□■□■□■□■□■□■□■□■□■□■□■□■□■□■ ===== 商売．笑売．盛売　勝売　それとも少売シリーズ　=== 【　商売シリーズ　美来医（ＭＩＲＡＩ　】 　　 □■□■□■□■□■□■□■□■□■□■□■□■□■□ ＧＮＯ の　中村憲司 永久保存　 レス―ペラトロール　フィリピン大学　論文データー 流石 フィリピン大学の　農学博士です　各セクション全て　細かな 論文で　全て　大切なデーターとして　使わせて頂きます 感謝 感謝の論文データー　で　このデーターと群馬県の産業支援センターの　 試験データーと　合わせて 東洋大の 下村教授と　打ち合わせ出来たら　最高と思い　準備して居ります この流れや考えは ビジネスや多くの　ビジネス　グループ等　全ての事に当てはまりますが 　 新規ビジネスを 立ち上げるのは　どれ程優れた　新製品や　新商品　が有るから 新規ビジネスとして　立ち上げて維持出来るのでは無くて オンリーワンで 他には無い　優れた新商品と言う　武器で戦って　例えヒット しても　その成功は　長くは　続きません フロントヤードで戦う武器と バックヤードで　戦う　商売の　武器は　異なりますし 戦いが 短期決戦の　1発屋で有るなら　１つだけの　製品で戦う事は可能ですが 製品に力が有れば　 一時的に　爆発するかも知れませんが　爆発しても　１つの商品だけでは 長くは　続きません 芸能界の 一発やと言われ　一世を風靡した方でも　短期間で　消えて行きます 大爆発した 製品に　続けて　爆発を続けられる　製品を出し続ける事が　出来て 初めて　ブランド品として　生き残れます　 フロントヤードで　集客して バックヤードで　　収益を上げると言う事は　ビジネスの基本ですが　 長年 基本を忠実に守る為には　沢山の　ビジネスアイテムを持たないと　 ブランド品は　産まれません　 曼荼羅図の 作れない　１つだけしかない武器では　ビジネスは　続かないで　消えて 行くのが　世の常と思ってます 沢山の 実戦で使える　強力な商売の武器は　ビジネスを継続させる為には必要です トヨタ自動車か どれだけの　車種を　世に送り出して居るのか　凄い数です　 ベンツと言う車も 毎年　少しずつ　少しずつ　形を変えて　世の中に　出してます 単品の 優れた　商品を開発しても　直ぐに　飽きられてしまいますので　続けるには　 沢山の　商品ラインが　必要と成ります この メリンジョの論文データーは　その中でも　核爆弾に匹敵する武器の １つとして　 今後　 何回でも　形を変えて　組み合わせを変えて　製品作りをしたら 戦場で使える　 凄い武器が　幾つも作れる　新商品開発の　凄い原動力に確実に　成ると 思ってますので 　 永久保存として　 他では　手に入らない　オリジナル新商品開発の　核爆弾として　残して 置きたいと思いまして　商売シリーズに　書き残します メリンジョから 産まれる　新商品は　数限りなく　作れますので　今後　ビジネス曼荼羅図で 増やして行きます　 ―――――――――――　大切な　永久保存メリンジョ　論文データー　―――――――――――― Evaluating belinjau (Gnetum gnemon L.) seed flour quality as a base for development of novel food products and food formulations • Rajeev Bhat, , , Nabilah binti Yahya , doi:10.1016/j.foodchem.2014.01.063 Abstract Belinjau (Gnetum gnemon L.) seed flour was evaluated for nutritional composition, antioxidant activity and functional properties. Seed flour was found to be rich in protein (19.0 g/100 g), crude fibre (8.66 g/100 g), carbohydrates (64.1%), total dietary fibre (14.5%) and encompassed adequate amounts of essential amino acids, fatty acids and minerals. Antioxidant compounds such as total phenols (15.1 and 12.6 mg GAE/100 g), tannins (35.6 and 16.1 mg CE/100 g) and flavonoids (709 and 81.6 mg CEQ/100 g) were higher in ethanolic extracts over aqueous extracts, respectively. Inhibition of DPPH was high in ethanol extracts (48.9%) compared to aqueous extracts (19.7%), whereas aqueous extracts showed a higher FRAP value compared to ethanol extracts (0.98 and 0.61 mmol Fe(II)/100 g, respectively). Results on functional properties revealed acceptable water and oil absorption capacities (5.51 and 1.98 g/g, respectively), emulsion capacity and stability (15.3% and 6.90%, respectively), and foaming capacity (5.78%). FTIR spectral analysis showed seed flour to encompass major functional groups such as: amines, amides, amino acids, polysaccharides, carboxylic acids, esters and lipids. As belinjau seed flour possesses a rich nutraceutical value, it has high potential to be used as a basic raw material to develop new low cost nutritious functional foods. Keywords • Belinjau seeds; Antioxidants; Nutritional features; Functional properties ________________________________________ 1. Introduction Hunger and poverty are the 2 vital issues the world is witnessing today. Another major problem faced in the developing and underdeveloped regions of the world is protein energy malnutrition, especially among lactating women and young children (Bhat and Karim, 2009 and Sridharan et al., 2012 ). Inadequate supply and enhanced demand for animal based proteins, accompanied by the ever increasing costs of commonly used plant based food stuffs has forced researchers to explore cheaper, reliable, and cost-effective sources of protein supply. Plants and their products have been traditionally used for their potential nutraceutical properties. Among the wide array of plant produce, legumes and seeds constitute a major portion and are capable of contributing substantially as a source of nutrition (Bhat, 2011). Research towards exploring inexpensive plant-based protein supplements, as well as developing new food products, has resulted in the investigation of the potential of underutilized dicotyledonous seeds (with substantial traditional knowledge) for humans, as well as for livestock consumption (Bhat, 2011 and Bhat and Sridhar, 2008). Almost 30 species of plants belonging to the genus Gnetum (Gnetaceae family) are reported to be widely distributed in the tropical regions of the world (Malaysia, Indonesia, Thailand, India) ( Lim, 2012 and Mialoundama, 1993). Gnetum gnemon L. (English name: Spanish joint fir) is popular in Malaysia as ‘belinjau’ or ‘bago’; as ‘daun melinjo’ or ‘belinjo’ in Indonesia and ‘melindjo’ in Singapore. Thai people refer to this as ‘pee sae’ or ‘phakmiang’, and in Vietnam, it is known as ‘rau danh’. The fruit encompasses a bulky nut which is usually 2–4 cm long covered by a thin external skin. The seeds of G. gnemon are regularly consumed after deep-frying (after removal of the outer shell) or are prepared as crackers, which are bitter in taste. Traditional preparations made from seeds in parts of Malaysia and Indonesia includes preparation of soup, crackers from ground flour, while in the Philippines they are used as a coffee substitute ( Lim, 2012). However, to our knowledge, except for a few scientific reports on the phytochemical constituents and their biological activities ( Berry, 1980, Kato et al., 2009 and Wazir et al., 2011), no detailed scientific investigations have been reported on the potentiality of this seed, especially in terms of nutritional qualities, functional properties and antioxidant activities. Hence, efforts have been made to decipher afore mentioned parameters in belinjau seed flour, the result of which are envisaged to be of practical use in the development of novel, low cost nutritious food products and food formulations. 