Effect of Fermentation and Oil Incorporation on the Retention of E Vitamers During Breadmaking Oliver Buddrick,† Oliver A. H. Jones, Paul D. Morrison, and Darryl M. Small ABSTRACT
Cereal Chem. 92(3):327–331
All forms of baking and processing cause a loss of nutrients, including vitamin E, but little is known about these occurrences or if they could be avoided. The objective of this research was to study the incorporation of palm oil and the stability of vitamin E in palm oil during breadmaking. Wheat meal and rye breads were baked with and without the addition of 0, 2, 5, or 8% palm oil. The eight E group vitamers (tocopherols and
tocotrienols) were extracted by using accelerated solvent extraction, freeze dried, and then analyzed with normal-phase HPLC. Compared with the controls, the inclusion of palm oil was found to increase the quantity of all forms of vitamin E in the final baked products. It is concluded that palm oil is effective in increasing the vitamin E content of whole grain bread.
Vitamin E is a term encompassing eight separate compounds (four tocopherols and four tocotrienols) collectively known as tocochromanols or tocols. These are lipid-soluble antioxidants with a chromanol ring and a hydrophobic side chain, and they play an essential role in human nutrition and health. The history and chemical structures of the eight vitamin E vitamers are well described, and details can be found in Kamal-Eldin and Appelqvist (1996). Vitamin E has many biological functions in humans, with the most important and best known being its antioxidant role. Other functions include regulating enzymatic activity and cell signaling (Zingg and Azzi 2004; Azzi 2007). The wheat grain is often regarded as an important dietary source of vitamin E, as are cereals in general (Sen et al. 2007). However, the total tocol content decreases during the mixing, fermentation, and baking stages of the breadmaking process (Dewettinck et al. 2008). Owing to the importance of vitamin E in the human diet, fats and oils are used as primary enriching ingredients in modern baking. Vegetable oils with high vitamin E content are widely used to fortify bakery products; when used in bread formulations, they improve the characteristics of the resulting dough by enhancing gas retention and thereby increasing the volume and softness of the final product (Cauvain 2003). The proportion of oil required varies according to the type of flour being used, with wholemeal flours requiring higher levels of oil in comparison with white, often as high as two to three times more (Williams and Pullen 1998). Palm oil is an excellent oil to use in baked products. It contains similar amounts of both unsaturated and saturated fatty acids, making it semisolid at room temperature. It is also heat stable with a high smoke point (232°C). These properties permit its use as a major component in baking and cooking without the need for hydrogenation and remove the problem of trans-fatty acids, generated by the latter process, which are detrimental to human health (Nor Aini and Miskandar 2007). In contrast to many other vegetable oils, palm oil also contains up to all eight vitamin E vitamers, and recent research has also demonstrated the benefits of the tocotrienol isomers of vitamin E as possessing potential blood cholesterol lowering and cardioprotective effects and a more efficient antioxidant activity in biological systems (Das et al. 2005, 2008; Nesaretnam et al. 2007; Nesaretnam 2008). Tocotrienols have also been shown to have greater anti-inflammatory
and antioxidant properties that could reduce the incidence of cancer and diabetes as well as cardiovascular and neurodegenerative diseases compared with tocopherols (Lau et al. 2007; Ryan et al. 2007; Akhtar and Ashgar 2011). Despite these potential benefits, there are few studies that investigate vitamin enrichment of bakery products with palm oil (Agnoletti et al. 2011; Akhtar and Ashgar 2011). One of the more detailed examples is provided by Al-Saqer et al. (2004), who prepared pan bread and sugar snap cookies with red palm olein and red palm shortening. The results showed that tocotrienols were the predominant form of vitamin E in cookies made with either red palm olein or red palm shortening. The same authors later demonstrated that a cookie recipe containing higher quantities of fat would provide lager amounts of vitamin E and other desirable phytochemicals (including b-carotene) than various bread formulations (Lenfant and Thyrion 1996; Al-Saqer et al. 2004). Research has consistently demonstrated the health benefits of wholemeal or whole grain products over those made from refined flour. There is also increased interest in rye flours for health benefits. Within the context of a broader study of the influence of fermentation conditions during baking, the objective of this study was to investigate the retention of E vitamers for wholemeal rye and wheat (whole grain meal in both cases) formulations. Specific objectives were the comparison of different temperature and fermentation times, along with a comparative evaluation of varying levels of incorporation of a palm oil as a potential source of the various E group vitamers to the final baked product.
