Australian Journal of Basic and Applied Sciences, 7(2): 728-734, 2013 ISSN 1991-8178

Effect of Debittering Process on Characterization of Egyptian Lupine Seeds Oil (Lupinus albus)

1Mostafa A.A. Awad-Allah and 2Haiam Elkatry

1Home Economics Department, Faculty of Specific Education, South Valley University, Qena, Egypt. 2Home Economics Department, Faculty of Specific Education, Ain Shams University, Cairo, Egypt.

Abstract: The aim of this study was to remove the alkaloids from bitter lupine (Lupinus albus) seeds and effect of debittering process on the characterization of lupine seed oil. After the debittering process, the total alkaloid content of seeds decreased significantly (p < 0.05) from 1.47±0.01 to 0.03±0.00 g/100 g. The values (on dry matter), protein, lipid, starch and ash content of seeds changed in a statistically significant manner (p < 0.05) after the debittering process. Lupine oils extracted from raw and debittered lupine seeds were investigated for their chemical properties. The oil fractions (% of total lipids), monoglycerides (3.68±0.03 %), hydrocarbons (4.72±0.04 %), diglycerides (6.70±0.05 %) and free fatty acids (7.48±0.24 %) were higher in debittered lupine whereas raw lupine seed oil had higher contents of steroids (6.21±0.03 %) and phospholipids (14.46±0.04 %). Lecithin (% of the phospholipids) was the main component in both phospholipid fractions of raw and debittered lupine seed oils (47.22±0.26 and 38.65±0.32 %), followed by phosphatidylethanolamine (15.24±0.07 and 13.67±0.07 %); respectively. The fatty acid profiles (% of the total fatty acids) of the lupine seed oils showed that the amounts of total unsaturated and essential fatty acids were higher than saturated fatty acids in both types of oils. The raw lupine seed oil had higher contents of essential fatty acids (40.44±0.03 %) as compared to debittered lupine seed oil (39.22±0.04 %). The predominant fatty acid in both oils was oleic acid. Its concentration in debittered oil was 46.11±0.10 % and in raw lupine seed oil was 43.82±0.05 %. Among tocopherols in both oil types, β fraction was not detected, while γ fraction dominated. The other fraction α tocopherol was significantly higher (5.81±0.05 mg/100g oil) in raw oil as compared to debittered one (4.91±0.02 mg/100g oil).

Key words: Lupine Seeds – Debittering process – Oil characteristics

INTRODUCTION

Lupine seeds are a valuable ancient legume which contains high amount of protein, dietary fiber, oil, minerals and different functional components. Lupine seeds are employed as a protein source for animal and human nutrition in various parts of the world, not only for their nutritional value (high in protein, lipids and dietary fiber), but also their adaptability to marginal soils and climates. Human consumption of lupine seeds has increased in recent years. Lupine flour is added for its nutritive value (high protein efficiency ratio) and also to provide functional properties in bakery and pastry products, protein concentrates and other industrial products, as well as for the elaboration of lactose-free milk and yoghurt analogues (Jiménez-Martínez et al., 2003; Lqari et al., 2002). The sweet lupines contain smaller amounts of toxic alkaloids than the bitter lupine varieties. The bitter lupine is an annual legume traditionally cultivated around the Mediterranean area (Huyghe, 1997). Bitter lupine seeds cannot be consumed directly since its high toxic alkaloid content. Cooking and soaking are effective processes for removing these toxic substances and anti-nutrients as phytic acid, trypsin inhibitors and oligosaccharides. The lupine oil can be used as human food because Lupinus albus and Lupinus mutabilis species contain an average oil content of 110 and 190 g/kg; respectively and are already identified as an oil sources (Gross et al., 1988; Oliveira and Ferreira, 2000). Williams (1989) reported that Lupinus mutabilis has intermediate values of oleic and linoleic acids as compared to the olive and sunflower oils. Consumption of lupine seeds based diets lowers plasma glucose, cholesterol, and triglycerides (Chango et al., 1998). Lupine oils are characterized by a balanced fatty acid composition with total saturated fatty acids of about 10 % and total unsaturated fatty acids around 90 % (Bhardwaj et al., 1998; Petterson, 2000). The fat level in lupine is ranked third after groundnut and soybean among different legumes (Cowling et al., 1998). Being one of the oil bearing plant materials, lupine seeds can be explored for the production of healthier dietary oil. The main purpose of this paper is to characterize the oils extracted from raw and debittering lupine seeds.