2. Materials and methods 2.1. Plant material Belinjau fruits (green and mature, see Fig. 1) used in this study were freshly harvested and purchased from a local wet market in Kepala Batas, Penang, Malaysia. Selection of green and mature fruits was based on the local traditional knowledge mainly owing to their consumption pattern and culinary use. Seeds were physically separated from their peel under an aseptic condition and freeze dried (Model 7754511, Labconco Corporation, Kansas City, Missouri, USA). The dried seeds were then ground to fine flour (mesh size 30) by using a commercial kitchen blender. The powdered seed samples were packed in air-tight polyethylene plastic pouches and were refrigerated at 4 °C, prior to analysis. Fig. 1. (a) Matured, green belinjau fruit; (b) length of a matured green fruit; (c) matured, semi-ripe fruit; (d) matured, fully ripened belinjau fruit; (e) belinjau in the natural habitat; and (f) cut open green belinjau fruit showing the seeds. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Figure options 2.2. Proximate analysis Moisture determination (of fresh sample) was completed by employing an infrared moisture analyser (Denver Instrument IR30, Colorado, USA). Fresh seeds were peeled out from their skin, chopped into smaller pieces under aseptic conditions and placed in a moisture dish at 105 °C until a constant reading was obtained. The readings were repeated for 3 independent samples. Standard methods of Association of Official Analytical Chemists (AOAC, 2000) were employed for determining the proximate composition of seed flour. Crude protein was determined based on micro-Kjeldahl method, while crude lipids were estimated by employing the Soxhlet apparatus method. Ash content was determined by igniting the samples up to 600 °C in an electric furnace (Thermolyne Sybran, Model 6000, USA), whereas crude fibre contents were estimated as outlined in AOAC Method 962.09 (AOAC, 2000). The total crude carbohydrate content (or nitrogen free extract) was estimated by difference (Müller & Tobin, 1980). Turn MathJaxon whereas, Gross energy was calculated based on the formula of Ekanayake, Jansz, and Nair (1999): Turn MathJaxon 2.3. Total dietary fibre Total dietary fibres in the samples were determined based on the enzymatic–gravimetric method (Method 985.29 of AOAC., 1997) by using α-amylase, protease and amylo-glucosidase enzymes. Results (values) obtained by the enzymatic method were corrected by determining the nitrogen content (using Kjeldahl method) and ashing at 525 °C. Turn MathJaxon Turn MathJaxon Turn MathJaxon 2.4. Mineral analysis Some of the vital minerals in seed flours such as: sodium, potassium, calcium, magnesium, copper, zinc, and manganese were determined by acid digestion employing the AOAC (1990) method. The digested samples were analysed for minerals by using an atomic absorption spectrophotometer (Perkin Elmer, Beaconfiend, UK) equipped with deuterium hallow cathode lamp background correcting system. 2.5. Amino acid analysis Amino acid content in the seed flour was determined based on previously reported methods with a slight modification (Bhat et al., 2008 and Huda et al., 2010). In brief, a known weight of the sample (0.1 g each in triplicates) taken in sealed glass tubes was treated with 5 ml of 6 N HCl and incubated in an oven operating at 110 °C for 24 h. The aliquots were added with AABA (alpha amino buteric acid, 0.4 ml of 50 mol/μ ml) as internal standards. This mixture was made up to 100 ml with distilled water and filtered through Whatman filter paper (No. 1) followed by filtration (Whatman syringe). Sulphur-containing amino acids such as methionine and cystine were analysed separately after oxidizing with chilled performic acid. Subsequent hydrolysis based on the aforementioned method was used to determine other amino acids. From the hydolysate, 10 μl was injected into HPLC (Waters 2475, US) system and run for 50 min and the peaks were identified by comparing with the retention time of known internal standards. 