F. Temelli, K. Stobbe, K. Rezaei, and T. Vasanthan
Samples of whole grain and 35% pearling flour of 20 different barley varieties grown in Alberta were analyzed for their lipid contents. Total lipid contents of whole grains were within 1.9% to 3.0% (w/w), whereas those of the 35% pearling flour were 4.3% to 7.9%. Lipids of 35% pearling flour fraction of Tercel barley were extracted using supercritical carbon dioxide (SC-CO2 ) at different pressures (24, 45, and 58 MPa) and temperatures (40 and 60 ◦ C) for 3 h. Lipid recoveries of 73% to 97% were achieved using SC-CO2 extraction under different operational conditions. Tocol contents and compositions of whole grain, 35% pearling flour, and SC-CO2 extracts were analyzed using HPLC. Tocol content of the whole grain was 53.8 to 124.9 μg/g and that of the pearling flour was 195 to 363 μg/g of flour. The hulless barley varieties were higher in tocols, with waxy, double waxy and Tercel varieties having the highest levels (P < 0.05). The ratios of total tocotrienols to total tocopherols varied within 1.6 to 3.9 range. Tocol concentrations of SC-CO2 extract fractions varied from 1171 to 4391 μg/g extract depending on the operational conditions. Barley oil is a good natural source of different tocol isomers rich in tocotrienols.
Abstract:
Keywords: barley, lipid, pearling, supercritical carbon dioxide, tocopherol, tocotrienol
Barley pearling flour can be a good source of a natural mixture of the family of vitamin E components, tocopherols and tocotrienols, which have been shown to have health benefits. Hulless barley varieties have higher lipid contents and are richer in tocols. Supercritical CO2 can be used to extract barley lipids.
Practical Application:
Introduction Tocopherols and tocotrienols (cumulatively referred to as tocols) are the family of vitamin E compounds found naturally in the plant tissues (Barnes 1983). They have been associated with numerous health benefits. More specifically, α-tocopherol has been found to reduce the risk of ischemic heart disease and cataracts, and also enhance immune system functionality (Gaby and Machlin 1991). Tocotrienols have antioxidant activity and reduce serum cholesterol levels in chicken, swine, and humans (Qureshi and others 1986, 1997). Chemical structures of tocols consist of a chroman ring and a long side chain, which is a saturated phytyl chain in the case of tocopherols but it is unsaturated in tocotrienols with 3 double bonds. As shown in Figure 1 (adopted from Christie 2011), each of the tocopherols and tocotrienols has 4 possible isomers as α, β, γ , and δ differing in the number and position of methyl substituents on the benzene ring (Ruperez and others 2001; Nielsen and Hansen 2008). Cereal grains and oilseeds are rich sources of tocols and are major sources of vitamin E (Wang and others 1993; Colombo and others 1998). Barley is unique since it contains a high concentration of tocols and the distribution of the biologically active isomers in barley is favorable (Moreau and others 2007a, 2007b). Barley grains are rich in tocotrienols; α-tocotrienol having the highest MS 20130010 Submitted 1/2/2013, Accepted 8/28/2013. Authors are with Dept. of Agricultural, Food and Nutritional Science, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2P5. Author Rezaei is also with the Dept. of Food Science, Engineering and Technology, Univ. of Tehran, Iran. Direct inquiries to author Temelli (E-mail: [email protected]).
R C 2013 Institute of Food Technologists
doi: 10.1111/1750-3841.12271 Further reproduction without permission is prohibited
concentration followed by α-tocopherol, γ -tocotrienol, and γ tocopherol (Barnes 1983; Moreau and others 2007a). Canada is the 2nd largest barley producer in the world (according to Statistics Canada, 8 million tons harvested in 2012) with the province of Alberta producing about half of Canada’s total annual crop. Different barley varieties are grown in Alberta for feed, malt, and food purposes. Up to 80% of the crop is utilized as feed for livestock. Recently, barley is also being considered as a feedstock for bioproducts, such as bioethanol production. Considering the nutritional and certain technological points of view, it is important to have a concise set of compositional data to maximize the utilization of different barley varieties. Lampi and others (2004) reported the lipid and phytosterol contents of hulless barley and rye cultivars. Moreau and others (2007a, 2007b) investigated the tocol composition of barley grains and fractions obtained by scarifying. More recently, Moreau and others (2012) showed that there were no significant differences in the levels of various components, including tocols, except for phytate for the 5 low- and 5 normal-phytate barley cultivars grown in 3 Idaho locations. In the current study, the tocol contents and tocol compositions of 20 different varieties grown in Alberta were investigated. Tocols are lipid-soluble and therefore can be extracted together with lipids. Lipids are concentrated in the outer layers and the germ of the barley kernel. Even though the lipid content of the whole barley grain is in the range of 1.8% to 3.0% that of the pearling flour can be as high as 12% depending on the barley variety and the level of pearling (Liu and Moreau 2008; Liu 2011). Therefore, the pearling flour removed during the pearling operation is a suitable starting material for lipid extraction.
Vol. 78, Nr. 11, 2013 r Journal of Food Science C1643
C: Food Chemistry
Tocol Composition and Supercritical Carbon Dioxide Extraction of Lipids from Barley Pearling Flour
Tocols of barley pearling flour . . .
C: Food Chemistry
There is a growing interest in the use of supercritical CO2 (SCCO2 ) for the extraction of natural biologically active components from plant and animal sources (Mitra and others 2011; Prado and others 2012; Domingues and others 2013). Carbon dioxide at pressure and temperature conditions above its critical point (31 ◦ C and 7.4 MPa) becomes a supercritical fluid and has a solvent power similar to those of organic solvents. There is no solvent residue left in the final product since CO2 is released as a gas after extraction. In CO2 environment, oxidation reactions are avoided. As well, extractions can be carried out at temperatures just above the ambient temperature minimizing the degradation of many heat-labile components. Despite the great potential for SC-CO2 extraction of barley lipids, including tocols, no studies have been reported to evaluate such possibilities. Therefore, the objectives of this study were: (1) to determine the lipid and tocol contents and tocol compositions of the whole grain and 35% pearling flour of 20 barley varieties grown in Alberta; (2) to extract lipids from barley pearling flour using SC-CO2 at different pressure and temperature conditions and to analyze the tocol content and composition of the barley oil extracted using SC-CO2 . Pearling level of 35% was selected to evaluate the compositions of 20 barley varieties to simulate the industrial practice employed for pearled barley production. In addition, 19% pearling flour of CDC Candle was also used for SC-CO2 extraction since in a parallel study (Lekhi 2004) it was shown that 19% pearling maximized β-glucan retention in the pearled grain while maximizing the lipid content in the pearling flour.
level of pearling (35%) was achieved. The pearling flour was separated from the pearled grain by screening (Burrows grain dockage slotted sieve, size 3/64 and 5/16) and weighed. The amount of pearling flour removed as a percentage of the starting whole grain weight is reported as degree of pearling. All samples were stored for a maximum of about 2 mo at –18 ◦ C until analysis. Pearling flour of CDC Candle barley at 19% pearling level was obtained at pilot scale according to Lekhi (2004). Pilot scale pearling of grains (1.0 to 1.5 kg), previously cleaned and passed through a grain sizer to obtain grains of 2.34 to 2.73 mm in size, was performed using a Westrup dehuller (Model 970218, Westrup Co., Sorovej, Denmark) at 3900 rpm. Then, pearling flour was separated using a SWECO Vibro-energy separator (Model 6033 E, SWECO Inc., Toronto, ON, Canada) equipped with a 1.76mm-size screen and weighed. Pearling percentage was reported as the weight of pearling flour over the total grain weight.
Solvent extraction of lipids Solvent extraction of lipids was performed according to the AACC method 30-25 (AACC 1982), using the Goldfisch extraction unit (Labconco, Kansas City, Mich., U.S.A.). Lipids were extracted from approximately 2.0 g of samples using petroleum ether (40 mL) for 6 h and collected in a preweighed Goldfisch extraction beaker. The solvent was then evaporated and the remaining lipid was weighed. All extractions were performed in duplicate to determine the lipid contents of the whole grain and 35% pearling flour fractions of the 20 barley varieties as well as the 19% pearling flour of CDC Candle.
Materials and Methods Samples of whole grain and 35% pearling flour of 20 different barley varieties (part of another trial) were provided by Dr. J. Helm, Field Crop Development Center, Alberta Agriculture and Rural Development, Lacombe, AB, Canada. Barley grains were first tempered in a controlled humidity chamber at 7 to 12 ◦ C to adjust the moisture content at 13% to 15% to minimize breakage during pearling. Grains were then pearled using a bench top pearler (Seedburo Equipment Co., Des Plaines, Ill., U.S.A.), following a protocol similar to that of Panfili and others (2008). The pearler was modified with a 0.5 hp DC variable motor to adjust speed, which was set at level 70, corresponding to approximately 1400 to 1500 rpm. A batch of 100 g barley sample was used for each run and pearling was continued until the targeted
SC-CO2 extraction of lipids Supercritical fluid extraction of lipids from barley pearling flour was performed using a laboratory scale system (Newport Scientific Inc., Jessup, Md., U.S.A.) described previously (Bozan and Temelli 2002). The feed material was placed in a sample basket (25 cm × 27 mm I.D.) and mixed with glass beads (3 mm in diameter) to improve the extraction process. Glass wool was inserted at both ends to hold the sample, and the basket was placed into the 300 mL extraction chamber. An o-ring placed between the basket and the chamber ensured CO2 flow through the sample inside the basket. Extraction temperatures were maintained within ±2 ◦ C using a thermocouple placed inside the extraction cell, a temperature controller and a heating jacket around the cell. A back-pressure
A α-tocopherol β-tocopherol δ-tocopherol γ-tocopherol
B α-tocotrienol β-tocotrienol δ-tocotrienol γ-tocotrienol
Figure 1–Chemical structures of tocopherols (A) and tocotrienols (B) (adopted from Christie 2011).