MATERIALS AND METHODS

Materials: Lupine seeds (Lupinus albus) were obtained from the Ministry of Agriculture, Giza, Egypt during 2011 season.

Corresponding Author: Mostafa A.A. Awad-Allah, Home Economics Department, Faculty of Specific Education, South Valley University, Qena, Egypt. E-mail: [email protected] 728

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Methods: Technological Methods: Debittering Process of Lupine Seeds: The debittering process for the lupine seeds consisted of cleaning, boiling, and debittering stages. Extraneous material and immature and damaged seeds were removed first. Twenty kg of chosen samples of lupine seeds were presoaked in running water for 12 hours, then the soaked seeds were boiled for one hour (1:3, seeds:water) to destroy thermo-labile anti-nutritional factors, such as trypsin inhibitors and to soften the seeds. The boiled seeds were debittered with tap water for 48 hours at room temperature (~25 ºC), the debittering water was changed every 2 hours. The samples were packed into 500 ml – glass jars and stored under refrigeration for analysis (Erbas, 2010).

Analytical Methods: Proximate Composition: Proximate composition was carried out according to ICC Standard Methods (ICC, 2001). Moisture content was determined by drying the samples at 105 °C to constant weight (ICC 109/01). Ash content was determined by calcinations at 900°C (ICC 104/01). Nitrogen content was determined by using Kjeldahl method with factor of 5.7 to determine protein content (ICC 105/02). The total lipid content was determined by extraction in the Soxhelt apparatus with hexane (ICC 136). The determination of starch content was assessed using a polarimetric method as described by Davidek et al. (1981). All the measurements of analyzed samples were made in triplicate and measurements were calculated on dry weight basis.

Extraction of Alkaloids for GC–MS Analyses: The seeds were air-dried for 4 – 6 days by exposing the wet seeds to the sun until the lupine seeds contained about 8 – 10 % moisture. The raw and treated lupine seed flours were obtained after grinding the seeds in a laboratory hammer mill until they could pass through a 250 μm screen. Extraction of the milled seed was done as described by (Muzquiz et al., 1993): 0.5 g of ground seeds were homogenized with 5 ml of 5 % trichloroacetic acid for 1 min. The mixture was then centrifuged for 15 min (10,000 g) and the supernatant separated. The extraction was repeated twice. The supernatants were collected in a decantation funnel and 0.8 ml of 10 M NaOH was added. Three extractions with 15 ml of dichloromethane were performed, and the organic phase was evaporated to dryness at room temperature. The residue was dissolved in 1 ml of methanol and 100 µl sparteine solution as an internal standard (final concentration of sparteine, 1 mg/ml) was added. 1 µl of this mixture was injected into capillary gas chromatograph.

Fractionation of Total Lipids: Total lipids were extracted from the lupine flour two times by chloroform: methanol mixture (2: 1 v/v) with intermittent stirring to extract the oil. The extracts were combined, filtered and dried over anhydrous sodium sulphate and the solvent was removed off by rotary evaporator under vacuum at 50°C. The oil sample was kept in well stopper dark containers at 0°C (to protect it from autoxidation) until use. The oil content was calculated on dry weight basis (AOCS, 1980). The oil was dissolved in chloroform. The total lipid samples were fractionated by the method of Stahl (1965) using silica gel, G 60 Merk type 5721 and 13 × 18 cm glass plates with 0.25 mm thickness. The developing solvent system was petroleum ether : diethyl ether : glacial acetic acid (80: 20: 1v/v).