2.6. Fatty acid analysis Fatty acid methyl ester (FAMEs) content of the seed flour was analysed based on the method described byIndarti, Majid, Hashim, and Chong (2005). A mixture of (4 ml) freshly prepared methanol, concentrated sulphuric acid and chloroform (1.7:0.3:2.0 v/v/v) was added to a known weight of the sample (20 mg weighed in a clean screw-top glass bottle). Bottles were closed firmly with Teflon caps and maintained inside a heating block at 90 °C (for 90 min). This mixture was cooled and 1 ml of distilled water was added, followed by vortex mixing (1 min). After the two phases separated, the lower phase was carefully transferred to a clean tube and dried over anhydrous sodium sulphite (Na2SO4) for 24 h to remove the presence of moisture. After this, a known volume of the dried fraction (0.5 ml) was transferred to a clean vial, which was pre-added with internal standards (as 1 μl of capric acid, C6: 0, methyl ester as pre-diluted in chloroform, 1:499 v/v). One microliter was injected into the GC (Automatic system XL of Perkin Elemer coupled with flame ionization detector-FID with a fused silica capillary Omega wax 250 column of 30 m × 0.25 mm ID, 0.25 μm film thickness; from Supelco, USA). The conditions sustained for GC analysis included an initial oven temperature of 50 °C (for 2 min) which was increased up to 220 °C at a rate of 4 °C/min and maintained at 220 °C (for 35 min). The injector temperature was maintained at 250 °C with the FID detector temperature being at 260 °C. The carrier gas used was Helium, which was controlled at 103.4 kPa. The compressed air for FID and Hydrogen was set at 275.6 kPa. Based on the retention times (RT) of FAMEs with the methyl ester mixture, the fatty acid components present in the sample were identified. 2.7. Antioxidant compounds and activity 2.7.1. Preparation of sample extract Sample extraction for determination of antioxidants was based on the method described by Wijekoon, Bhat, and Karim (2011) with slight modifications. Briefly, 1 g of freeze dried, powdered samples were mixed individually with 50 ml of selected solvents (aqueous and ethanol) and soaked overnight (16 h) in the dark, for better extraction of the antioxidant compounds. After this time period, the extracts were thoroughly mixed in an orbital shaker (200 rpm for 20 min.), followed by centrifugation at 3000×g for 15 min. The final supernatant collected was transferred into the reagent bottle, covered with aluminium foil (to avoid light exposure) and immediately used for determination of total phenolics, tannins, flavonoids and other antioxidant activities. 2.7.2. Determination of total phenolics content Total phenolics in the extracts were determined using the Folin–Ciocalteu (FC) assay as described bySingleton and Rossi (1965), with slight modifications. For 40 μl of extracts, 2.0 ml of pre-diluted (10 times) FC reagent (R & M Chemicals, Essex, UK) was added and mixed thoroughly and the mixture was allowed to stand for 5–6 min. at room temperature (25 ± 1 °C). Followed by this, 1.8 ml of (7.5% w/v) sodium carbonate solution was added. The solutions were mixed thoroughly and allowed to stand for 30 min at room temperature, followed by measurement of the absorbance at 765 nm using a UV–visible spectrophotometer (Shimadzu UV-160A spectrophotometer, Kyoto, Japan). A calibration curve was prepared, using a standard solution of Gallic acid (Sigma–Aldrich, St. Louis, MO, USA) (20, 40, 60, 80 and 100 mg/L, r2 = 0.998). Results were expressed as mg Gallic acid equivalents (GAE) per 100 g of dry sample. 2.7.3. Determination of tannin content For determination of tannins, the vanillin–HCl method described by earlier researchers was employed (Bhat et al., 2007 and Broadhurst and Jones, 1978). The content of tannins in the sample was expressed as mg Catechin (Sigma–Aldrich, St. Louis, MO, USA). equivalent (CE) per 100 g dry sample. 2.7.4. Determination of total flavonoids Total flavonoids in the extracts were determined based on the method described by Zhishen, Mengcheng, and Jianming (1999). In brief, 1 ml of sample extract was thoroughly mixed with 4 ml of distilled water. Initially (at zero time), 0.3 ml of 5% (w/v) NaNO2 was added. This mixture was allowed to stand for 5 min after which 0.3 ml of AlCl3 was added (10% w/v). For this, (at 6 min), 2 ml of 1 M NaOH were added and the volume was immediately made up to 10 ml by addition of 2.4 ml of distilled water. This mixture was vortex mixed and the absorbance was read at 510 nm using a UV–visible spectrophotometer (Shimadzu UV-160A PC, Shimadzu Corporation, Kyoto, Japan). A suitable calibration curve was prepared by using standard solution of Catechin (20, 40, 60, 80, 100 and 120 mg/L, r2 = 0.997) and the results obtained were expressed as mg Catechin (Sigma–Aldrich, St. Louis, MO, USA) equivalents (CEQ) per 100 g of dry sample. 2.7.5. Ferric reducing antioxidant power assay (FRAP assay) FRAP assay was pursued based on the method described by Benzie and Strain (1999). Concisely, to a known volume of the extract (40 μl aliquot), freshly prepared FRAP reagent (3 ml) was added. This reaction mixture was incubated for 4 min at 37 °C. Followed by this, the absorbance was determined at 593 nm against a blank (prepared using distilled water and incubated for 1 h). Initially, care was taken to prepare a FRAP reagent fresh under be pre-warmed conditions at 37 °C by mixing 2.5 ml of a 10 mM 2,4,6-tris (1-pyridyl)-5-triazine (TPTZ, Fluka company, Switzerland) solution in 40 mM HCl with 2.5 ml of 20 mM FeCl3·6H2O and 25 ml of 0.3 M acetate buffer (pH). A suitable calibration curve was prepared, using aqueous solution of ferrous sulphate FeSO4·7H2O (200, 400, 600, 800 and 1000 L M, r2 = 0.997) and the FRAP values were expressed as micromoles of ferrous equivalent Fe(II) per 100 g of sample. 