide with a similar amino acid sequence pattern (SARQQWELQGDRRCQS, m/z 649.9785, charge 3+). In another study, the peptides at m/z 807.2451 (CCNELNEFENNQR; 2+) and m/z 949.8894 (CMCEALQQIMENQSDR; 2+ of the cysteine oxidised form) were selected for Ara h2 (Careri et al., 2007). However, we did not observe any peptide with this sequence for Ara h2. It may be possible that peptides containing 15 amino acids are too short to be identied as Ara h 2 specic cleavage products. On the other hand, some peptides comprising 14 hydrophilic amino acids (e.g. K, R, E, D, etc.) might have been lost because they bind only loosely to the C18 trapping column of the LC-MS/MS system. Some large hydrophobic peptides (>40 amino acids) may ionise with poor efciency and are therefore hard to detect. The high TIA in the AF (Table 3) may be another indicator for the presence of Ara h2 because it acts as a trypsin inhibitor (Maleki et al., 2003). Therefore, it is possible that most of the proteins having Ara h2 were extracted in the AF, thus leaving a small portion in the R. In the case of Ara h3, the peptide (RSVNELDLPIL, m/z 634.8632, 2+) was found to be the most abundant. Chassaigne et al. (2007)

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reported two specic peptides, SPDIYNPQAGSLK (m/z 695.35, charge 2+) and SQSENFEYVAFK (m/z 724.84, charge 2+) for Ara h3. However, on the basis of signal intensity, retention time repeatability, lack of missed cleavages, specicity, the peptides at m/z 648.8109 (AHYQVVDSNGDR, 2+), m/z 695.3468 (SPDIYNPQAGSLK, 2+) were reported as APM for Ara h 3/4 (Careri et al., 2007). However, we did not detect any peptide of this characteristic which may be due to the intensive hydrolysis of enzyme during EAE. Concerning Ara h 6, Ara h7, Ara h8 and Ara h8 isoforms, the peptides at m/z 564.8124 (KRELMNLPQ, 2+), m/z 435.2453 (ELRNLPQ, 2+), m/z 494.2535 (KPDEEELK, 2+), and m/z 686.8682 (KATVVDGDELTPK, 2+), respectively, were identied as APMs. To the best of our knowledge no previous data are available to compare these APMs. The presence of these APMs in the protein fraction obtained through EAE suggested that the enzymatic treatment could not eliminate these peanut allergens. 4. Conclusions A signicant amount of protein from peanut seeds can be recovered during extraction of the oil by using EAE. An improved amino acid prole was observed for the protein in the aqueous fraction (AF) obtained through EAE as compared with the residue obtained by solvent extraction (Rs) or cold extraction (Rc). The phytate content was dramatically reduced in AF as compared to that in Rs. However, trypsin inhibitor activity was reduced in the residue obtained by EAE (R). MALDI-TOF scan revealed AF as a potential source of peptides. Based on allergen peptide markers (APMs) allergens (Ara h1, h3, and h68) proteins were identied by nano-LCESI-MS/MS. However, it is recommended that their allergic potential should be the subject of further evaluation. The peptide rich AF obtained by the EAE may be used in various foods or feed applications. Acknowledgements Authors are indebted to the Alexander von Humboldt Foundation, Germany for providing nancial support to Dr. Sajid Latif. References
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