Determination of Phospholipids: The method recommended for phospholipids extraction by Kates (1972) was used in this work. The phospholipids class was separated on silica gel G 60-coated plates using a developing solvent comprising a mixture of chloroform, methanol and water (65: 25: 4, v/v). The separated fractions of total lipids and phospholipids were visualized by exposure to iodine vapor in a closed chamber after drying. All lipid fractions were identified on thin layer plates by comparing their relative retention times with those of known lipid standards. For quantitative analysis, the different lipid fractions were scanned using Shimadzu-TLC-Scanner (C-S- 910). The area under each peak was measured by the triangulation method as described by Kates (1972). The percentage of each component was calculated with regard to the total area as follows: % component = (area of each peak / total peaks area) × 100.

Estimation of Fatty Acids: Fatty acid contents were determined as described in the AOAC (2007). The fatty acids methyl esters were prepared from aliquots of total lipids of raw and debittered lupine seed oils using 5 ml 3 % H2SO4 in absolute methanol and 2 ml benzene. The contents were heated for methanolysis at 90°C for 90 min. After cooling, phase separation was performed by addition of 2 ml distilled water and methyl esters were extracted with 2 aliquots of

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5 ml hexane each. The organic phase (upper water) was removed, then transferred through a small amount of anhydrous Na2SO4 and concentrated using a rotary evaporator. For identification of fatty acids methyl esters, gas liquid chromatography (Perkin Elemer Gas Chromatograph "GC-4 CM Shimadzu" with a flame ionizing detector "FID" in the presence of nitrogen as a carrier gas) was used at the National Research Center, Cairo. The separation was carried out at 190 – 230°C (temperature rate 4°C / min) on a (3.0 m × 0.3 mm) glass column, packed with silar 5CP on Chromo Q 11, 80 – 100 mesh. The injector and detector temperatures were 270°C. The nitrogen, hydrogen and air flow rates were 20.0, 1.0 and 0.5 ml / min; respectively. The chart speed was 5 mm / min. The peaks were identified by comparison with pure methyl ester standards by means of their relative retention times under identical conditions. The proportion of each fatty acid present in investigated samples was calculated by the triangulation method as follows: % fatty acid = (area of each peak / total peaks area) × 100.

Determination of Tocopherols: HPLC analysis of tocopherols (T), in the sample as well as the standard tocopherols mixture, was carried out using Toyo-Soda-CCPM HPLC instrument. An oil sample (10 g) was dissolved in n-hexane to make 10 % solution and 10 µl was injected into silica column (YMC-A-012, 6.0 × 150 mm). Isocratic elution was conducted using n-hexane: isopropyl alcohol (100 : 0.5, v/v) as mobile phase, at a flow rate of 1-2 ml/min. Hitachi-650-10S fluorescence detector was used. Spectral absorption was set at excitation and emission wavelength of 295 and 325 nm; respectively. The conditions were optimized to elute δ- T after 10 min; the results were automatically recorded as peak areas percentages by electronic integrator. From the peak area and the corresponding weight of each individual standard T, the weight of each individual T in the oil can be calculated (El-Mallah et al., 1994; El-Shami et al., 1994).

Statistical Analysis: Analysis of variance (ANOVA) was carried out using SPSS program (version. 16.0). The effect of debittering process on oil characteristics of lupine seeds were analyzed using ANOVA. When the treatment factor effect was found significant, indicated by a significant F-test (p< 0.05), differences between the respective means were determined using least significant difference (LSD) and considered significant when p < 0.05. Means ± standard deviations of three replicates were used (Pallant, 2005).

