Effect of Refining on the Physico-chemical Properties and Nutritional Composition of Solvent Extraction-derived Flaxseed Oil

122 2010, Vol. 31, No. 16 Effect of Refining on the Physico-chemical Properties and Nutritional Composition of Solvent Extraction-derived Flaxseed Oil REN Hai-wei ( College of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China) Abstract The changes in physico-chemical properties, fatty acid composition and vitamin E content of solvent extractionderived flaxseed oil during the refining process consisting of degumming, alkaline treatment and bleaching, deodorization were measured in order to address the effect of refining on the physico-chemical properties and nutritional composition of solvent extraction-derived flaxseed oil. Over the whole refining process, the content of phospholipids and acid value markedly decreased, from 1.541 to 0.163 g/100 g and from 2.826 to 0.635 mg KOH/g, respectively. However, no obvious change in iodine value, saponification value and refraction index was observed. The capillary column gas chromatographic analysis revealed that solvent extraction-derived flaxseed oil contained 4 saturated fatty acids (mainly hexadecanoic acid and stearic acid) and 6 unsaturated fatty acids (mainly linolenic acid, linoleic acid and oleic acid). Refining had little effect on the fatty acid composition and total fatty acid content in solvent extraction-derived flaxseed oil, but the relative content of linolenic acid presented an obvious decrease, especially at the stages of bleaching and deodorization. The refining process gave a vitamin E loss of up to 49.19%, indicating that the nutritional composition of solvent extraction-derived flaxseed oil was greatly affected by the refining process. Key words solvent extraction flaxseed oil refining physico-chemical properties nutritional components ( 730050) VE 1.541g/ 100g 0.163g/100g 2.826mg KOH/g 0.635mg KOH/g 4 ( ) 6 ( ) - 49.19% TS224.6 A 1002-6630(2010)16-0122-06 Flax (Linum usitatissimum L.), a member of the Linaceae family, is an economically important oilseed crop. Flaxseed (also called linseed) contains approximately 40% oil, 30% dietary fiber, 20% protein, 6% moisture and 4% ash [1]. Apart from its traditional usage as a raw material in oil production, flaxseed is a functional food ingredient because of its potential to reduce the risk of cardiovascular diseases, cancers, inflammations of the mucous membranes and gastrointestinal disorders [2]. Flaxseed oil is obtained by either mechanical pressing or solvent extracting the flaxseed, which is separated during milling flaxseed to flour. Flaxseed oil is an excellent source of tocopherols (Vitamin E) and polyunsaturated fatty acids (PUFAs), which including -6 and -3, defined by the position of the double bond closest to the methyl at 2009-09-10 (1983 ) E-mail rhw52571119@163.com

2010, Vol. 31, No. 16 123 end of the molecule. -linolenic acid (ALA) is an essential fatty acids, and its metabolites EPA and DHA may exert protective effects against some common cancers, especially cancers of the breast, colon, prostate and other cancer illnesses. Tocopherols are important lipid oxidation inhibitors in food and biological system. Therefore, flaxseed oil is recognized as a healthful, nutritional edible oil and often consumed in the main flaxseed production area of China. Traditionally, the purpose of refining oil was to eliminate all impurities that could be harmful to health and obtain good quality oils, such as good flavor and appearance. However, -6 or -3 PUFAs and tocopherols are sensitive to heat, oxygen or light, and will be drastically reduced during different refining stages [3]. Therefore, refining process is very important to quality characteristic, because the loss of highly desirable PUFAs and tocopherols could reduce oxidative stability and nutritional value of flaxseed oil. Now, the nutritional quality of edible oil means that refining has the purpose of not only eliminating impurities such as phospholipids, free fatty acids(ffas), peroxides, polymers, pigments and secondary oxidation products but also minimizing tocopherols loss and high retention of PUFAs. The objective of this study is to monitor changes of physicochemical properties, tocopherols contents and fatty acid compositions during and after the classic refining process. 