C1644 Journal of Food Science r Vol. 78, Nr. 11, 2013
R’ CH3 CH3 H H
R” CH3 H H CH 3
R’ CH3 CH3 H H
R” CH3 H H CH3
regulator was used to maintain the desired pressure. A 35% pearling flour of Tercel barley (5.0 g) was used as the feed material and the extraction was carried out at different pressure (24, 45, and 58 MPa) and temperature (40 and 60 ◦ C) conditions. Extractions were continued for 3 h with intermittent collection of the extract fractions. CO2 flow rate was maintained at 0.5 L/min (measured at ambient conditions). After passing through a rotameter and gas meter, CO2 was vented since the lab unit did not have any recycle capability. SC-CO2 extraction was also performed using 19% pearling flour of CDC Candle at 60 ◦ C and 55 MPa. All extractions were performed in duplicate and lipid extracts were collected in glass tubes sitting in a refrigerated bath (–10 ◦ C) upon depressurization of SC-CO2 . The extract tubes were equilibrated at ambient conditions for about 20 min prior to gravimetric quantification. Cumulative amounts of extracts were reported as a function of time. For analysis purposes, the tubes containing the extracts were washed with hexane to ensure quantitative transfer of the extracts into glass vials. Hexane was then evaporated under a gentle flow of nitrogen and the samples were stored at –18 ◦ C until analysis. Prior to the high-performance liquid chromatography (HPLC) analysis of samples, each extract was redissolved in 1 mL hexane for the determination of tocol content and composition.
HPLC analysis of tocols Determination of the tocol contents and compositions of the whole grain, 35% pearling flours and SC-CO2 extracts was performed using the HPLC procedure described by Peterson and Qureshi (1993). Prior to the HPLC analysis, lipids containing the tocols were extracted using methanol (Peterson and Qureshi 1993). All samples were prepared in triplicate. HPLC-grade methanol (3.0 mL) was added to the flour samples (0.4 g), followed by gentle shaking of the samples for 15 min and centrifugation at 3000 rpm for 15 min. An aliquot (1.0 mL) of the supernatant was taken and its solvent was evaporated. The solid precipitate was re-extracted using additional 3.0 mL methanol following the same procedure. One milliliter aliquot of the supernatant from this stage was combined with the lipid sample from the 1st extraction step and again the solvent was evaporated. Samples were stored at –18 ◦ C until the HPLC analysis, when the samples were redissolved in HPLC-grade hexane (200 μL), stirred and centrifuged for 1 min. The supernatant was then transferred into an HPLC vial for tocol analysis. The HPLC system was a Varian 9010 unit (Walnut Creek, Calif., U.S.A.) equipped with a Supelco LC-Diol column (25 cm × 4.6 cm × 5 μm, Supelco Inc., Bellefonte, Pa., U.S.A.) and a Shimadzu RF535 fluorescence detector (Shimadzu Scientific Instruments Inc., Columbia, Md., U.S.A.). A Hewlett Packard 1050 autosampler with a 25-μL sample loop was used for sample injection. The conditions reported by Peterson and Qureshi (1993) were employed. The mobile phase consisted of 0.6% (v/v) iso-propanol in hexane (both HPLC grade from Fisher Scientific, Fair Lawn, N.J., U.S.A.) flowing at 1.0 mL/min. The detector was set at 298 nm for the excitation and 330 nm for the emission. Total run time for each sample was 30 min. Standard solutions containing all 8 tocol isomers were also prepared at 5 different concentration levels and were run at the beginning, in the middle and at the end of each sample set. The standards, α-, δ-, and γ -tocopherols were obtained from Sigma-Aldrich Inc. (St. Louis, Mo., U.S.A.), β-tocopherol from Chromatographic Specialties Inc. (Brockville, ON, Canada), and α-, β-, δ-, and γ -tocotrienols from CalBiochem (Darmstadt, Germany). These standards were used for peak identification and
quantification of sample components according to the external standard calibration method. The peaks for all 8 tocol isomers were completely separated with retention times of 7.8, 8.7, 15.0, 16.2, 17.6, 18.9, 23.1, and 27.5 min for α-tocopherol, α-tocotrienol, β- and γ -tocopherol, β- and γ -tocotrienol, δ-tocopherol and δtocotrienol, respectively. All data were collected and handled by a Shimadzu Class-VP Chromatography automated software system.
Statistical analysis Analysis of variance of the results was performed using General Linear Model procedure of SAS Statistical Software, Version 8 (SAS Inst. Inc., Cary, N.C., U.S.A.). Multiple comparison of means was performed by least significant difference test at α = 0.05 level.
Results and Discussion Lipid contents and tocol compositions of barley varieties Total lipid contents of whole grain and 35% pearling flour of 20 different barley varieties used in this study are presented in Table 1. The lipid content of whole grains varied from 1.8% to 3.0% (w/w), whereas that of 35% pearling flour was in the range 4.3% to 7.9%. The variety HB340 (CDC Alamo), CDC Candle, and HB 335 (CDC McGwire) had the highest lipid contents in the whole grain whereas HB340 (CDC Alamo) was the highest in the 35% pearling flour. This finding is in agreement with the results of Moreau and others (2012) who also found the lipid content of CDC Alamo to be higher than that of the other varieties they studied. The lipid content of the 35% pearling flour was substantially lower than that observed in a separate study conducted in our lab focusing on different levels of pearling (unpublished data), where a lipid content of 11.7% was obtained for 15% pearling flour of a different hulless double waxy barley variety (SB94794). This demonstrates the impact of pearling level and the dilution of lipids with starch at higher levels of pearling as more of the starch-rich endosperm is removed together with the outer bran layers. Such a dilution effect was also reported by Moreau and others (2007b) where the oil content of pearling flour obtained from hulless barley after 60 s of scarification was lower than that obtained after 20 s. Of the total lipids present in the whole grain, on average, 90.9% was present in the 35% pearling flour of the hulless varieties, whereas it was 80.6% for the hulled varieties, again indicating the dilution effect of the hulls (Table 1). In general, the lipid contents of hulless varieties were higher especially in the 35% pearling flour fraction (5.2% to 7.9% for hulless compared with 4.3% to 5.0% for hulled varieties, Table 1). Similarly, the oil contents of the scarification fines obtained from hulless varieties by Moreau and others (2007a) were higher than that of hulled varieties. Tocol contents and compositions of the whole grain and 35% pearling flour of the 20 barley varieties are presented in Tables 2 and 3, respectively. In the whole grain, total tocols ranged from 53.8 to 124.9 μg/g of flour (Table 2). These levels are similar to those reported by Peterson and Qureshi (1993) (42 to 80 μg/g) and by Moreau and others (2007a) (84.7 to 151.1 mg/kg) for other barley varieties. Following the general trends in the total lipid content, the hulless varieties exhibited higher levels of total tocols, with the waxy (CDC Candle), double waxy (HB340 – CDC Alamo) and Tercel barley varieties having the highest levels (P < 0.05). In terms of the individual tocol isomers in the whole grain, α-tocotrienol was the most abundant one making up 38.6% to 50.1% (w/w) of the total tocols, followed by α-tocopherol (17.7% to 33.9%), γ -tocotrienol (10.4% to 20.2%), γ -tocopherol (1.9% to 9.2%), Vol. 78, Nr. 11, 2013 r Journal of Food Science C1645
C: Food Chemistry
Tocols of barley pearling flour . . .
Tocols of barley pearling flour . . . Table 1–Total lipid contents (%, w/w, as is basis) of whole grain and 35% pearling flour of 20 barley varieties determined by solvent extraction using petroleum ether (n = 2). Lipid content (%, w/w) Variety
C: Food Chemistry
Whole grain
35% pearling flour
% of lipids from whole grain present in the pearling flour
2-row malting 2-row malting 2-row malting 6-row malting 2-row feed 2-row feed 2-row feed 2-row malting 2-row malting 2-row malting 2-row malting
2.0cde 1.9ef 2.5b 1.9def 1.8f 2.2cd 2.1cde 2.1cde 2.0def 2.1cde 2.0cde 2.0 ± 0.2
4.8gh 4.6ij 4.9g 4.7hi 4.3k 4.4jk 4.7hi 4.4k 4.7hi 5.0g 4.7hi 4.7 ± 0.2
84.1 84.8 68.7 86.6 83.7 71.6 78.4 81.1 82.3 83.4 82.3 80.6 ± 5.6
2-row waxy 2-row 6-row 2-row 2-row double waxy 6-row 6-row 2-row 2-row
2.9a 2.2cd 2.2c 2.6a 3.0a 2.2c 2.3c 2.2cd 2.1cde 2.4 ± 0.4
6.7b 6.2c 5.4e 6.1c 7.9a 5.8d 5.6d 6.1c 5.2f 6.1 ± 0.8
80.9 98.7 82.2 82.2 92.2 92.3 95.1 98.7 95.9 90.9 ± 7.2
Type
Hulled AC Axbow AC Metcalf B-1215 B-1602 CDC Dolly CDC Fleet CDC Guardian CDC Kendall CDC Stratus Harrington Manley Average (mean ± std. dev.) Hulless CDC Candle CDC Dawn Falcon HB-335∗ HB-340∗ Jaeger Peregrine Phoenix Tercel Average (mean ± std. dev.) ∗ HB335 a–k
is CDC McGwire and HB340 is CDC Alamo. In each column, means identified with the same letter are not significantly different (P > 0.05).