2.7.6. DPPH free radical-scavenging assay The method described by Sanchez-Moreno, Larrauri, and Saura-Calixto (1998) was adopted to determine the capacity of the seed flour extracts to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH., Fluka, Switzerland) radical. Results were expressed as percentage inhibition of DPPH using the following formula: Turn MathJaxon where, Abs control is the absorbance of the DPPH solution without extract and the Abs sample is the absorbance of the sample with DPPH solution. 2.8. Determination of functional properties 2.8.1. Water and oil absorption capacity Water and oil absorption capacities (WAC and OAC) were determined based on the method reported byBeuchat (1977) with slight modifications. Briefly, 2.5 g of the seed powder was mixed with 25 ml of distilled water or refined oil (Sunflower oil, Seri Rasa Brand, Malaysia) in a 50 ml centrifuge tube. This mixture was centrifuged at 3000×g (Model: Kubota 4000 Tokyo, Japan) for 15 min and left at room temperature for 30 min, followed by weighing of the residue. The WAC and OAC were calculated using the following formula: Turn MathJaxon 2.8.2. Emulsifying properties and foaming capacity The emulsifying activity (EA) and emulsion stability (ES) was determined based on the method described by earlier researchers (Neto et al., 2001 and Sathe et al., 1983). Emulsifying activity and emulsifying stability (ES) were calculated as: Turn MathJaxon Turn MathJaxon Foaming capacity (FC) of the seed flour was determined based on the method reported by Seena and Sridhar (2005), and was calculated as follows: Turn MathJaxon where, V0 = volume before whipping; V1 = volume after whipping; V2 = volume after standing 2.9. Functional group analysis Fourier transform infrared (FTIR) spectrometry (System 2000, Perkin Elmer, Wellesly, MD, USA) was used for identification of the functional groups present in the seed flour. The spectra obtained from the sample on potassium bromide (KBr) pellets were in the range of 4000–400 cm−1. 2.10. Statistical analysis Results presented in this study are the mean values of three independent determinants with standard deviation. Statistical significance of the obtained results were analysed by One-way analysis of variance (ANOVA) and Tukey’s HSD post hoc test using the SPSS software – SPSS 12.01 (SPSS Inc., Wacker Drive, Chicago, IL, USA). Values were considered to be significantly different from each other at a significance level of p < 0.05. 3. Results and discussion 3.1. Proximate composition, dietary fibre content and essential minerals Results obtained for proximate composition, dietary fibre content and essential minerals of belinjau seed flour are depicted in Table 1. The crude protein content (on dry wt. basis) in the seed flour was found to be 19.0 g/100 g, which was much higher compared to wheat (8.55 g/100 g) and comparable to some of the wild and underutilized legumes/pulses (range: 12–20 g/100 g) (Arinathan et al., 2003, Bhat et al., 2008,Bhat and Karim, 2009, Zia-Ul-Haq, Cavar et al., 2013 and Zia-Ul-Haq, Ahmad et al., 2013). High protein in the seed indicates the potential of the flour to be an important source of nutrient, whereas high carbohydrate content can constitute a vital source of energy which can be useful to prevent marasmus, especially when infant nutrition is concerned. Belinjau seed flour showed a high carbohydrate content (64.11%), which is comparable to kidney beans and cowpea (60–70% carbohydrate) (Khattab, Arntfield, & Nyachoti, 2009). Low fat content recorded in belinjau seed flour (2.76 g/100 g) is comparable to winged beans (1.7 g/100 g) and Canadian peas (2.43 g/100 g) (Khattab et al., 2009 and Ningombam et al., 2012). However, it should be noted that proximate composition can vary depending on the plant variety, agronomic practices, actual stage of collection of seeds and also on the climatic and geological conditions of the area from where seeds are collected. Determining proximate composition is an important indicator and an vital initiating step for nutritional evaluation of seeds and it can influence further studies on components which are rather more interesting (Zia-Ul-Haq et al., 2012 and Zia-Ul-Haq et al., 2011). Table 1. Proximate composition, dietary fibre content and minerals of G. gnemon seed flour. Component Composition Moisture (%) 82.5 ± 0.7 Ash (g/100 g) 5.50 ± 0.1 Crude fat (g/100 g) 2.76 ± 0.1 Crude protein (g/100 g) 19.0 ± 0.4 Crude fibre (g/100 g) 8.66 ± 0.8 Carbohydrate (%) 64.1 ± 0.4 Gross energy (kcal) 1434 ± 0.0 Soluble dietary fibre (%) 1.47 ± 0.3 Insoluble dietary fibre (%) 13.0 ± 0.9 Total dietary fibre (%) 14.5 ± 0.9 Calcium (mg/100 g) 157 ± 0.7 Sodium (mg/100 g) 14.0 ± 0.2 Copper (mg/100 g) 1.07 ± 0.0 Potassium (mg/100 g) Below detection limit Magnesium (mg/100 g) 26.4 ± 0.3 Zinc (mg/100 g) 4.12 ± 0.1 ∗Mean of triplicates (n = 3 ± SD) expressed on dry weight basis (Except for moisture, which is expressed on wet weight basis). Table options With regard to total soluble dietary fibre (SDF), total insoluble dietary fibre (IDF) and total dietary fibre (TDF) in the seed flour, these were found to be 1.47%, 13.0% and 14.5%, respectively. Overall, the dietary fibre composition of belinjau seed can be considered as moderate, as the daily intake in a