1 Materials and Methods 1.1 Materials, reagents and instruments Flaxseed in the present study was donated by the Agricultural Bureau of Jingle County in Shanxi Province, China. Hexane used in the extraction was analytical reagent grade and was purchased from the Nanjing Chemical Company. All the other chemicals were of analytical grade. Agilent 6890N gas chromatograph, equipped with an Agilent 7683 series autosampler (Agilent Technologies Inc., Wilmington, DE, USA); HPLC system consisting of a Varian 9010 solvent delivery system (Varian Associates, Inc., Walnut Creek, CA, USA); Ultraspec 3300 pro spectrophotometer (Amersham Pharmacia Biotech, Uppsala, Sweden). 1.2 Analytical methods The Chinese official methods for the hygienic standard analysis of edible oils were used to determine the Peroxide value (PV, GB/T 5009.37 2003), Acid value(av, GB/T 5530 2005), Iodine value (IV, GB/T 5532 1995), phospholipids contents (GB/T 5537 1985), specific gravity (GB/T 5526 85) and saponification value (SV, GB/T 5534 1995). The refractive index was determined by methods for determination of refractive index (GB/T 5527 1985). 1.3 Hexane extraction of flaxseed oil After being cleaned by hand carefully to remove the foreign materials such as other seeds, stones and small stalks, flaxseed were dried at 50 for 12 h in an oven, and then crushed into powder in a grinder with a size range of 0.55 1.0 mm. The resulted powder was kept in a vacuum dryer until use. Flaxseed ground samples were mixed with hexane (1:10, m/v) in flask, which was covered with Parafilm over the top and aluminum foil at the sides to keep out air and light. The mixture was stirred for 1.5 h and then filtered through filter paper under vacuum. The solids were rinsed once with hexane. The acquired oil was evaporated using a rotary evaporator to remove the residual hexane, and then transferred to airtight brown flask immediately. 1.4 Degumming of flaxseed oil During refining stage of degumming, phospholipids are eliminated by water degumming with acid pretreatment. After a number of trials, the following degumming procedure was chosen. Phosphoric acid (0.20%, V/V) was added to the heated crude oil (60 ), and the mixture was vigorously stirred (260 r/min) for 5 min. Distilled water(3.0%, V/V) was then added and the mixture was stirred at a higher shear rate (200 r/min) for 8 min. The mixing speed was gradually reduced to 60 r/min, followed by a 60 r/min (0.1% phosphoric acid,70 agitating-holding period of 20 min, according to the rate of gum formation and aggregation. The insoluble, hydrated gums were allowed to standing overnight for further separation as a sludge and the supernatant oil was decanted. The degummed oil was stored and proceed to the alkali refining step. 1.5 Alkali treatment or neutralizing of flaxseed oil The optimum quantity of alkali needed was calculated based on AV of oil plus 0.12% excess to ensure the displacement of the reaction toward the formation of soapstock as described by [4]. The degummed oil was preheated to 40, and sodium hydroxide solution(26 B ) was added with stirring to neutralize the FFAs and redundant phosphoric acid. The mixture was gently stirred with a glass rod for 10 min until soapstock was separated. The mixture was transferred to a water bath maintained at 85 for 8 h, subsequently, the precipitated soapstock was then separated by centrifugation at 4000 r/min for 5 min. To eliminate traces of soapstock dissolved in the oil and the remains of the mineral reagents added, the deacidified oil was mixed with hot, soft water and