Table 2–Tocol contents and tocol compositions of the whole grain of the 20 barley varieties investigated (n = 3). Tocols extracted with methanol according to Peterson and Qureshi (1993) prior to HPLC analysis. Sample
α -T
α - T3
β -T
γ -T
Hulled AC Axbow AC Metcalf B-1215 B-1602 CDC Dolly CDC Fleet CDC Guardian CDC Kendall CDC Stratus Harrington Manley Average (mean ± std. dev.)
18.4 20.3 17.6 21.0 15.2 20.0 19.6 15.4 20.3 21.0 19.5
39.9 44.1 45.2 43.1 38.1 40.5 39.3 40.4 44.9 44.2 43.1
1.4 1.6 1.1 1.3 1.2 1.0 1.3 0.8 0.9 0.7 0.9
3.4 2.5 3.7 4.0 4.5 3.3 7.9 3.2 4.1 4.2 4.2
31.9 10.6 32.5 21.0 32.3 29.4 34.0 23.2 26.3
49.9 26.9 44.6 46.7 51.0 45.6 46.5 44.7 46.3
1.9 0.6 1.2 1.1 2.1 1.5 1.4 1.1 1.8
7.0 2.8 2.0 5.8 8.8 4.1 1.9 4.0 9.9
β - T3 (μg/g flour)
γ -T3
δ-T
δ - T3
5.4 6.7 7.7 4.2 4.2 4.2 2.6 4.8 4.7 5.2 4.3
9.4 12.4 20.1 13.9 11.8 12.3 12.5 13.8 14.5 17.7 12.1
0.6 0.8 0.7 1.0 0.9 0.6 1.4 0.6 0.8 0.6 0.7
1.3 1.4 3.2 1.6 1.2 1.2 1.2 1.9 2.0 2.4 1.5
79.7 89.8 99.2 90.0 77.1 83.1 85.8 81.0 92.1 96.0 86.4 87.3
± ± ± ± ± ± ± ± ± ± ± ±
4.9gh 1.6gdfceh 5.2dce 0.1gdfceh 11.4h 0.6gfh 1.7gfeh 2.7gfh 2.5gdfceh 0.6dfce 4.7gdfce 6.9
70.2 72.0 76.7 69.7 71.7 70.1 64.9 75.2 71.7 72.4 70.7 71.4 ± 3.0
4.5 3.3 3.6 7.7 6.0 3.4 2.8 4.7 9.0
19.2 7.9 10.0 15.9 20.4 16.2 11.7 13.2 20.6
1.3 0.5 0.6 1.0 1.7 0.7 0.5 0.9 1.8
1.5 1.3 1.3 2.2 2.5 1.9 0.8 1.6 4.4
117.3 53.8 95.7 101.5 124.9 102.8 99.5 93.3 120.1 101.0
± ± ± ± ± ± ± ± ± ±
2.2ab 13.8i 0.2dfce 2.6dc 1.5a 1.4bc 0.4dce 0.9gdfce 7.5a 21.0
64.1 73.1 62.2 71.5 64.0 65.2 62.1 68.8 66.9 66.4 ± 4.0
Total tocols
% T3
Hulless CDC Candle CDC Dawn Falcon HB335 HB340 Jaeger Peregrine Phoenix Tercel Average (Mean ± std. dev.) ∗ T a–i
denotes tocopherols and T3 denotes tocotrienols. Means with the same letter are not significantly different (P > 0.05).
C1646 Journal of Food Science r Vol. 78, Nr. 11, 2013
Tocols of barley pearling flour . . . Table 3–Tocol contents and tocol compositions of 35% pearling flour of the 20 barley varieties investigated (n = 3). Tocols extracted with methanol according to Peterson and Qureshi (1993) prior to HPLC analysis. Sample
α -T
α - T3
β -T
γ -T
β - T3
γ -T3
δ-T
δ - T3
Total tocols∗
% T3
Hulled AC Axbow AC Metcalf B-1215 B-1602 CDC Dolly CDC Fleet CDC Guardian CDC Kendall CDC Stratus Harrington Manley Average (mean ± std. dev.) Hulless CDC Candle CDC Dawn Falcon HB335 HB340 Jaeger Peregrine Phoenix Tercel Average (mean ± std. dev.) ∗
37.8 28.5 44.3 41.8 51.0 39.2 35.3 30.1 41.4 39.3 32.1
118 107 147 133 139 123 107 118 124 120 107
12.4 8.7 12.5 10.6 16.7 8.5 8.4 6.1 6.1 5.4 5.9
8.0 5.8 11.0 12.0 25.5 9.1 17.5 6.5 10.5 10.2 9.4
17.4 16.1 30.6 13.9 23.7 14.6 7.6 15.0 14.4 14.8 11.0
27.1 26.2 69.5 40.7 58.7 36.1 38.7 36.8 41.1 45.4 28.3
2.0 0.3 2.3 2.3 2.4 2.0 2.9 1.9 0.7 0.5 0.6
3.6 2.0 9.6 4.6 4.2 3.7 3.0 4.7 4.3 4.3 2.4
226 195 336 250 321 236 211 219 243 240 197 243.1
± ± ± ± ± ± ± ± ± ± ± ±
3ih 4k 8b 1feg 6bcd 4hg 2ij 2i 9fg 11hg 5kj 46.0
73.4 77.7 76.5 77.0 70.2 75.1 73.7 79.6 75.8 77.0 75.5 75.6 ± 2.5
45.5 52.2 53.1 33.8 54.3 53.2 87.6 54.0 63.1
133 142 114 130 145 130 147 133 139
12.0 15.1 9.1 7.0 14.6 11.6 15.0 14.5 17.7
16.2 22.4 5.7 12.1 24.6 7.9 7.6 24.4 31.0
10.9 27.8 10.6 21.2 16.7 9.9 11.9 18.7 32.0
41.3 57.1 25.5 38.3 51.0 38.7 43.2 51.5 66.7
1.2 3.3 0.3 2.2 2.1 1.9 0.3 3.3 3.0
2.2 8.6 2.4 4.9 4.8 4.4 2.0 6.7 11.0
263 328 220 263 313 258 315 306 363 292.1
± ± ± ± ± ± ± ± ± ±
4e 9cb 6i 6e 4cd 6fe 10cd 4d 9a 44.1
69.4 68.3 64.9 69.1 69.4 71.5 71.0 73.9 68.6 69.6 ± 2.5
T denotes tocopherols and T3 denotes tocotrienols. Means with the same letter are not significantly different (P > 0.05).
a–k
and β-tocotrienol (2.9% to 7.8%). β-Tocopherol, δ-tocopherol, and δ-tocotrienol made up 2.7% to 6.7% of total tocols. The ratio of tocotrienols to tocopherols ranged from 1.6 to 3.3. On average, tocotrienols comprised 71.4 ± 3.0% and 66.4 ± 4.0% of the total tocols in the hulled and hulless varieties, respectively (Table 2). Similarly, Moreau and others (2007a) reported that tocotrienols made up 70.6% to 75.7% of the total tocols in the whole kernels of the 2 hulled and 2 hulless barley varieties they investigated. Such a distribution of tocol isomers is quite favorable since αand γ -tocotrienol are especially receiving increasing attention in terms of their health benefits (Papas 1999). In the 35% pearling flours, tocol contents and compositions followed similar trends to those observed in the whole grain with hulless varieties having higher levels of tocols. Total tocol content in the 35% pearling flours varied from 195 to 363 μg/g of pearling flour (Table 3) with Tercel variety having the highest level (P < 0.05). Moreau and others (2007a) reported that the tocol content of the scarifying fines obtained after 60 s was 153.4 to 448.8 mg/kg. This extent of scarifying corresponded to 11.6% to 15.2% pearling by weight, which was lower than the 35% pearling used in the current study, where the tocols in the pearling flour were diluted by other components. α-Tocotrienol in the 35% pearling flours was at somewhat higher levels than that in the whole grain, making up 38.1% to 55.0% (w/w) of the total tocols. This was followed by α-tocopherol (13.2% to 27.8%), γ -tocotrienol (11.6% to 20.7%), γ -tocopherol (2.4% to 8.5%), and β-tocotrienol (3.6% to 9.1%). The relative proportion of β-tocopherol (2.3% to 5.5%) was higher than that in the whole grain. δ-Tocopherol and δtocotrienol together contributed to 0.05).