RESULTS AND DISCUSSION

The loss of dry matter from the seeds during the debittering process was ~ 23 g/100 g. This loss was a result of the loss of fiber (~ 5 g/100 g), which occurred because of separation of the softened husks and soluble material (~18 g/100 g) during the debittering process. The detached husks could be observed as small particles in all the changes of extraction liquid during debittering process. In addition, the extraction liquids became yellowish in color because of the presence of pigments. Jimenez-Martinez et al. (2001) have reported that during the process of debittering, 120–270 g/kg of solids were lost, which depended on the specific treatment that was used. The other changes in the chemical properties of the seeds, which depended on the processing, are shown in Table (1). Dry matter was decreased by the introduction of water into the seeds and the loss of soluble components and detached husks. During the debittering process, the components of the seeds were modified at different rates. The protein and starch contents of seeds increased by 7.70 and 2.02 g/100 g; respectively. These increases may have been caused by the fact that oligosaccharides, minerals, alkaloids, flavonoids and fiber were removed when the debittering liquid was changed. The ash content of the seeds decreased as a result of the decrease in crude fiber content and the removal of minerals by the extraction process. The common decrease in fat content that was observed during debittering process may have been caused by removal of the lipid-rich embryo together with the detached husks.

Table 1: Effect of debittering process on the proximate composition* (g/100 g) of lupine seeds**. Components Raw Debittered Dry matter 89.19±0.28 a 66.46±0.40 b Protein 38.60±0.20 b 46.30±0.52 a Ash 3.41±0.02 a 2.11±0.01 b Fat 9.94±0.03 a 8.25±0.08 b Starch 1.98±0.07 b 4.00±0.11 a Crude fiber 44.60±0.39 a 39.30±0.65 b Total alkaloids 1.47±0.01 a 0.03±0.00 b * = Mean ± standard deviation of three replicates. ** = on dry weight basis except dry matter. Mean values in the same row showing the different letter are significantly different (p < 0.05)

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In the raw seeds, the total concentration of alkaloids was 1.47±0.01 g/100 g, and this was reduced to below the detection limit (0.03±0.00 g/100 g) by the debittering process. It has been reported that the total alkaloid content of Turkish bitter landraces of Lupinus albus is 19.1 g/kg (Muzquiz et al., 1994). The changes in the levels of seed components that occurred during debittering process in this study were similar to those that were described by other researchers (Jiménez-Martínez et al., 2001; Torres et al., 2005). The protein content of the debittered lupine seeds was determined to be 46.30±0.52 g/100 g, and it was similar to other lupine species point of view protein abundant. Furthermore, the crude fiber and lipid contents were similar to those found in the seeds of other lupine species (Jiménez-Martínez et al., 2001). Sujak et al. (2006) have reported that the protein, lipid, crude fiber and ash contents of Lupinus albus seeds are 363, 115, 144 and 34 g/kg; respectively. Raw lupine seeds are low in starch (1.98±0.07 g/100 g), whereas the seeds of other common legumes have high starch content (Cerning-Beroad and Filiatre, 1976). Therefore, the debittered and defatted lupine seeds may be a potential source of material for the production of dietetic foods because they have a high protein and dietary fiber content and low starch content as compared with other legumes and cereals. Also, lupine seeds could be used to enrich different types of food products with respect to protein and dietary fiber. Data presented in Table (2) showed that the two types of lupine seeds oils differed significantly in their oil fractions. The debittered lupine seed oil had significantly higher contents of monoglycerides (3.68±0.03 %), hydrocarbons (4.72±0.04 %), diglycerides (6.70±0.05 %) and free fatty acids (7.48±0.24 %) whereas raw lupine seed oil had higher contents of steroids (6.21±0.03 %) and phospholipids (14.46±0.04 %). The results were agreement with those of Alamri (2012) who reported that the sweet lupine seed oil had significantly higher contents of hydrocarbons and free fatty acids, whereas bitter lupine seed oil had higher contents of steroids and phospholipids. While, the results are disagreement with to the findings of Hamama and Bhardwaj (2004) who reported that 2.6 to 2.8 % phospholipids were found in seven varieties of white lupine seed oils. Phospholipids can also serve as an emulsifier in different food products. The presence of higher phospholipids content in lupine seed oil observed in present studies can also be an added benefit while using these oils.