healthy individual aged above 20 years is recommended to be in the range of 25–35 g/day (Dietary Reference Intakes., 2001). Hence, in addition to consumption of this seed or the flour preparations, intakes of other natural source of dietary fibre (from fruits vegetables, cereals, pulses) are also recommended. In belinjau seed flour, adequate amounts of essential minerals such as sodium, calcium, magnesium, copper and zinc were detected (see Table 1). The presence of these minerals will not only be beneficial for fulfilling the daily minimal dietary requirements, but will also be of use wherein bio-fortification of the seed flours can be tried along with other cereal flours used in the bakery industry (such as with wheat or refined wheat flour which are generally deficient of the essential minerals). 3.2. Amino acids and fatty acids Nutritional qualities of proteins are based on their amino acid composition (essential and non-essential amino acids). In Table 2, amino acids composition of belinjau seed flour has been compared with soybean and FAO/WHO pattern (1991). Essential amino acids (EAA) such as threonine, valine, leucine, tyrosine + phenylalanine, and lysine were present in ample amounts and were comparable either with soybeans or FAO/WHO pattern. Compared to popular legume such as soybeans (Bau et al., 1994) or FAO/WHO pattern, seed flour had higher amount of sulphur amino acids (methionine + cysteine). Presence of adequate amount of amino acids in belinjau seed flour can be considered advantageous, as most of the amino acids derived from various food stuffs have been shown to posses antioxidant, antimicrobial and anti-inflammatory properties. Additionally, amino acids can be immune stimulating agents and a source of muscle energy (Bernal et al., 2011 and Bhat et al., 2008). Table 2. Amino acid composition in G. gnemon seed flour compared with Soybean and FAO/WHO pattern. Composition (g/100 g protein) ________________________________________ G. gnemon seed Soybean a FAO/WHO b Pattern Essential amino acid Histidine 1.20 ± 0.2 2.50 1.9 Threonine 3.32 ± 0.5 3.76 3.4 Valine 4.32 ± 0.4 4.59 3.5 Cysteine 8.28 ± 5.9 1.70 2.5c Methionine 0.58 ± 0.3 1.22 Lysine 4.53 ± 0.6 6.08 5.8 Isoleusine 2.86 ± 0.3 4.62 2.8 Leucine 4.89 ± 0.5 7.72 6.6 Phenylalanine 2.69 ± 0.5 4.84 6.3 Tryptophan ND 3.39 1.1 Non-essential amino acid Aspartic acid 7.07 ± 0.4 Serine 5.41 ± 0.9 Glutamic acid 11.6 ± 0.3 Glysine 4.55 ± 1.5 Arginine 3.35 ± 0.9 Alanine 6.59 ± 0.4 Proline 5.69 ± 0.2 Tyrosine 2.48 ± 0.6 ∗Mean of triplicate determinations (n = 3 ± SD) expressed on dry weight basis. ND, not detected. a Bau et al. (1994). b FAO/WHO (1991) pattern. c Methionine + Cysteine. Table options Lipid or the oil quality depends on the quantity of fatty acids and their interactions with other major food components or macromolecules (James & Leonard, 2002). Polyunsaturated fatty acids (PUFA) content of belinjau seed were low compared to saturated fatty acids (SFA). However, the overall ratio was in an acceptable range (Table 3). Nevertheless, some of the saturated fatty acids like myristic acid and palmitic acid are an integral part of cell membrane structure in humans. These fatty acids are also useful in sustaining the optimal function of the kidneys (Monserrat, Curtin, & Coll, 2000). Also, medium chain fatty acids like that of caprylic acid are known to boost human immune system as well as possess antimicrobial and anti-protozoal properties (Chomchalow, 2011). As the ratio of PUFA:SFA is useful to evaluate the nutritional value of fats, further common/conventional cooking methods are expected to improve the quality by reducing the SFA contents. Behenic acid, an important antinutrient fatty acid (Bhat & Sridhar, 2008) was present in a lower amount, whereas cyclopropene fatty acids (CPFA) which were earlier reported to be present in ample amounts in the seed oil (Berry, 1980) were not detectable. Being a biological sample of plant origin, many characteristic features such as actual stage of maturity, genetic factors or genotype diversity and environmental conditions can affect the composition of the seed. Hence, this might explain why CPFA was not detected in this study. Table 3. Fatty acids composition of G. gnemon seed flour. Fatty acids Composition (mg/g lipid) Saturated fatty acids Caprylic acid (C8:0) 0.0862 ± 0.00 Capric acid (C10:0) 0.152 ± 0.01 Lauric acid (C12:0) 0.0014 ± 0.00 Tridecanoic acid (C13:0) 0.0241 ± 0.00 Myristic acid (C14:0) 0.0189 ± 0.00 Pentadecanoic acid (C15:0) 0.0062 ± 0.01 Palmitic acid (C16:0) 0.0426 ± 0.03 Heptadecanoic acid (C17:0) 0.0209 ± 0.03 Stearic acid (C18:0) 0.0495 ± 0.04 Arachidic acid (C20:0) 0.0035 ± 0.00 Heneicosanoic acid (C21:0) 0.0033 ± 0.00 Behenic acid (C22:0) 0.0004 ± 0.00 Tricosanoic acid (C23:0) 0.0062 ± 0.00 Unsaturated fatty acids Cis-7-hexadecenoic acid (C16:1) 0.0028 ± 0.00 Cis-10-heptadecanoic acid (C17:1) 0.0012 ± 0.00 Oleic acid (C18:1) 0.0471 ± 0.01 Linoleic acid (C18:2) 0.0085 ± 0.00 Eicosenoic acid (C20:1) 0.0027 ± 0.00 Erucic acid (C22:1) 0.0027 ± 0.00 Total saturated fatty acids 0.413 ± 0.04 Total unsaturated fatty acids 0.0651 ± 0.01 P/S ratio 6.34 ∗Mean of triplicate determinations (n = 3 ± SD) expressed on dry weight