C1648 Journal of Food Science r Vol. 78, Nr. 11, 2013
However, an increase in pressure also results in a decrease in the diffusivity of the solutes in SC-CO2 , which has a negative impact on the mass transfer kinetics. On the other hand, an increase in temperature causes a reduction in the CO2 density but enhances the vapor pressure of the solutes as well as their diffusivity in the supercritical medium (Rezaei and Temelli 2000). The extent of these changes is pressure dependent, leading to the well-known cross-over behavior of solubility isotherms (Guclu-Ustundag and Temelli 2000). Above the cross-over pressure, the oil solubility in SC-CO2 increases with temperature. The extraction yield can be increased further with an increase in solvent-to-feed ratio or the CO2 flow rate, which was kept at a minimum level in this study to maximize contact between the feed material and CO2 . Tocol concentration of each extracted oil fraction obtained over different time intervals was analyzed using HPLC and the data are presented in Figure 3. Total tocol concentration varied from 1171 to 4391 μg/g extract. It seems that at 40 ◦ C tocols were extracted to a greater extent in the 1st 20 min of extraction but then the tocol concentration in the extracts decreased. However, this was not the case consistently at all the pressures tested at 60 ◦ C, since the later fractions seemed to get more concentrated in tocols at 58 MPa. With the SC-CO2 extraction of lipids, the tocol concentration increased from 120.1 μg/g of whole grain flour of Tercel barley to 1171 to 4391 μg/g extract, resulting in 10- to 37-fold enrichment of tocols in these extracted fractions. SC-CO2 extraction of barley lipids was also carried out using the 19% pearling flour of CDC Candle. CDC Candle was selected for this purpose because of its high β-glucan content and our parallel initiatives on the concentration of this health benefiting soluble fiber component (Vasanthan and Temelli 2009). Pearling at a level of 19% instead of 35% exhibited greater β-glucan retention in the pearled grain, which could be fractionated further (Lekhi 2004). Total lipid content of the 19% pearling flour of CDC Candle was 8.7%. After 3 h of extraction using SC-CO2 at 55 MPa and 60 ◦ C, the recovery of oil was 84%. Total tocol content of the oil extracted using SC-CO2 was 1565 μg/g oil for CDC Candle resulting in a 13-fold enrichment, considering the 117.3 μg/g tocols in the whole grain (Table 2). For the development of a potential biorefinery based on the full utilization of barley grains, pearling can be used as the 1st unit operation for dry separation of the grain tissues, where the outer layers including the bran and germ would be concentrated in the pearling flour and endosperm would be left as the pearled grain, enriched in starch and β-glucan. As demonstrated in this study, pearling flour can be a good viable starting material for the recovery of barley oil, rich in tocols with a natural mixture of all eight tocopherol and tocotrienol isomers and a favorable ratio of tocotrienols to tocopherols. Similarly, SC-CO2 extraction is a viable alternative to the traditional hexane extraction of oil, especially for oils rich in such bioactive components as tocols. With the latest developments in supercritical technology (Temelli 2009), SC-CO2 extraction has become mainstream, offering numerous advantages over hexane extraction at a time when hexane use is facing greater safety concerns. It has also been demonstrated that the process economics can become comparable to that of the conventional processes when the capacity of supercritical plants is increased to an appropriate level using optimized process design (Perrut 2000).
Conclusions Lipid contents of the 35% pearling flour of 20 different barley varieties grown in Alberta were greater than those of the whole
grain flour with the hulless varieties exhibiting higher levels of lipids than the hulled varieties. This demonstrates that the lipids are mainly concentrated in the outer layers of the barley grain and further optimization of pearling level could result in pearling flour containing high levels of lipids. Tocol content also followed a similar pattern whereby tocols were concentrated in higher amounts in the pearling flour compared to the whole grain. Hulless varieties also contained higher levels of tocols in the pearling flour than did the hulled varieties. The analysis of tocol composition indicated that α-tocotrienol was the most abundant isomer followed by α-tocopherol and γ -tocotrienol as the most favorable isomers for achieving health benefits. SC-CO2 extraction of the 35% pearling flour at the various pressure and temperature conditions of this study indicated that the most favorable condition to yield the highest amount of extract was 45 MPa and 60 ◦ C. Barley pearling flour can be an appropriate starting material for the recovery of lipids that are rich in tocols with a favorable distribution of tocopherols and tocotrienols.
Acknowledgments We are grateful to the Alberta Barley Commission and the Alberta Agricultural Research Inst. for financial support of this project and Dr. James Helm, Alberta Agriculture and Rural Development for providing the barley samples.
References AACC. 1982. Approved methods of the American Association of Cereal Chemists. Method 30–25: crude fat in wheat and soy flour, feeds and cooked feeds. St. Paul, Minn.: AACC. Barnes PJ. 1983. Non-saponifiable lipids in cereals. In: Barnes PJ, editor. Lipids in cereal technology. New York: Academic Press. p 33–55. Bozan B, Temelli F. 2002. Supercritical CO2 extraction of flaxseed. J Am Oil Chem Soc 79:231–5. Christie WW. 2011. Tocopherols and tocotrienols – structure, composition, biology and analysis. In: Harwood JL, Weselake RJ, editors. Lipid chemistry, biology, technology & analysis. American Chemical Society. Available from: http://lipidlibrary.aocs.org/. Accessed on April 5, 2013. Colombo ML, Corsini A, Mossa A, Sala L, Stanca M. 1998. Supercritical carbon dioxide extraction, fluorimetric and electrochemical high performance liquid chromatrographic detection of vitamin E from Hordeum vulgare L. Phytochem Anal 9(4):192–5. Domingues RMA, de Melo MMR, Oliveira ELG, Neto CP, Silvestre AJD, Silva CM. 2013. Optimization of the supercritical fluid extraction of triterpenic acids from Eucalyptus globulus bark using experimental design. J Supercrit Fluids 74:105–14. Gaby WK, Machlin LJ. 1991. Vitamin E. In: Gaby SK, Bendich A, Singh V, Machlin L, editors. Vitamin intake and health: a scientific review. New York: Marcel Dekker. p 71–101. Guclu-Ustundag O, Temelli F. 2000. Correlating the solubility behavior of fatty acids, mono-, di-, and triglycerides, and fatty acid esters in supercritical carbon dioxide. Ind Eng Chem Res 39(12):4756–66. Joki´c S, Nagy B, Zekovi´c Z, Vidovi´c S, Bili´c M, Veli´c D, Sim´andi B. 2012. Effects of supercritical CO2 extraction parameters on soybean oil yield. Food Bioprod Process 90(4):693–9. Lampi AM, Moreau RA, Piironen V, Hicks KB. 2004. Pearling barley and rye to produce phytosterol-rich fractions. Lipids 39:783–7. Lekhi AK. 2004. Extraction and functional properties of barley proteins [M.Sc. thesis]. Edmonton, AB, Canada: Univ. of Alberta. Liu KS. 2011. Comparison of lipid content and fatty acid composition and their distribution within seeds of 5 small grain species. J Food Sci 76(2):C334–42. Liu KS, Moreau RA. 2008. Concentrations of functional lipids in abraded fractions of hulless barley and effect of storage. J Food Sci 73(7):C569–76. Mitra P, Barman PC, Chang KS. 2011. Coumarin extraction from cuscuta reflexa using supercritical fluid carbon dioxide and development of an artificial neural network model to predict the coumarin yield. Food Bioprocess Technol 4(5):737–44. Moreau RA, Flores R, Hicks KB. 2007a. Composition of functional lipids in hulled and hulless barley in fractions obtained by scarification and in barley oil. Cereal Chem 84:1–5. Moreau RA, Wayns KE, Flores RA, Hicks KB. 2007b. Tocopherols and tocotrienols in barley oil prepared from germ and other fractions from scarification and sieving of hulless barley. Cereal Chem 84:587–92. Moreau RA, Bregitzer P, Liu KS, Hicks KB. 2012. Compositional equivalence of barleys differing only in low and normal phytate levels. J Agric Food Chem 60:6493–8. Nielsen MM, Hansen A. 2008. Rapid high-performance liquid chromatography determination of tocopherols and tocotrienols in cereals. Cereal Chem 85(2):248–51. Panfili G, Fratianni A, Di Criscio T, Marconi E. 2008. Tocol and β-glucan levels in barley varieties and in pearling by-products. Food Chem 107:84–91. Papas AM. 1999. Vitamin E: tocopherols and tocotrienols. In: Papas AM, editor. Antioxidant status, diet, nutrition and health. Boca Raton, Fla.: CRC Press LLC. Perrut M. 2000. Supercritical fluid applications: industrial developments and economic issues. Ind Eng Chem Res 39:4531–5. Peterson DM, Qureshi AA. 1993. Genotype and environment effects on tocols of barley and oats. Cereal Chem 70(2):157–62.
Vol. 78, Nr. 11, 2013 r Journal of Food Science C1649
C: Food Chemistry
Tocols of barley pearling flour . . .
Tocols of barley pearling flour . . .
C: Food Chemistry
Prado JM, Dalmolin I, Carareto NDD, Basso RC, Meirelles AJA, Oliveir JV, Batista EAC, Meireles MAA. 2012. Supercritical fluid extraction of grape seed: process scale-up, extract chemical composition and economic evaluation. J Food Eng 109(2):249–57. Qureshi AA, Burger WC, Peterson DM, Elson CE. 1986. The structure of an inhibitor of choloesterol biosynthesis isolated from barley. J Biol Chem 261:10544–50. Qureshi AA, Bradlow BA, Salser WA, Brace LD. 1997. Novel tocotrienols of rice bran modulate cardiovascular disease risk parameters of hypercholesterolemic humans. J Nutr Biochem 8: 290–8. Rezaei KA, Temelli F. 2000. Using supercritical fluid chromatography to determine the binary diffusion coefficient of lipids in supercritical CO2 . J Supercrit Fluids 17:35–44.
C1650 Journal of Food Science r Vol. 78, Nr. 11, 2013
Ruperez FJ, Martin D, Herrera E, Barbas C. 2001. Chromatographic analysis of α-tocopherol and related compounds in various matrices. J Chromatogr A 935:45–69. Temelli F. 2009. Perspectives on supercritical fluid processing of fats and oils. J Supercrit Fluids 47:583–90. Vasanthan T, Temelli F. 2009. Grain fractionation methods and products. U.S. Patent 7,566, 470 B2. Wang L, Xue Q, Newman RK, Newman CW. 1993. Enrichment of tocopherols, tocotrienols, and oil in barley fractions by milling and pearling. Cereal Chem 70(5):499–501.