Table 2: The total lipid fractions* of raw and debittered lupine seed oils (% of the total lipids). Components Raw Debittered Triglycerides 63.26±0.30 a 60.30±0.25 b Diglycerides 5.03±0.01 b 6.70±0.05 a Monoglycerides 2.30±0.03 b 3.68±0.03 a Steroids 6.21±0.03 a 4.60±0.07 b Hydrocarbons 4.47±0.05 b 4.72±0.04 a Free fatty acids 4.26±0.02 b 7.48±0.24 a Phospholipids 14.46±0.04 a 12.53±0.06 b * = Mean ± standard deviation of three replicates. Mean values in the same row showing the different letter are significantly different (p < 0.05)

The qualitative and quantitative phospholipid composition of the investigated oils is presented in Table (3). The obtained results show that the qualitative phospholipid composition is varied. Lecithin was the main component (47.22±0.26 and 38.65±0.32 %) in both phospholipid fractions in raw and debittered lupine seed oils, followed by phosphatidylethanolamine (15.24±0.07 and 13.67±0.07 %); respectively. The debittered lupine seed oil had significantly higher contents of phosphatidylglycerol and lysolecithin were found to be 18.00±0.05 and 6.94±0.04 %; respectively. On the other hand, the content of sphingomyline (10.48±0.15 %) was considerably higher in raw lupine seed oil.

Table 3: Phospholipid fractions* of raw and debittered lupine seed oils (% of the phospholipids). Fractions Raw Debittered Lysolecithin 5.02±0.02 b 6.94±0.04 a Sphingomyline 10.48±0.15 a 8.34±0.07 b Lecithin 47.22±0.26 a 38.65±0.32 b Phosphatidylserine 12.05±0.04 b 14.42±0.06 a Phosphatidylethanolamine 15.24±0.07 a 13.67±0.07 b Phosphatidylglycerol 10.00±0.03 b 18.00±0.05 a * = Mean ± standard deviation of three replicates. Mean values in the same row showing the different letter are significantly different (p < 0.05)

Fatty acid composition of raw and debittered lupine seed oils is presented in Table (4). There was a significant variation between two types of oils with respect to their fatty acid profiles. The dominant fractions in both oils on descending were oleic, linoleic, palmitic and linolenic acids among other fatty acids. The oleic and linolenic acids were significantly higher in debittered lupine oil (46.11±0.10 and 7.51±0.02 %; respectively) than raw ones (43.82±0.05 and 6.27±0.01 %; respectively). Raw lupine seed oil had higher contents of linoleic (32.19±0.02 %) and palmitic (8.84±0.01 %) as compared to debittered lupine seed oil in which these fractions were 28.40±0.02 and 6.19±0.01 %; respectively. The content of linolenic acid in both types of lupine seed oils are far below (less than 8 %) than the contents reported in flaxseed oils, i.e., above 47 % by (Zhang et al., 2011).

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This can be a good indicator of the better stability of lupine seed oil as the presence of unsaturated fatty acids may cause problems during the long term storage of oil containing food products. The amounts of total unsaturated and essential fatty acids were higher than saturated fatty acids in both types of oils. The raw lupine seed oil had higher contents of essential fatty acids (40.44±0.03 %) as compared to debittered lupine seed oil (39.22±0.04 %). The results are comparable with an earlier study on lupine by Bhardwaj et al. (1998) who reported 51 % linoleic acid, 23 % oleic acid, 10 % palmitic acid, 7 % linolenic acid and 14 % saturated fatty acids. The results further indicate that ratio of linoleic over linolenic was good in both raw and debittered lupine seed oils (5.13:1 and 3.78:1; respectively). The medical research has shown that the excessive contained in 1 g of oil (Firestone, 1996; ISO, 1996). Level of linoleic acid relative to linolenic acid may increase the probabilities of a number of diseases (Hibbeln et al., 2006). However, it has been suggested by Hu (2001) that linoleic to linolenic acid ratio of 10: 1 or less results in reduction of cardiovascular diseases. The saturated fatty acid contents of the raw and debittered lupine seed oils analyzed in the present study are lower (i.e., 11.64±0.01 and 9.93±0.01 %; respectively) as compared to earlier reported by Fitzpatrick and Scarth (1998) in soybean oil (14 %). These findings can be helpful to recommend the use of lupine seed oil as a replacement of soybean oil due to its lower contents of saturated fatty acids.