basis. Table options 3.3. Antioxidant compounds and activity Free radicals generated during a normal metabolic process or from extrinsic factors can lead to development of various physiological and pathological abnormalities, such as cardiovascular diseases and ageing. Therefore, to prevent this, high intake of natural antioxidants preferably available from natural plant resources are required. Evaluating the levels of health promoting antioxidant compounds and their activity in a plant produce is essential while developing healthy food formulations. Apart from one report bySiswoyo, Mardiana, Lee, and Hoshokawa (2011), who have compared the antioxidant activities of two protein fractions of G. gnemon (popular as ‘melinjo’ in Indonesia), there are no detailed reports available on the antioxidant potential of belinjau seed. Recovery of polyphenols or antioxidant compounds from a plant produce can be directly influenced by their solubility in a particular solvent (polar and non-polar solvents) used during the extraction process. In the present study, distilled water and ethanol were used as extracting solvents, as both of these can be considered as food grade solvents and have acceptability for human consumption. Additionally, results generated by using these two solvents will be of better use when developing healthy novel food products. Results obtained for antioxidant compounds and antioxidant capacity is depicted in Table 4. Table 4. Antioxidant compounds, antioxidant capacity and functional properties of G. gnemon seed flour. A Extraction solvents ________________________________________ Aqueous extracts Ethanol extracts Antioxidants Total phenolics (mg GAE/100 g) 12.6 ± 5.06a 15.1 ± 2.19a Tannins (mg CE/100 g) 16.1 ± 6.17a 35.6 ± 3.81b Flavonoids (mg CEQ/100 g) 81.6 ± 20.49a 709 ± 79.9b FRAP value (mmol Fe(II)/100 g) 0.98 ± 0.29a 0.61 ± 0.52a Inhibition of DPPH (%) 19.7 ± 13.17a 48.9 ± 38.9b Functional properties Water absorption capacity (WAC) (g/g) 5.51 ± 0.99 Oil absorption capacity (OAC) (g/g) 1.98 ± 0.10 Emulsion capacity (%) 15.3 ± 0.53 Emulsion stability (%) 6.90 ± 0.24 Foaming capacity (%) 5.78 ± 2.00 GAE, gallic acid equivalents; CE, catechin equivalent; CEQ, catechin equivalents. A Mean of triplicate determinations expressed on dry weight basis (n = 3 ± SD). Values followed by identical letters within a column have no significantly different at p < 0.05. Table options 3.3.1. Total phenolics, tannins and total flavonoids Overall, ethanolic extracts revealed higher total phenolics, tannins and flavonoids compared to aqueous extracts. Total phenolics were 12.6 and 15.1 mg GAE/100 g in aqueous and ethanol extracts, respectively.Wazir et al. (2011) have reported the total phenol content of G. gnemon seeds (extracted in hot water) to be 10.7 mg GAE/g. However, contrary to this, in this study the amount of phenolics recovered varied, which can be attributed to the preparation of sample extraction as well as variation in the sampling place and genetic diversity. Tannins present in the seed flour extracts were 16.1 and 35.6 mg CE/100 g in aqueous and ethanol extracts, respectively. Compared to ethanol, water extraction did not yield a high tannin content. This is because tannins exhibit hydrophobicity mainly due the presence of benzene rings in the molecules. Further, presence of many hydroxyl groups during water extraction is unable to extract more tannin effectively and the extraction solvent requires having both a hydrophilic as well as a hydrophobic character (Wijekoon et al., 2011). Apart from this, recovery of tannins can also be affected by variations in the sample particle size, wherein smaller the particles size, higher is the recovery of the tannins extracted (Deshpande & Cheryan, 1985). Our results on phenolics and tannins are comparable to some of the underutilized legumes such asCanavalia ensiformis, Canavalia gladiata, Entada scandens, and Vigna umbellata ( Katoch, 2013,Sasipriya and Siddhuraju, 2012 and Vadivel et al., 2012). However, in most these reports the extracting solvent and the standard used to express results are different. Presence of adequate amounts of phenolics and tannins in the seed flour are desirable, as these compounds can act as natural antioxidants (rather these are potential free radical scavengers), and are capable of reducing the oxidative damage associated with many degenerative diseases like cancer, arteriosclerosis or cardiovascular diseases. With regard to flavonoids, the contents were recorded to be 81.6 and 709 mg CEQ/100 g in aqueous and ethanol extracts, respectively. As per our knowledge, there is not much reported work on flavonoids in legumes. Presence of flavonoids in high amounts in belinjau seed flour is an acceptable attribute and can be of use while developing novel functional food. These flavonoids are the largest group of naturally occurring phenols that exhibit rich antioxidant properties. They are capable of interacting and effectively scavenging free radicals, which are known to damage cell membranes and biological molecules like that of DNA (Paniwnyk, Beaufoy, Lorimer, & Mason, 2001). As plant based phenolics are one of the most important classes of bioactive compounds responsible for varied biological activities, further studies are warranted to evaluate, identify and quantify the amount of phenolic compounds present in belinjau seed flour by employing modern techniques (e.g. HPLC, LC–MS, etc.), thus allowing understanding of their structure and activity. 