Samples of whole grain and 35% pearling flour of 20 different barley varieties grown in Alberta were analyzed for their lipid contents. Total lipid contents of whole grains were within 1.9% to 3.0% (w/w), whereas those of the 35% pearling flour were 4.3% to 7.9%. Lipids of 35% pearling flour fraction of Tercel barley were extracted using supercritical carbon dioxide (SC-CO2 ) at different pressures (24, 45, and 58 MPa) and temperatures (40 and 60 ◦ C) for 3 h. Lipid recoveries of 73% to 97% were achieved using SC-CO2 extraction under different operational conditions. Tocol contents and compositions of whole grain, 35% pearling flour, and SC-CO2 extracts were analyzed using HPLC. Tocol content of the whole grain was 53.8 to 124.9 μg/g and that of the pearling flour was 195 to 363 μg/g of flour. The hulless barley varieties were higher in tocols, with waxy, double waxy and Tercel varieties having the highest levels (P < 0.05). The ratios of total tocotrienols to total tocopherols varied within 1.6 to 3.9 range. Tocol concentrations of SC-CO2 extract fractions varied from 1171 to 4391 μg/g extract depending on the operational conditions. Barley oil is a good natural source of different tocol isomers rich in tocotrienols.
Abstract:
Keywords: barley, lipid, pearling, supercritical carbon dioxide, tocopherol, tocotrienol
Barley pearling flour can be a good source of a natural mixture of the family of vitamin E components, tocopherols and tocotrienols, which have been shown to have health benefits. Hulless barley varieties have higher lipid contents and are richer in tocols. Supercritical CO2 can be used to extract barley lipids.
Practical Application:
Introduction Tocopherols and tocotrienols (cumulatively referred to as tocols) are the family of vitamin E compounds found naturally in the plant tissues (Barnes 1983). They have been associated with numerous health benefits. More specifically, α-tocopherol has been found to reduce the risk of ischemic heart disease and cataracts, and also enhance immune system functionality (Gaby and Machlin 1991). Tocotrienols have antioxidant activity and reduce serum cholesterol levels in chicken, swine, and humans (Qureshi and others 1986, 1997). Chemical structures of tocols consist of a chroman ring and a long side chain, which is a saturated phytyl chain in the case of tocopherols but it is unsaturated in tocotrienols with 3 double bonds. As shown in Figure 1 (adopted from Christie 2011), each of the tocopherols and tocotrienols has 4 possible isomers as α, β, γ , and δ differing in the number and position of methyl substituents on the benzene ring (Ruperez and others 2001; Nielsen and Hansen 2008). Cereal grains and oilseeds are rich sources of tocols and are major sources of vitamin E (Wang and others 1993; Colombo and others 1998). Barley is unique since it contains a high concentration of tocols and the distribution of the biologically active isomers in barley is favorable (Moreau and others 2007a, 2007b). Barley grains are rich in tocotrienols; α-tocotrienol having the highest MS 20130010 Submitted 1/2/2013, Accepted 8/28/2013. Authors are with Dept. of Agricultural, Food and Nutritional Science, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2P5. Author Rezaei is also with the Dept. of Food Science, Engineering and Technology, Univ. of Tehran, Iran. Direct inquiries to author Temelli (E-mail: [email protected]).
R C 2013 Institute of Food Technologists
doi: 10.1111/1750-3841.12271 Further reproduction without permission is prohibited
concentration followed by α-tocopherol, γ -tocotrienol, and γ tocopherol (Barnes 1983; Moreau and others 2007a). Canada is the 2nd largest barley producer in the world (according to Statistics Canada, 8 million tons harvested in 2012) with the province of Alberta producing about half of Canada’s total annual crop. Different barley varieties are grown in Alberta for feed, malt, and food purposes. Up to 80% of the crop is utilized as feed for livestock. Recently, barley is also being considered as a feedstock for bioproducts, such as bioethanol production. Considering the nutritional and certain technological points of view, it is important to have a concise set of compositional data to maximize the utilization of different barley varieties. Lampi and others (2004) reported the lipid and phytosterol contents of hulless barley and rye cultivars. Moreau and others (2007a, 2007b) investigated the tocol composition of barley grains and fractions obtained by scarifying. More recently, Moreau and others (2012) showed that there were no significant differences in the levels of various components, including tocols, except for phytate for the 5 low- and 5 normal-phytate barley cultivars grown in 3 Idaho locations. In the current study, the tocol contents and tocol compositions of 20 different varieties grown in Alberta were investigated. Tocols are lipid-soluble and therefore can be extracted together with lipids. Lipids are concentrated in the outer layers and the germ of the barley kernel. Even though the lipid content of the whole barley grain is in the range of 1.8% to 3.0% that of the pearling flour can be as high as 12% depending on the barley variety and the level of pearling (Liu and Moreau 2008; Liu 2011). Therefore, the pearling flour removed during the pearling operation is a suitable starting material for lipid extraction.
Vol. 78, Nr. 11, 2013 r Journal of Food Science C1643
C: Food Chemistry
Tocol Composition and Supercritical Carbon Dioxide Extraction of Lipids from Barley Pearling Flour
Tocols of barley pearling flour . . .
C: Food Chemistry
There is a growing interest in the use of supercritical CO2 (SCCO2 ) for the extraction of natural biologically active components from plant and animal sources (Mitra and others 2011; Prado and others 2012; Domingues and others 2013). Carbon dioxide at pressure and temperature conditions above its critical point (31 ◦ C and 7.4 MPa) becomes a supercritical fluid and has a solvent power similar to those of organic solvents. There is no solvent residue left in the final product since CO2 is released as a gas after extraction. In CO2 environment, oxidation reactions are avoided. As well, extractions can be carried out at temperatures just above the ambient temperature minimizing the degradation of many heat-labile components. Despite the great potential for SC-CO2 extraction of barley lipids, including tocols, no studies have been reported to evaluate such possibilities. Therefore, the objectives of this study were: (1) to determine the lipid and tocol contents and tocol compositions of the whole grain and 35% pearling flour of 20 barley varieties grown in Alberta; (2) to extract lipids from barley pearling flour using SC-CO2 at different pressure and temperature conditions and to analyze the tocol content and composition of the barley oil extracted using SC-CO2 . Pearling level of 35% was selected to evaluate the compositions of 20 barley varieties to simulate the industrial practice employed for pearled barley production. In addition, 19% pearling flour of CDC Candle was also used for SC-CO2 extraction since in a parallel study (Lekhi 2004) it was shown that 19% pearling maximized β-glucan retention in the pearled grain while maximizing the lipid content in the pearling flour.
level of pearling (35%) was achieved. The pearling flour was separated from the pearled grain by screening (Burrows grain dockage slotted sieve, size 3/64 and 5/16) and weighed. The amount of pearling flour removed as a percentage of the starting whole grain weight is reported as degree of pearling. All samples were stored for a maximum of about 2 mo at –18 ◦ C until analysis. Pearling flour of CDC Candle barley at 19% pearling level was obtained at pilot scale according to Lekhi (2004). Pilot scale pearling of grains (1.0 to 1.5 kg), previously cleaned and passed through a grain sizer to obtain grains of 2.34 to 2.73 mm in size, was performed using a Westrup dehuller (Model 970218, Westrup Co., Sorovej, Denmark) at 3900 rpm. Then, pearling flour was separated using a SWECO Vibro-energy separator (Model 6033 E, SWECO Inc., Toronto, ON, Canada) equipped with a 1.76mm-size screen and weighed. Pearling percentage was reported as the weight of pearling flour over the total grain weight.
Solvent extraction of lipids Solvent extraction of lipids was performed according to the AACC method 30-25 (AACC 1982), using the Goldfisch extraction unit (Labconco, Kansas City, Mich., U.S.A.). Lipids were extracted from approximately 2.0 g of samples using petroleum ether (40 mL) for 6 h and collected in a preweighed Goldfisch extraction beaker. The solvent was then evaporated and the remaining lipid was weighed. All extractions were performed in duplicate to determine the lipid contents of the whole grain and 35% pearling flour fractions of the 20 barley varieties as well as the 19% pearling flour of CDC Candle.
Materials and Methods Samples of whole grain and 35% pearling flour of 20 different barley varieties (part of another trial) were provided by Dr. J. Helm, Field Crop Development Center, Alberta Agriculture and Rural Development, Lacombe, AB, Canada. Barley grains were first tempered in a controlled humidity chamber at 7 to 12 ◦ C to adjust the moisture content at 13% to 15% to minimize breakage during pearling. Grains were then pearled using a bench top pearler (Seedburo Equipment Co., Des Plaines, Ill., U.S.A.), following a protocol similar to that of Panfili and others (2008). The pearler was modified with a 0.5 hp DC variable motor to adjust speed, which was set at level 70, corresponding to approximately 1400 to 1500 rpm. A batch of 100 g barley sample was used for each run and pearling was continued until the targeted
SC-CO2 extraction of lipids Supercritical fluid extraction of lipids from barley pearling flour was performed using a laboratory scale system (Newport Scientific Inc., Jessup, Md., U.S.A.) described previously (Bozan and Temelli 2002). The feed material was placed in a sample basket (25 cm × 27 mm I.D.) and mixed with glass beads (3 mm in diameter) to improve the extraction process. Glass wool was inserted at both ends to hold the sample, and the basket was placed into the 300 mL extraction chamber. An o-ring placed between the basket and the chamber ensured CO2 flow through the sample inside the basket. Extraction temperatures were maintained within ±2 ◦ C using a thermocouple placed inside the extraction cell, a temperature controller and a heating jacket around the cell. A back-pressure
A α-tocopherol β-tocopherol δ-tocopherol γ-tocopherol
B α-tocotrienol β-tocotrienol δ-tocotrienol γ-tocotrienol
Figure 1–Chemical structures of tocopherols (A) and tocotrienols (B) (adopted from Christie 2011).