124 2010, Vol. 31, No. 16 again centrifugally separated to remove residual soapstock. The water washed, deacidified oil, containing traces of moisture, was dried in a rotary evaporator under vacuum prior to bleaching. 1.6 Bleaching of flaxseed oil Bleaching of the deacidified oil involves the removal of pigments that were either dissolved in the oil or presented in the form of colloidally dispersed particles. Vacuum absorptive bleaching could use less activated clay, operate at lower temperatures, and minimize oxidation by reducing exposure to air and produce high quality bleached oil. Bleaching parameters (including activated clay type and amount, temperature, time) were initially evaluated by pilot test. According to the preliminary experiment results, 5% (m/v) activated clay, 80 and 15 min was adequate for the maximum reduction observed in oil color. Alkali-refined oil was preheated fleetly to 80, subsequently, activated clay (2.5%, m/v) was added to the heated oil, and the mixture was rotated (100 r/min) on rotary evaporator under vacuum (0.60 MPa) for 15 min. And then, the mixture was allowed to cool rapidly to 25 and centrifuged at 3000 r/min for 10 min until clear, bright bleached oil was obtained. 1.7 Deodorization of flaxseed oil Deodorization was an important step in oil refining process. During traditional technology, steam was injected into the oil to eliminate FFAs, aldehydes, unsaturated hydrocarbons and ketones, which caused undesirable odors and flavors in the oil. However, N2 was used as stripping gas for deodorizing olive, sunflower and soybean oil recently [5], there were certain advantages to using N2 vs. steam, such as recovery of high-quality deodorizer distillates. Therefore, the bleached oil was deodorized mildly using N2 as described previously by Wang et al [6]. 1.8 Fatty acid compositions of flaxseed oil Fatty acid compositions were determined using capillary-column gas chromatography (GC) of fatty acids methyl esters (FAME). FAME was prepared according to the method of Dan [7] with some modifications. Approximately, 0.35 g of oil sample was weighed into a glass tube and 4 ml of 0.5 mol/l methanolic sodium hydroxide was added. The mixture was incubated at 60 for 20 min and then cooled to room temperature. About 2 ml of hexane, 3 ml of water were added and mixed well by tilting the capped tube with no shaking of the tube. Hexane was then discarded and the aqueous layer was backwashed with 2 ml of hexane and then transferred to a new tube. About 1 ml of 14% BF3-methanol solution was added and the mixture was incubated for 20 min. About 2 ml of saturated sodium chloride solution was then added. 1 ml of the upper hexane layer containing FAME was pipetted into a 2 ml sample vial and stored at 20 for later analysis. The FAME solution was quantified on an GC-2010 gas chromatograph, equipped with a flame ionization detector (FID). The FAME in hexane (1 L) was injected into the column with a split ratio of 100:1. The injector and detector temperature were set at 230 and 250, respectively. Hydrogen was used as the carrier gas at a flow rate of 2 ml/min. Separation was carried out on a Restek @ -1MS capillary column (30 mm 0.25 mm) with a film thickness of 0.25 m. The column temperature was programmed from 100 to 160 at 2 /min and then to 250 at 4 /min and finally held at 250 for 20 min. The weights of the individual FAME were calculated on the basis of their relative peak area compared with that of internal standard, and then they were corrected using the corresponding GC response factors for each fatty acid. 1.9 Tocopherol compositions of flaxseed oil The contents of total and individual tocopherol at different refining stages were determined by high-performance liquid chromatography (HPLC) according to GB/T 5009.82 2003. Oil samples (10 ml) were analyzed by HPLC system consisting of a solvent delivery system with an injector of 10 ml loop size, a diode array detector set at 300 nm and analyzing software. An analytical prepacked column (4 mm 4.5 cm), ultrasphere ODS10 m, was used with methanol in water (98:2, V/V) as the mobile phase. The system was operated isocratically at a flow rate of 1.7 ml/min. Typically, a 10 min equilibration period was used between samples, requiring about 40 min/ sample. Tocopherols peaks were identified by comparison with the retention times of the respective standards. Quantification was based on an internal standard method. 1.10 Statistical analysis Data were interpreted by analysis of variance (ANOVA), with Duncan s multiple-range test using SAS software package. The statistical significance was evaluated at P 0.01 level. All analyses were carried out for three times. 2 Results and Discussion 2.1 Effect of refining conditions on physicochemical characteristics In general case, oil refining process was very important to the quality characteristics, functionality and economic