3.3.2. FRAP and DPPH free radical scavenging assays As there are varied groups of antioxidant compounds present in a plant produce, measuring each of the compounds is difficult and time consuming. Many methods have been developed to determine the antioxidant capacity in plant materials (Guo et al., 2003), which are able to measure the ability of antioxidants to scavenge specific radicals by inhibiting lipid peroxidation or chelating metal ions. In the present study, two commonly employed methods (FRAP assay and DPPH free radical-scavenging assay) were used to evaluate the antioxidant capacity of belinjau seed flour extracts. In the FRAP assay, the antioxidant capacity of sample extracts are determined by the ability of the antioxidants in these extracts to reduce ferric iron to ferrous in FRAP reagent, resulting in the formation of a blue product (Ferrous–TPTZ complex) which is measured at 593 nm. In this study, aqueous extracts (0.98 mmol Fe(II)/100 g) showed a higher FRAP value compared to ethanol extracts (0.61 mmol Fe(II)/100 g), but were statistically insignificant (p > 0.05). In the DPPH assay, reaction occurrs between a specific antioxidant with a stable free radical like that of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) resulting in the reduction of DPPH concentration leading to changes in the colour, which can be measured at a known wavelength. In the present study, percent inhibition of DPPH was found to be high in ethanol extracts (48.9%) compared to aqueous extracts (19.70%), which is an indication that solvents tend to extract antioxidant compounds more effectively. Our results on inhibition of the DPPH radical is comparable to other legumes such as: C. ensiformis (56.8%), Vigna radiata (25%) and Soybeans (44%) ( Boateng et al., 2008, Randhir et al., 2004 and Vadivel et al., 2012). 3.4. Determination of functional properties Results obtained for various functional properties evaluated for belinjau seed flour are depicted in Table 4. Providing adequate information on the functional properties assumes importance as these can affect the overall organoleptic features and can influence consumer acceptance when a new food product needs to be developed. 3.4.1. Water and oil absorption capacities In the present study, water absorption capacity (WAC) of the seed flour was found to be 5.51 g/g, while oil absorption capacity (OAC) was found to be 1.98 g/g dry sample. Results of WAC are comparable with soybeans (3 g/g), cowpea (5.1 g/g) and lima beans (1.2–1.92 g/g), whereas results of OAC are comparable to soybeans (1.95 g/g) (Ekpo & Ugbenyen, 2011). Withholding of liquid in seed flours can be a vital index to determine the capacity of the protein to absorb and retain water and/or oil. This retention can be directly correlated wherein it can influence the texture and mouth-feel of foods especially for those of baked products as well as meats and meat analogues. In addition, OAC can be a vital parameter which can influence on how a food stuff can act as flavour retainer and affect the palatability of foods (Bhat & Sridhar, 2008). Additionally, retention of a liquid in the seed flour can be influenced by the dietary fibre content, protein content as well as gelation of carbohydrates. 3.4.2. Emulsion properties In the present investigation, belinjau seed flour showed the emulsion capacity and stability to be 15.3% and 6.90%, respectively. Emulsion capacity indicates the maximum amount of oil that is emulsified by protein dispersion, while emulsion stability reflects the ability of an emulsion with specific composition that remains unaltered (emulsion being a two phase system influenced by surface activities of protein present). Additionally, adsorption kinetics (of oil or water phase) can influence the emulsifying property, which is directly dependent on concentration. Emulsion stability is an important parameter of functional properties of seed flour which helps to identify fat/water phase stability in most of the baked food products. In the present study, emulsion stability was quite low and needs to be improved by addition of other food additives. 3.4.3. Foaming capacity Foaming capacity is an important parameter for seed flour that is used in the production of leavening food products like cakes, biscuits and other bakery products. In the present study, the foaming capacity was recorded to be 5.78% with a negligible amount of stability. These results are comparable to germinated beans (6%) of C. ensiformis and Pigeon pea ( Akpapunam and Sefa-Dedeh, 1997 and Olalekan and Bosede, 2010). The foam formations are generally affected by factors such as transportation, penetration and reorganization of molecules at air/water interface and the capacity of the molecules. Protein concentration of the seed flour can highly influence the foam capacity as increase in viscosity renders the formation of a multilayer, cohesive protein film at the interface. Also, foaming properties of belinjau seed flours can be enhanced by addition of common salt (sodium chloride), as this is known to increase the ionic strength of water as well as the solubilised protein. 