C1644 Journal of Food Science r Vol. 78, Nr. 11, 2013
R’ CH3 CH3 H H
R” CH3 H H CH 3
R’ CH3 CH3 H H
R” CH3 H H CH3
regulator was used to maintain the desired pressure. A 35% pearling flour of Tercel barley (5.0 g) was used as the feed material and the extraction was carried out at different pressure (24, 45, and 58 MPa) and temperature (40 and 60 ◦ C) conditions. Extractions were continued for 3 h with intermittent collection of the extract fractions. CO2 flow rate was maintained at 0.5 L/min (measured at ambient conditions). After passing through a rotameter and gas meter, CO2 was vented since the lab unit did not have any recycle capability. SC-CO2 extraction was also performed using 19% pearling flour of CDC Candle at 60 ◦ C and 55 MPa. All extractions were performed in duplicate and lipid extracts were collected in glass tubes sitting in a refrigerated bath (–10 ◦ C) upon depressurization of SC-CO2 . The extract tubes were equilibrated at ambient conditions for about 20 min prior to gravimetric quantification. Cumulative amounts of extracts were reported as a function of time. For analysis purposes, the tubes containing the extracts were washed with hexane to ensure quantitative transfer of the extracts into glass vials. Hexane was then evaporated under a gentle flow of nitrogen and the samples were stored at –18 ◦ C until analysis. Prior to the high-performance liquid chromatography (HPLC) analysis of samples, each extract was redissolved in 1 mL hexane for the determination of tocol content and composition.
HPLC analysis of tocols Determination of the tocol contents and compositions of the whole grain, 35% pearling flours and SC-CO2 extracts was performed using the HPLC procedure described by Peterson and Qureshi (1993). Prior to the HPLC analysis, lipids containing the tocols were extracted using methanol (Peterson and Qureshi 1993). All samples were prepared in triplicate. HPLC-grade methanol (3.0 mL) was added to the flour samples (0.4 g), followed by gentle shaking of the samples for 15 min and centrifugation at 3000 rpm for 15 min. An aliquot (1.0 mL) of the supernatant was taken and its solvent was evaporated. The solid precipitate was re-extracted using additional 3.0 mL methanol following the same procedure. One milliliter aliquot of the supernatant from this stage was combined with the lipid sample from the 1st extraction step and again the solvent was evaporated. Samples were stored at –18 ◦ C until the HPLC analysis, when the samples were redissolved in HPLC-grade hexane (200 μL), stirred and centrifuged for 1 min. The supernatant was then transferred into an HPLC vial for tocol analysis. The HPLC system was a Varian 9010 unit (Walnut Creek, Calif., U.S.A.) equipped with a Supelco LC-Diol column (25 cm × 4.6 cm × 5 μm, Supelco Inc., Bellefonte, Pa., U.S.A.) and a Shimadzu RF535 fluorescence detector (Shimadzu Scientific Instruments Inc., Columbia, Md., U.S.A.). A Hewlett Packard 1050 autosampler with a 25-μL sample loop was used for sample injection. The conditions reported by Peterson and Qureshi (1993) were employed. The mobile phase consisted of 0.6% (v/v) iso-propanol in hexane (both HPLC grade from Fisher Scientific, Fair Lawn, N.J., U.S.A.) flowing at 1.0 mL/min. The detector was set at 298 nm for the excitation and 330 nm for the emission. Total run time for each sample was 30 min. Standard solutions containing all 8 tocol isomers were also prepared at 5 different concentration levels and were run at the beginning, in the middle and at the end of each sample set. The standards, α-, δ-, and γ -tocopherols were obtained from Sigma-Aldrich Inc. (St. Louis, Mo., U.S.A.), β-tocopherol from Chromatographic Specialties Inc. (Brockville, ON, Canada), and α-, β-, δ-, and γ -tocotrienols from CalBiochem (Darmstadt, Germany). These standards were used for peak identification and
quantification of sample components according to the external standard calibration method. The peaks for all 8 tocol isomers were completely separated with retention times of 7.8, 8.7, 15.0, 16.2, 17.6, 18.9, 23.1, and 27.5 min for α-tocopherol, α-tocotrienol, β- and γ -tocopherol, β- and γ -tocotrienol, δ-tocopherol and δtocotrienol, respectively. All data were collected and handled by a Shimadzu Class-VP Chromatography automated software system.
Statistical analysis Analysis of variance of the results was performed using General Linear Model procedure of SAS Statistical Software, Version 8 (SAS Inst. Inc., Cary, N.C., U.S.A.). Multiple comparison of means was performed by least significant difference test at α = 0.05 level.
Results and Discussion Lipid contents and tocol compositions of barley varieties Total lipid contents of whole grain and 35% pearling flour of 20 different barley varieties used in this study are presented in Table 1. The lipid content of whole grains varied from 1.8% to 3.0% (w/w), whereas that of 35% pearling flour was in the range 4.3% to 7.9%. The variety HB340 (CDC Alamo), CDC Candle, and HB 335 (CDC McGwire) had the highest lipid contents in the whole grain whereas HB340 (CDC Alamo) was the highest in the 35% pearling flour. This finding is in agreement with the results of Moreau and others (2012) who also found the lipid content of CDC Alamo to be higher than that of the other varieties they studied. The lipid content of the 35% pearling flour was substantially lower than that observed in a separate study conducted in our lab focusing on different levels of pearling (unpublished data), where a lipid content of 11.7% was obtained for 15% pearling flour of a different hulless double waxy barley variety (SB94794). This demonstrates the impact of pearling level and the dilution of lipids with starch at higher levels of pearling as more of the starch-rich endosperm is removed together with the outer bran layers. Such a dilution effect was also reported by Moreau and others (2007b) where the oil content of pearling flour obtained from hulless barley after 60 s of scarification was lower than that obtained after 20 s. Of the total lipids present in the whole grain, on average, 90.9% was present in the 35% pearling flour of the hulless varieties, whereas it was 80.6% for the hulled varieties, again indicating the dilution effect of the hulls (Table 1). In general, the lipid contents of hulless varieties were higher especially in the 35% pearling flour fraction (5.2% to 7.9% for hulless compared with 4.3% to 5.0% for hulled varieties, Table 1). Similarly, the oil contents of the scarification fines obtained from hulless varieties by Moreau and others (2007a) were higher than that of hulled varieties. Tocol contents and compositions of the whole grain and 35% pearling flour of the 20 barley varieties are presented in Tables 2 and 3, respectively. In the whole grain, total tocols ranged from 53.8 to 124.9 μg/g of flour (Table 2). These levels are similar to those reported by Peterson and Qureshi (1993) (42 to 80 μg/g) and by Moreau and others (2007a) (84.7 to 151.1 mg/kg) for other barley varieties. Following the general trends in the total lipid content, the hulless varieties exhibited higher levels of total tocols, with the waxy (CDC Candle), double waxy (HB340 – CDC Alamo) and Tercel barley varieties having the highest levels (P < 0.05). In terms of the individual tocol isomers in the whole grain, α-tocotrienol was the most abundant one making up 38.6% to 50.1% (w/w) of the total tocols, followed by α-tocopherol (17.7% to 33.9%), γ -tocotrienol (10.4% to 20.2%), γ -tocopherol (1.9% to 9.2%), Vol. 78, Nr. 11, 2013 r Journal of Food Science C1645
C: Food Chemistry
Tocols of barley pearling flour . . .
Tocols of barley pearling flour . . . Table 1–Total lipid contents (%, w/w, as is basis) of whole grain and 35% pearling flour of 20 barley varieties determined by solvent extraction using petroleum ether (n = 2). Lipid content (%, w/w) Variety
C: Food Chemistry
Whole grain
35% pearling flour
% of lipids from whole grain present in the pearling flour
2-row malting 2-row malting 2-row malting 6-row malting 2-row feed 2-row feed 2-row feed 2-row malting 2-row malting 2-row malting 2-row malting
2.0cde 1.9ef 2.5b 1.9def 1.8f 2.2cd 2.1cde 2.1cde 2.0def 2.1cde 2.0cde 2.0 ± 0.2
4.8gh 4.6ij 4.9g 4.7hi 4.3k 4.4jk 4.7hi 4.4k 4.7hi 5.0g 4.7hi 4.7 ± 0.2
84.1 84.8 68.7 86.6 83.7 71.6 78.4 81.1 82.3 83.4 82.3 80.6 ± 5.6
2-row waxy 2-row 6-row 2-row 2-row double waxy 6-row 6-row 2-row 2-row
2.9a 2.2cd 2.2c 2.6a 3.0a 2.2c 2.3c 2.2cd 2.1cde 2.4 ± 0.4
6.7b 6.2c 5.4e 6.1c 7.9a 5.8d 5.6d 6.1c 5.2f 6.1 ± 0.8
80.9 98.7 82.2 82.2 92.2 92.3 95.1 98.7 95.9 90.9 ± 7.2
Type
Hulled AC Axbow AC Metcalf B-1215 B-1602 CDC Dolly CDC Fleet CDC Guardian CDC Kendall CDC Stratus Harrington Manley Average (mean ± std. dev.) Hulless CDC Candle CDC Dawn Falcon HB-335∗ HB-340∗ Jaeger Peregrine Phoenix Tercel Average (mean ± std. dev.) ∗ HB335 a–k
is CDC McGwire and HB340 is CDC Alamo. In each column, means identified with the same letter are not significantly different (P > 0.05).