Table 4: Fatty acids composition* of raw and debittered lupine seed oils (% of the total fatty acids). Fatty acids Carbon chain Raw Debittered a b Myristic C14:0 0.68±0.01 0.11±0.00 a b Palmitic C16:0 8.84±0.01 6.19±0.01 b a Stearic C18:0 2.12±0.00 3.63±0.01 b a Oleic C18:1 43.82±0.05 46.11±0.10 a b Linoleic C18:2 32.19±0.02 28.40±0.02 b a Linolenic C18:3 6.27±0.01 7.51±0.02 b a Eicosanoic C20:3 4.10±0.03 4.74±0.04 b a Arachidonic C20:4 1.98±0.01 3.31±0.01 Saturated fatty acids (SFA) 11.64±0.01 a 9.93±0.01 b Unsaturated fatty acids (USFA) 88.36±0.04 b 90.07±0.01 a USFA:SFA 7.59:1 9.07:1 Linoleic: Linolenic 5.13:1 3.78:1 Essential fatty acids 40.44±0.03 a 39.22±0.04 b * = Mean ± standard deviation of three replicates. Mean values in the same row showing the different letter are significantly different (p < 0.05)

Tocopherols are the most popular plant antioxidants in the food industry. The natural antioxidants alpha-, beta-, gamma- and delta-tocopherols are compounds present in the green parts of plants and in their seeds (Sheppard et al., 1993). Each of these four types has different biological and antioxidant activities (Kamal-Eldin and Appelqvist, 1996) but they act as the main antioxidants in cell membranes (Bramley et al., 2000). Tocopherol contents of raw and debittered lupine seed oils are shown in Table (5). Raw lupine seed oil has significantly higher α- (5.81±0.05 mg/100 g oil) and δ-tocopherols (5.11±0.04 mg/100 g oil) contents than debittered lupine seed oil. The total tocopherol contents were higher (84.75±0.42 mg/100 g oil) in raw lupine seed oil when compared to the oil of debittered lupine seed (70.83±0.30 mg/100 g oil). The results suggest that lupine vitamin E content is similar to that of soybean oil. Lampart-Szczapa et al. (2003) also reported the three tocopherols in Lupinus albus as alpha- (2.8 %), beta- (86.1 %) and delta (11.1 %), and in Lupinus luteus as alpha- (3.2 %), beta- (88.5 %) and delta-tocopherols (8.3 %). But according to Hatzold et al. (1983) only two fractions, i.e, alpha- (3.8 %) and gamma-tocopherols (96.2 %) were present in lupine seed oil.

Table 5: Tocopherol fractions content* of raw and debittered lupine seed oils (mg/ 100 g oil). Fractions Raw Debittered α- Tocopherol 5.81±0.05 a 4.91±0.02 b β- Tocopherol ND ND γ- Tocopherol 73.83±0.44 a 61.52±0.3 b δ- Tocopherol 5.11±0.04 a 4.4±0.02 b Total Tocopherol 84.75±0.42 a 70.83±0.30 b * = Mean ± standard deviation of three replicates. ND = Not detected. Mean values in the same row showing the different letter are significantly different (p < 0.05)

Conclusion: Based on the present study conducted to characterize the lupine seed oil of raw and debittered one, it can be concluded that the debittering process had positive effect on the proximate composition and properties of lupine seed oil. Hence, lupine seed oil can be used as a healthy replacement of other traditional oils used for dietary purposes. The balanced fatty acid profile and presence of antioxidants makes these oils suitable for their utilization in different food products as additives.

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