2010, Vol. 31, No. 16 125 Table 1 Determination results of physicochemical properties of flaxseed oil during refining process steps Characteristic Crude oil Degumming oil Alkali-refined oil Bleaching oil Deodorization oil NY/T 751 2006 IV/(g iodine/100 g) 204.69 1.26 a 203.65 1.10 a 203.65 2.05 a 202.65 1.36 a 201.65 1.55 a 175 198 Refractive index (n 20 ) 1.476 0.00 a 1.474 0.01 a 1.475 0.03 a 1.476 0.00 a 1.477 0.02 a 1.471 1.476 SV/(mg KOH/g) 193.65 1.52 a 192.65 1.12 a 191.65 1.12 a 190.25 1.36 a 190.73 1.08 a 188 195 PV/(meq/kg) 1.365 0.12 a 1.350 0.06 a 1.355 0.12 a 1.068 0.12 b 0.982 0.10 b 1.576 Phospholipids/(g/100g) 1.541 0.12 a 1.23 0.12 b 0.598 0.12 c 0.358 0.12 d 0.163 0.02 e AV/(mg KOH/g) 2.826 0.12 a 2.960 0.10 a 1.233 0.05 b 1.389 0.02 b 0.635 0.00 c 1.0 Specific gravity (d 0 ) 0.936 0.01 a 0.935 0.01 a 0.933 0.01 a 0.935 0.01 a 0.934 0.01 a 0.928 0.938 Note: Values with different superscript letters within a row are significantly different at P 0.01. value. Table 1 shows the physicochemical characteristics of oil during and after every refining stage. Trials confirmed that the phosphorus content was significantly reduced to the very low level required after refining steps; however, degumming did not reduce phosphorus as effectively as did neutralizing (P 0.01). This was because oil was treated with phosphoric acid, so that the metal-phospholipid complexes were dissociated into insoluble metal salts and in alkali refining, by adding to alkali, the phospholipids were converted into Na salts, which were more hydratable and therefore more easily removed from the oil. In addition, the soapstock formed during alkali refining may act as a good adsorbent for the phospholipids and other undesired components. Experimental evidence indicates that, although AV decreased totally in overall refining stages, AV of degummed oil and bleached oil increased distinguishingly, because phosphoric acid was added to the oil during degumming, and active clay was treated with sulfuric acid, the excess mineral acid lead to the increasing of AV. PV was most commonly used as an indicator of the oxidation of oils during storage or refining. PV was not significantly affected by degumming and neutralizing (P 0.01), but was significantly reduced after bleaching (P 0.01). The possible reasons were that the bleaching clay accelerated the oxidation of oil and high temperatures and long residence times in the stage of deodorization. There were no significant differences of IV, refractive index, specific gravity and SV during refining steps, refining stages had little effect on these properties (P 0.01). In a word, it was found that oil refining process had a great influence on the quality characteristics of oil product. Moreover, compared with the quality specification of NY/T 751 2006, different index of refining flaxseed oil were in good agreement with the requirements of the national standard. 2.2 Effect of refining on the component and content of fatty acids Table 2 shows a summary of the fatty acid compositions, percent of UFAs and ratios of -3 to -6 fatty acids at different refining stages. Analysis of the data showed that the ratio of -3 to -6 fatty acids of crude flaxseed oil was 3.69, so flaxseed oil was one good way to increase -3 fatty acids in the diet, because most countries nowadays are known to generally consume plenty of -6 fatty acids which riched in margarine and vegetable oils, and the absolute amounts of -3 fatty acids in the diet are too low. 10 fatty acids were identified, which consisted of 4 SFAs (7.10%) and 6 UFAs (92.72%). The main SFAs are palmitic acid (4.52%) and stearic acid(2.31%); the main UFAs are ALA (57.87%), linoleic acid (15.69%) and oleic acid (16.54%). These results don, t in accord with Wang et al [7] who reported that the contents of above mentioned UFAs were 53.06%, 16.22% and 20.18% in pressing flaxseed