Table 2–Tocol contents and tocol compositions of the whole grain of the 20 barley varieties investigated (n = 3). Tocols extracted with methanol according to Peterson and Qureshi (1993) prior to HPLC analysis. Sample
α -T
α - T3
β -T
γ -T
Hulled AC Axbow AC Metcalf B-1215 B-1602 CDC Dolly CDC Fleet CDC Guardian CDC Kendall CDC Stratus Harrington Manley Average (mean ± std. dev.)
18.4 20.3 17.6 21.0 15.2 20.0 19.6 15.4 20.3 21.0 19.5
39.9 44.1 45.2 43.1 38.1 40.5 39.3 40.4 44.9 44.2 43.1
1.4 1.6 1.1 1.3 1.2 1.0 1.3 0.8 0.9 0.7 0.9
3.4 2.5 3.7 4.0 4.5 3.3 7.9 3.2 4.1 4.2 4.2
31.9 10.6 32.5 21.0 32.3 29.4 34.0 23.2 26.3
49.9 26.9 44.6 46.7 51.0 45.6 46.5 44.7 46.3
1.9 0.6 1.2 1.1 2.1 1.5 1.4 1.1 1.8
7.0 2.8 2.0 5.8 8.8 4.1 1.9 4.0 9.9
β - T3 (μg/g flour)
γ -T3
δ-T
δ - T3
5.4 6.7 7.7 4.2 4.2 4.2 2.6 4.8 4.7 5.2 4.3
9.4 12.4 20.1 13.9 11.8 12.3 12.5 13.8 14.5 17.7 12.1
0.6 0.8 0.7 1.0 0.9 0.6 1.4 0.6 0.8 0.6 0.7
1.3 1.4 3.2 1.6 1.2 1.2 1.2 1.9 2.0 2.4 1.5
79.7 89.8 99.2 90.0 77.1 83.1 85.8 81.0 92.1 96.0 86.4 87.3
± ± ± ± ± ± ± ± ± ± ± ±
4.9gh 1.6gdfceh 5.2dce 0.1gdfceh 11.4h 0.6gfh 1.7gfeh 2.7gfh 2.5gdfceh 0.6dfce 4.7gdfce 6.9
70.2 72.0 76.7 69.7 71.7 70.1 64.9 75.2 71.7 72.4 70.7 71.4 ± 3.0
4.5 3.3 3.6 7.7 6.0 3.4 2.8 4.7 9.0
19.2 7.9 10.0 15.9 20.4 16.2 11.7 13.2 20.6
1.3 0.5 0.6 1.0 1.7 0.7 0.5 0.9 1.8
1.5 1.3 1.3 2.2 2.5 1.9 0.8 1.6 4.4
117.3 53.8 95.7 101.5 124.9 102.8 99.5 93.3 120.1 101.0
± ± ± ± ± ± ± ± ± ±
2.2ab 13.8i 0.2dfce 2.6dc 1.5a 1.4bc 0.4dce 0.9gdfce 7.5a 21.0
64.1 73.1 62.2 71.5 64.0 65.2 62.1 68.8 66.9 66.4 ± 4.0
Total tocols
% T3
Hulless CDC Candle CDC Dawn Falcon HB335 HB340 Jaeger Peregrine Phoenix Tercel Average (Mean ± std. dev.) ∗ T a–i
denotes tocopherols and T3 denotes tocotrienols. Means with the same letter are not significantly different (P > 0.05).
C1646 Journal of Food Science r Vol. 78, Nr. 11, 2013
Tocols of barley pearling flour . . . Table 3–Tocol contents and tocol compositions of 35% pearling flour of the 20 barley varieties investigated (n = 3). Tocols extracted with methanol according to Peterson and Qureshi (1993) prior to HPLC analysis. Sample
α -T
α - T3
β -T
γ -T
β - T3
γ -T3
δ-T
δ - T3
Total tocols∗
% T3
Hulled AC Axbow AC Metcalf B-1215 B-1602 CDC Dolly CDC Fleet CDC Guardian CDC Kendall CDC Stratus Harrington Manley Average (mean ± std. dev.) Hulless CDC Candle CDC Dawn Falcon HB335 HB340 Jaeger Peregrine Phoenix Tercel Average (mean ± std. dev.) ∗
37.8 28.5 44.3 41.8 51.0 39.2 35.3 30.1 41.4 39.3 32.1
118 107 147 133 139 123 107 118 124 120 107
12.4 8.7 12.5 10.6 16.7 8.5 8.4 6.1 6.1 5.4 5.9
8.0 5.8 11.0 12.0 25.5 9.1 17.5 6.5 10.5 10.2 9.4
17.4 16.1 30.6 13.9 23.7 14.6 7.6 15.0 14.4 14.8 11.0
27.1 26.2 69.5 40.7 58.7 36.1 38.7 36.8 41.1 45.4 28.3
2.0 0.3 2.3 2.3 2.4 2.0 2.9 1.9 0.7 0.5 0.6
3.6 2.0 9.6 4.6 4.2 3.7 3.0 4.7 4.3 4.3 2.4
226 195 336 250 321 236 211 219 243 240 197 243.1
± ± ± ± ± ± ± ± ± ± ± ±
3ih 4k 8b 1feg 6bcd 4hg 2ij 2i 9fg 11hg 5kj 46.0
73.4 77.7 76.5 77.0 70.2 75.1 73.7 79.6 75.8 77.0 75.5 75.6 ± 2.5
45.5 52.2 53.1 33.8 54.3 53.2 87.6 54.0 63.1
133 142 114 130 145 130 147 133 139
12.0 15.1 9.1 7.0 14.6 11.6 15.0 14.5 17.7
16.2 22.4 5.7 12.1 24.6 7.9 7.6 24.4 31.0
10.9 27.8 10.6 21.2 16.7 9.9 11.9 18.7 32.0
41.3 57.1 25.5 38.3 51.0 38.7 43.2 51.5 66.7
1.2 3.3 0.3 2.2 2.1 1.9 0.3 3.3 3.0
2.2 8.6 2.4 4.9 4.8 4.4 2.0 6.7 11.0
263 328 220 263 313 258 315 306 363 292.1
± ± ± ± ± ± ± ± ± ±
4e 9cb 6i 6e 4cd 6fe 10cd 4d 9a 44.1
69.4 68.3 64.9 69.1 69.4 71.5 71.0 73.9 68.6 69.6 ± 2.5
T denotes tocopherols and T3 denotes tocotrienols. Means with the same letter are not significantly different (P > 0.05).
a–k
and β-tocotrienol (2.9% to 7.8%). β-Tocopherol, δ-tocopherol, and δ-tocotrienol made up 2.7% to 6.7% of total tocols. The ratio of tocotrienols to tocopherols ranged from 1.6 to 3.3. On average, tocotrienols comprised 71.4 ± 3.0% and 66.4 ± 4.0% of the total tocols in the hulled and hulless varieties, respectively (Table 2). Similarly, Moreau and others (2007a) reported that tocotrienols made up 70.6% to 75.7% of the total tocols in the whole kernels of the 2 hulled and 2 hulless barley varieties they investigated. Such a distribution of tocol isomers is quite favorable since αand γ -tocotrienol are especially receiving increasing attention in terms of their health benefits (Papas 1999). In the 35% pearling flours, tocol contents and compositions followed similar trends to those observed in the whole grain with hulless varieties having higher levels of tocols. Total tocol content in the 35% pearling flours varied from 195 to 363 μg/g of pearling flour (Table 3) with Tercel variety having the highest level (P < 0.05). Moreau and others (2007a) reported that the tocol content of the scarifying fines obtained after 60 s was 153.4 to 448.8 mg/kg. This extent of scarifying corresponded to 11.6% to 15.2% pearling by weight, which was lower than the 35% pearling used in the current study, where the tocols in the pearling flour were diluted by other components. α-Tocotrienol in the 35% pearling flours was at somewhat higher levels than that in the whole grain, making up 38.1% to 55.0% (w/w) of the total tocols. This was followed by α-tocopherol (13.2% to 27.8%), γ -tocotrienol (11.6% to 20.7%), γ -tocopherol (2.4% to 8.5%), and β-tocotrienol (3.6% to 9.1%). The relative proportion of β-tocopherol (2.3% to 5.5%) was higher than that in the whole grain. δ-Tocopherol and δtocotrienol together contributed to 0.05).