3.5. Fourier transform infrared spectroscopy (FTIR) analysis To categorize major functional groups present in the belinjau seed flour, FTIR analysis was performed. The absorption bands and the wave number (cm−1) of dominant peaks obtained from absorption spectrum are presented in Table 5. FTIR is a rapid, sensitive, reliable, non-destructive, and a cost-efficient technique that can be used for characterizing the chemical composition or identifying functional groups present in material. The observed bands for amines, amides, amino acids indicate the presence of protein, whereas other absorption bands indicated the presence of bio-molecules like polysaccharides, carboxylic acids and lipids. Table 5. Functional groups detected in G. gnemon seed flour based on FTIR analysis. Compound Functional group Wave number (cm−1) Amines N–H stretching 3418.71, 2361.35 N–H bending 1639.75 C–N stretching 1077 Amides N–H stretching 3418.71, 2849.23, 2361.35 C–O stretching 1152.60, 1382.16 Amino acids N–H stretching 3418.71, 2849.23, 2361.35 N–H bending 1639.75 C–O stretching 1152.60, 1382.16 Carbohydrates N–H wagging 765.90 Polysaccharides C–O–C stretching 1241.25,1152.60,765.9 Lipid, alkenes C–H stretching 2927.13 Alkenes C C stretching 1639.75 C–H out-of plane bending 765.9 Sulfonyl and sulfonate S O stretching 1077.52, 1021.23 Aromatics C–H out-of plane bending 765.9 Alcohols, esters, anhydrides C–O stretching 1152.6 Ethers C–H stretching 2849.23 Carboxylic acids O–H stretching C O stretching 2927.13, 1410.33 1241.25 Table options 4. Conclusions Results of the present study revealed belinjau seed flour to be a rich source of nutrients exhibiting good antioxidant activities with acceptable functional properties. Seed flour showed appreciable levels of crude protein, carbohydrates and dietary fibre with the presence of adequate amounts of essential minerals (sodium, calcium, magnesium, copper and zinc) and essential amino acids (threonine, valine, leucine, tyrosine + phenylalanine, and lysine). The Polyunsaturated fatty acids (PUFA) content of belinjau seed was low compared to saturated fatty acids (SFA). However, the overall ratio of PUFA: SFA was in an acceptable range. Results on antioxidant compounds and inhibition of DPPH radical revealed ethanolic extracts to exhibit higher total phenolics, tannins and flavonoids content compared to aqueous extracts. Results on FTIR showed the presence of amines, amides, amino acids, polysaccharides, carboxylic acids and lipids. These results are a clear indication that the seed flour can be efficiently used for developing novel functional foods, especially those of bakery products. Further studies are warranted to evaluate the presence of anti-nutrients and undertake toxicity studies (if any), characterize the phenolics compounds, as well study the effects of physical or chemical treatments, which can be aimed towards modifying or improving the overall functional qualities of the seed flour. Acknowledgements Funding received from Universiti Sains Malaysia, is gratefully acknowledged. Authors gratefully acknowledge anonymous referees for constructive comments which were useful in upgrading the quality of this manuscript. References 1. M.A. Akpapunam, S. Sefa-Dedeh. Some physiological properties and antinutritional factors of raw, cooked and germinated jack bean (Canavalia ensiformis). Food Chemistry, 59 (1997), pp. 121–125 2. AOAC, Official methods of analysis. (15th ed.)Association of Official Analytical Chemists, Washington, DC (1990) pp. 1–1230 3. AOAC, Official methods of analysis of AOAC International. 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De Feo o Compositional studies, antioxidant and antidiabetic activities of Capparis decidua (Forsk.) Edgew o International Journal of Molecular Sciences, 12 (2011), pp. 8846–8861 54. o Zia-Ul-Haq, Cavar et al., 2013 o M. Zia-Ul-Haq, S. Cavar, M. Qayum, I. Khan, S. Ahmad o Compositional studies and antioxidant potential of Acacia leucophloea Roxb o Acta Botanica Croatica, 72 (2013), pp. 27–31 1. Source: Bhat, N and N. Yahya 2014. Evaluating belinju (Gnetum gnemon) seed flour quality as a base for development of novel food products and food formulations. Food chemistry 156:42-49. 2. Proximate composition, dietary fibre content and minerals of G. gnemon seed flour. Component Composition Moisture (%) 82.5 ± 0.7 Ash (g/100 g) 5.50 ± 0.1 Crude fat (g/100 g) 2.76 ± 0.1 Crude protein (g/100 g) 19.0 ± 0.4 Crude fibre (g/100 g) 8.66 ± 0.8 Carbohydrate (%) 64.1 ± 0.4 Gross energy (kcal) 1434 ± 0.0 Soluble dietary fibre (%) 1.47 ± 0.3 Insoluble dietary fibre (%) 13.0 ± 0.9 Total dietary fibre (%) 14.5 ± 0.9 Calcium (mg/100 g) 157 ± 0.7 Sodium (mg/100 g) 14.0 ± 0.2 Copper (mg/100 g) 1.07 ± 0.0 Potassium (mg/100 g) Below detection limit Magnesium (mg/100 g) 26.4 ± 0.3 Zinc (mg/100 g) 4.12 ± 0.1 ■■■■■■■■■■■■■■■■■■■■■■■■ 以前の　物お読みになります場合のバックナンバーは 　http://www3.wind.ne.jp/~temis/ml/cgmmm.cgi _/_/_/_/_/_/_/_/_/_/_/_/_/_/Temis Network_/_/_/_/_/ 【商売・笑売・盛売 製作】中村憲司　temis@mail.wind.ne.jp