oil, respectively. This may be due to different preparation methods of crude flaxseed oil. Although fatty acid compositions and total percentage of UFAs (all over 90.00%) varied slightly during overall refining(p 0.01), there were wide variations in the content of individual fatty acids such as ALA and linoleic acid. The dominant ALA, 57.87%, found in crude oil was decreased significantly to 51.80% during the overall refining process, especially bleaching and deodorizing stages (P 0.01). The possible reason was bleaching clay absorbed ALA and the removal of deodorizer distillate that usually contained more ALA. Another possible reason was that, ALA was very susceptible to chemical reactions, light and heating, so as to lead to rapid photo-oxidation, lipid oxidation and deterioration. However, this may only contribute a small part to the contents change of ALA. Analysis of the data also showed that the ratio of -3 to -6 fatty acids decreased in different refining samples because of the increasing of linoleic acid content and the decreasing of ALA content correspondingly. In a word, if the loss of fatty acids would be avoided during refining, the refining conditions should be modified to

126 2010, Vol. 31, No. 16 Table 2 Summary of fatty acid compositions, UFAs (%) and ratio of -3 to -6 fatty acids Fatty acid Crude oil Degummed oil Alkali-refined oil Bleached oil Deodorized oil Palmitic acid(c16:0) 4.52 0.14 a 4.83 0.06 b 4.93 0.20 a 4.67 0.07 a 4.48 0.09 a Palmitoleic acid (C16:1) 0.08 0.22 a 0.07 0.01 a 0.09 0.02 a 0.09 0.01 a 0.10 0.01 b Stearic acid (C18:0) 2.31 0.47 a 2.85 0.04 b 3.09 0.26 b 3.84 0.33 b 3.92 0.28 b Oleic acid (C18:1) 16.54 0.21 a 16.22 0.16 a 16.62 0.08 a 16.66 0.14 a 16.21 0.89 a Linoleic acid (C18:2) 15.69 0.23 a 17.45 0.05 b 17.17 0.31 b 18.31 0.30 c 19.77 0.09 d Linolenic acid (C18:3) 57.87 2.59 a 56.25 0.29 a 55.45 1.30 b 53.11 3.03 c 51.80 1.38 d Arachidic acid(c20:0) 0.15 0.02 a 0.15 0.01 a 0.17 0.03 b 0.16 0.04 a 0.16 0.01 a Eicosanoic acid (C20:1) 1.26 0.01 a 1.16 0.01 b 1.13 0.01 b 1.15 0.00 b 1.20 0.02 a Behenic acid (C22:0) 0.12 0.02 a 0.14 0.00 a 0.14 0.01 a 0.14 0.01 a 0.13 0.02 a Lignoceric acid (C22:1) 1.28 0.02 a 1.20 0.01 a 1.20 0.01 a 1.20 0.01 a 1.29 0.01 a UFAs 92.72 0.31 a 91.35 0.08 a 91.66 0.06 a 90.52 0.11 a 90.27 0.42 a -3: -6 fatty acids 3.69 0.14 a 3.22 0.03 b 3.23 0.01 b 2.91 0.02 c 2.62 0.10 d Note: Values with different superscript letters within a row are significantly different at P 0.01. % minimize loss of fatty acids, especially PUFAs with high nutritional value. 2.3 Effect of refining on Tocopherol Tocopherol(,,, ), which was physiologically active as vitamin E, was a major natural lipophilic antioxidants and was used for protection of oils from atmospheric oxidation. Increased tocopherols intake has been associated with lower risk of cardiovascular and coronary heart diseases and cancer [8]. Table 3 shows total and individual tocopherol contents and losses during different refining steps. It showed that crude oil was rich in tocopherol and the total tocopherol content was 57.59 mg/100 g oil. Tocopherol were three different forms: -, - and -tocopherol, tocopherol was dominant representing 91.86% of the total tocopherols. Flaxseed oil also contained -tocopherol (4.71%) and -tocopherol (3.44%) at relatively low concentration. The -tocopherol was not detected. According to the results (Table 3), the total tocopherols content was significantly reduced(p 0.01) until the end of the refining. The total tocopherols content, 57.59 mg/100 g, decreased to a value of 29.26 mg/100 g gradually, this corresponded to about 49.19% loss. This is because the volatilization and oxidation of tocopherol at the deodorization stage might affect the levels, in contrast to the others stages. In addition, tocopherol was adsorbed and oxidized by the bleaching clay in the bleaching stage, which also promote general oxidation process of tocopherol. In a word, optimal conditions were important to maintain constant tocopherol contents. For individual tocopherol, there were higher total losses in -tocopherol contents when compared with that of and -tocopherol. There were considerable losses