C1648 Journal of Food Science r Vol. 78, Nr. 11, 2013
However, an increase in pressure also results in a decrease in the diffusivity of the solutes in SC-CO2 , which has a negative impact on the mass transfer kinetics. On the other hand, an increase in temperature causes a reduction in the CO2 density but enhances the vapor pressure of the solutes as well as their diffusivity in the supercritical medium (Rezaei and Temelli 2000). The extent of these changes is pressure dependent, leading to the well-known cross-over behavior of solubility isotherms (Guclu-Ustundag and Temelli 2000). Above the cross-over pressure, the oil solubility in SC-CO2 increases with temperature. The extraction yield can be increased further with an increase in solvent-to-feed ratio or the CO2 flow rate, which was kept at a minimum level in this study to maximize contact between the feed material and CO2 . Tocol concentration of each extracted oil fraction obtained over different time intervals was analyzed using HPLC and the data are presented in Figure 3. Total tocol concentration varied from 1171 to 4391 μg/g extract. It seems that at 40 ◦ C tocols were extracted to a greater extent in the 1st 20 min of extraction but then the tocol concentration in the extracts decreased. However, this was not the case consistently at all the pressures tested at 60 ◦ C, since the later fractions seemed to get more concentrated in tocols at 58 MPa. With the SC-CO2 extraction of lipids, the tocol concentration increased from 120.1 μg/g of whole grain flour of Tercel barley to 1171 to 4391 μg/g extract, resulting in 10- to 37-fold enrichment of tocols in these extracted fractions. SC-CO2 extraction of barley lipids was also carried out using the 19% pearling flour of CDC Candle. CDC Candle was selected for this purpose because of its high β-glucan content and our parallel initiatives on the concentration of this health benefiting soluble fiber component (Vasanthan and Temelli 2009). Pearling at a level of 19% instead of 35% exhibited greater β-glucan retention in the pearled grain, which could be fractionated further (Lekhi 2004). Total lipid content of the 19% pearling flour of CDC Candle was 8.7%. After 3 h of extraction using SC-CO2 at 55 MPa and 60 ◦ C, the recovery of oil was 84%. Total tocol content of the oil extracted using SC-CO2 was 1565 μg/g oil for CDC Candle resulting in a 13-fold enrichment, considering the 117.3 μg/g tocols in the whole grain (Table 2). For the development of a potential biorefinery based on the full utilization of barley grains, pearling can be used as the 1st unit operation for dry separation of the grain tissues, where the outer layers including the bran and germ would be concentrated in the pearling flour and endosperm would be left as the pearled grain, enriched in starch and β-glucan. As demonstrated in this study, pearling flour can be a good viable starting material for the recovery of barley oil, rich in tocols with a natural mixture of all eight tocopherol and tocotrienol isomers and a favorable ratio of tocotrienols to tocopherols. Similarly, SC-CO2 extraction is a viable alternative to the traditional hexane extraction of oil, especially for oils rich in such bioactive components as tocols. With the latest developments in supercritical technology (Temelli 2009), SC-CO2 extraction has become mainstream, offering numerous advantages over hexane extraction at a time when hexane use is facing greater safety concerns. It has also been demonstrated that the process economics can become comparable to that of the conventional processes when the capacity of supercritical plants is increased to an appropriate level using optimized process design (Perrut 2000).
Conclusions Lipid contents of the 35% pearling flour of 20 different barley varieties grown in Alberta were greater than those of the whole
grain flour with the hulless varieties exhibiting higher levels of lipids than the hulled varieties. This demonstrates that the lipids are mainly concentrated in the outer layers of the barley grain and further optimization of pearling level could result in pearling flour containing high levels of lipids. Tocol content also followed a similar pattern whereby tocols were concentrated in higher amounts in the pearling flour compared to the whole grain. Hulless varieties also contained higher levels of tocols in the pearling flour than did the hulled varieties. The analysis of tocol composition indicated that α-tocotrienol was the most abundant isomer followed by α-tocopherol and γ -tocotrienol as the most favorable isomers for achieving health benefits. SC-CO2 extraction of the 35% pearling flour at the various pressure and temperature conditions of this study indicated that the most favorable condition to yield the highest amount of extract was 45 MPa and 60 ◦ C. Barley pearling flour can be an appropriate starting material for the recovery of lipids that are rich in tocols with a favorable distribution of tocopherols and tocotrienols.
Acknowledgments We are grateful to the Alberta Barley Commission and the Alberta Agricultural Research Inst. for financial support of this project and Dr. James Helm, Alberta Agriculture and Rural Development for providing the barley samples.
References AACC. 1982. Approved methods of the American Association of Cereal Chemists. Method 30–25: crude fat in wheat and soy flour, feeds and cooked feeds. St. Paul, Minn.: AACC. Barnes PJ. 1983. Non-saponifiable lipids in cereals. In: Barnes PJ, editor. Lipids in cereal technology. New York: Academic Press. p 33–55. Bozan B, Temelli F. 2002. Supercritical CO2 extraction of flaxseed. J Am Oil Chem Soc 79:231–5. Christie WW. 2011. Tocopherols and tocotrienols – structure, composition, biology and analysis. In: Harwood JL, Weselake RJ, editors. Lipid chemistry, biology, technology & analysis. American Chemical Society. Available from: http://lipidlibrary.aocs.org/. Accessed on April 5, 2013. Colombo ML, Corsini A, Mossa A, Sala L, Stanca M. 1998. Supercritical carbon dioxide extraction, fluorimetric and electrochemical high performance liquid chromatrographic detection of vitamin E from Hordeum vulgare L. Phytochem Anal 9(4):192–5. Domingues RMA, de Melo MMR, Oliveira ELG, Neto CP, Silvestre AJD, Silva CM. 2013. Optimization of the supercritical fluid extraction of triterpenic acids from Eucalyptus globulus bark using experimental design. J Supercrit Fluids 74:105–14. Gaby WK, Machlin LJ. 1991. Vitamin E. In: Gaby SK, Bendich A, Singh V, Machlin L, editors. Vitamin intake and health: a scientific review. New York: Marcel Dekker. p 71–101. Guclu-Ustundag O, Temelli F. 2000. Correlating the solubility behavior of fatty acids, mono-, di-, and triglycerides, and fatty acid esters in supercritical carbon dioxide. Ind Eng Chem Res 39(12):4756–66. Joki´c S, Nagy B, Zekovi´c Z, Vidovi´c S, Bili´c M, Veli´c D, Sim´andi B. 2012. Effects of supercritical CO2 extraction parameters on soybean oil yield. Food Bioprod Process 90(4):693–9. Lampi AM, Moreau RA, Piironen V, Hicks KB. 2004. Pearling barley and rye to produce phytosterol-rich fractions. Lipids 39:783–7. Lekhi AK. 2004. Extraction and functional properties of barley proteins [M.Sc. thesis]. Edmonton, AB, Canada: Univ. of Alberta. Liu KS. 2011. Comparison of lipid content and fatty acid composition and their distribution within seeds of 5 small grain species. J Food Sci 76(2):C334–42. Liu KS, Moreau RA. 2008. Concentrations of functional lipids in abraded fractions of hulless barley and effect of storage. J Food Sci 73(7):C569–76. Mitra P, Barman PC, Chang KS. 2011. Coumarin extraction from cuscuta reflexa using supercritical fluid carbon dioxide and development of an artificial neural network model to predict the coumarin yield. Food Bioprocess Technol 4(5):737–44. Moreau RA, Flores R, Hicks KB. 2007a. Composition of functional lipids in hulled and hulless barley in fractions obtained by scarification and in barley oil. Cereal Chem 84:1–5. Moreau RA, Wayns KE, Flores RA, Hicks KB. 2007b. Tocopherols and tocotrienols in barley oil prepared from germ and other fractions from scarification and sieving of hulless barley. Cereal Chem 84:587–92. Moreau RA, Bregitzer P, Liu KS, Hicks KB. 2012. Compositional equivalence of barleys differing only in low and normal phytate levels. J Agric Food Chem 60:6493–8. Nielsen MM, Hansen A. 2008. Rapid high-performance liquid chromatography determination of tocopherols and tocotrienols in cereals. Cereal Chem 85(2):248–51. Panfili G, Fratianni A, Di Criscio T, Marconi E. 2008. Tocol and β-glucan levels in barley varieties and in pearling by-products. Food Chem 107:84–91. Papas AM. 1999. Vitamin E: tocopherols and tocotrienols. In: Papas AM, editor. Antioxidant status, diet, nutrition and health. Boca Raton, Fla.: CRC Press LLC. Perrut M. 2000. Supercritical fluid applications: industrial developments and economic issues. Ind Eng Chem Res 39:4531–5. Peterson DM, Qureshi AA. 1993. Genotype and environment effects on tocols of barley and oats. Cereal Chem 70(2):157–62.
Vol. 78, Nr. 11, 2013 r Journal of Food Science C1649
C: Food Chemistry
Tocols of barley pearling flour . . .
Tocols of barley pearling flour . . .
C: Food Chemistry
Prado JM, Dalmolin I, Carareto NDD, Basso RC, Meirelles AJA, Oliveir JV, Batista EAC, Meireles MAA. 2012. Supercritical fluid extraction of grape seed: process scale-up, extract chemical composition and economic evaluation. J Food Eng 109(2):249–57. Qureshi AA, Burger WC, Peterson DM, Elson CE. 1986. The structure of an inhibitor of choloesterol biosynthesis isolated from barley. J Biol Chem 261:10544–50. Qureshi AA, Bradlow BA, Salser WA, Brace LD. 1997. Novel tocotrienols of rice bran modulate cardiovascular disease risk parameters of hypercholesterolemic humans. J Nutr Biochem 8: 290–8. Rezaei KA, Temelli F. 2000. Using supercritical fluid chromatography to determine the binary diffusion coefficient of lipids in supercritical CO2 . J Supercrit Fluids 17:35–44.
C1650 Journal of Food Science r Vol. 78, Nr. 11, 2013
Ruperez FJ, Martin D, Herrera E, Barbas C. 2001. Chromatographic analysis of α-tocopherol and related compounds in various matrices. J Chromatogr A 935:45–69. Temelli F. 2009. Perspectives on supercritical fluid processing of fats and oils. J Supercrit Fluids 47:583–90. Vasanthan T, Temelli F. 2009. Grain fractionation methods and products. U.S. Patent 7,566, 470 B2. Wang L, Xue Q, Newman RK, Newman CW. 1993. Enrichment of tocopherols, tocotrienols, and oil in barley fractions by milling and pearling. Cereal Chem 70(5):499–501.