in -, and -tocopherols levels during overall refining stages, which were about 68.36%, 47.94% and 51.01%, respectively. Moreover, - and -tocopherols had better antioxidant activities than -tocopherol at high temperatures (50 100 ), -tocopherols was chemically and biologically the most active and the most effective at preventing oxidation at below 50 [9]. In addition, -tocopherol was able to improve some established quality criteria and inhibit the formation of oxidation products. However, the important natural antioxidant was drastically decreased and markedly reduced during bleaching, the relative proportion of -tocopherol in crude oil, 4.70%, decreased significantly to a value of 2.90% after deodorizing stage. These changes must be taken into account the refining process and the refining parameters should be carefully evaluated to reduce the loss of -tocopherol. Table 3 Total and individual tocopherol contents after different Oil sample Note: Values in each column with different letters are significantly different (P 0.01); ND. not detected. refining process steps Tocopherol/(mg/100 g of oil) -T Loss(%) -T -T Loss(%) -T Loss(%) Total Loss(%) Crude oil 2.71 a N D 52.90 a 1.98 a 57.59 a Degummed oil 2.23 b 17.71 N D 53.50 a 1.23 b 38.88 56.96 a 1.09 Alkali-refined oil 2.13 b 4.48 N D 52.70 a 1.49 1.04 b 15.44 55.87 a 1.91 Bleached oil 1.10 c 48.36 N D 48.40 b 8.16 1.01 b 2.88 52.56 b 5.92 Deodorized oil 0.85 d 22.73 N D 27.54 c 43.10 0.87 c 13.86 29.26 c 44.33 Total 68.63 47.94 51.01 49.19 3 Conclusions The typical method of refining oil inv olves degumming, neutralizing, bleaching and deodorization,

2010, Vol. 31, No. 16 127 which had profound effects on physicochemical properties, tocopherol retention and fatty acid compositions. The phospholipids content and AV were reduced obviously after overall refining stages, but refining process had little effect on IV, refractive index, specific gravity and SV. Although fatty acid compositions and total percentage of UFAs varied slightly during overall refining, -linolenic acid was decreased significantly during overall refining process. It was necessary to take into account the fact that a significant amount of tocopherol in crude oil was removed or destroyed drastically during the refining process. Moreover, the optimal processing conditions should be applied in order to retain both the nutritive value and the oxidative stability. In a word, the characteristics of flaxseed oil in terms of physicochemical properties, its fatty acid profile and tocopherol contents play significant roles in the quality of the oil. Hence monitoring changes in these parameters during refining is important as they define the quality and functionality. Reference [1] CHOO W S, BIRCH E J, DUFOUR J P. Physicochemical and quality characteristics of cold-pressed flaxseed oils[j]. Journal of Food Composition and Analysis, 2007, 20(3/4): 202-211. [2] CHOO W S, BIRCH E J, DUFOUR J P. Physicochemical and stability characteristics of flaxseed oils during pan-heating[j]. Amer Oil Chem Soc, 2007, 84(8): 735-740. [3] ARANZAZU G M, VICTORIA R M, ROMERO C P, et al. Effect of refining on the phenolic composition of crude olive oils[j]. Amer Oil Chem Soc, 2006, 83(2): 159-164. [4] TONG W, LAWRENCE A J. Refining high-free fatty acid wheat germ oil[j]. Amer Oil Chem Soc, 2001, 78(1): 71-76. [5] RUIZ-MNDEZ M V, MRQUEZ-RUIZ G, DOBARGANES M C. Comparative performance of steam and nitrogen as stripping gas in physical refining of edible oils[j]. Amer Oil Chem Soc, 1996, 73(12): 1641-1645. [6] WANG Xi, WANG Tong, JOHNSON L A. Composition and sensory qualities of minimum-refined soybean oils[j]. Amer Oil Chem Soc, 2002, 79(12): 1207-1214. [7] WANG Changqing, REN Haiwei, ZHANG Guohua. Change of fatty acid and VE in the refining process of flaxseed oil[j]. China Oils and Fats, 2008, 33(3):14-16. [8] MURAT T, MEHMET D. Total and individual tocopherol contents of sunflower oil at different steps of refining[j]. Eur Food Res Technol, 2005, 220(3/4): 251-254. [9] WU Shimin, WU Moucheng, ZHANG Qiaozhong. Changes of tocopherol during the refining process of rapeseed oil[j]. Acta Nutrimenta Sinica, 2003, 25(4): 418-420.