† Corresponding
author. Phone: +61 (03) 9925 2124. E-mail:
[email protected]
School of Applied Sciences, P.O. Box 2476, Royal Melbourne Institute of Technology, Melbourne, VIC 3001, Australia. http://dx.doi.org/10.1094/CCHEM-07-14-0159-R © 2015 AACC International, Inc.
MATERIALS AND METHODS We have recently reported on procedures for the analysis of vitamin E by normal-phase HPLC with heptane rather than hexane for the separation of the eight vitamer forms in wheat bread (Buddrick et al. 2013); similar protocols were used in the present study, and the method is outlined here. Bread Preparation. Milling. Wholemeal flours were freshly milled on the day of baking. Organically grown wheat grain of mixed variety and rye (11.7 and 11.5% protein, respectively) were obtained from the Laucky Flour Mill (Bridgewater, VIC, Australia). A benchtop mill (Grain Master whisper mill, Retsel, Dandenong, VIC, Australia), which uses upright blades spinning at high speed (10,000 rpm) was used to generate a fine grain meal with a large surface area. Wheat Flour Fermentation and Baking. The wheat fermentation involved yeast and a bulk fermentation for periods of 3, 5, and 7 h and temperatures of 23, 30, and 37°C. To prepare the dough, 100% wheat meal, 70% water, 5% red palm oil, 2% salt, 0.2% instant dry yeast (all percentage values are relative to the total flour weight) were first mixed with a bench mixer (heavy duty, 5KPM50, KitchenAid, Benton Harbor, MI, U.S.A.) at slow speed (speed Vol. 92, No. 3, 2015
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setting 2) for 4 min and followed by fast speed (speed setting 4) for 6 min until full dough development was achieved (taken as the point when the dough no longer stuck to the dough hook or mixing bowl). At this point, the dough was weighed, and a 180 g subsample of each type was placed into bread tins. The next stage was the final proofing, for which the dough (in the tins) was placed in an incubator at 37°C for 45 min. Baking was then performed at 230°C for 10 min, followed by 15 min at 200°C. This method resulted in an evenly baked loaf with a normal, golden brown crust. Rye Flour Fermentation and Baking. Baking bread with ryebased meal or flour is a different process than the corresponding wheat-based process. When using rye, the first step is a sourdough fermentation in which 35% of the flour is fermented with 10% starter culture. The production of such a starter culture is performed by incubating a rye meal slurry (a 1:1 mix of rye meal and water) for 24 h. After this time, 1% of the slurry is added to a fresh 1:1 mix slurry. This process was repeated every 24 h for at least three days to allow the formation of the necessary microbial population (Ercolini et al. 2013). Once the starter was ready, a rye bread formulation consisting of 90% rye meal, 10% wheat meal, 100% water, 0, 2, 5, or 8% red palm oil, 2% salt, and 1% instant dry yeast was prepared and mixed with the starter culture (again, all percentage values are relative to the total flour weight) and mixed for 10 min at slow speed. The resulting dough was placed into bread tins for a final proofing stage of 37°C for 45 min. Baking was then performed at 230°C for 10 min, followed by a further 15 min at 200°C, as previously described (Buddrick et al. 2013). All breads (wheat and rye) were prepared in triplicate. Freeze Drying. Following baking, samples were allowed to cool to room temperature, sliced, and frozen at –40°C with a freeze dryer (VirTis, SP Industries, Gardiner, NY, U.S.A.) to obtain lowmoisture-content samples for further analysis (Buddrick et al. 2013). The freeze-dried samples were then ground with a mortar and pestle to form a fine powder, which was then placed in an airtight glass jar, wrapped in foil (to prevent light degradation), and stored at –18°C prior to analysis. Vitamin E Extraction. Accelerated solvent extraction was used to extract the total vitamin E content of each sample. This involved first taking a 2.0 g subsample of the previously prepared powdered bread. The sample was mixed with 0.05 g of ascorbic acid and 1.9 g of HydroMatrix Celite absorbent medium (SigmaAldrich) as a drying agent. The mixture was placed in a 22 mL stainless steel extraction cell, which was sealed and placed in the sample tray of a Dionex ASE 200 accelerated solvent extractor (Thermo Fisher Scientific, Scoresby, VIC, Australia) equipped with a solvent controller. The vitamin E was extracted with an in-house extraction program. The cell was filled with 90% hexane and 10% ethyl acetate, heated to a temperature of 80°C, and pressurized at 1,600 psi. These conditions were maintained for 5 min, followed by two static cycles for 10 min. After that, the cell was flushed for 60 s and purged for 120 s via the onboard control software. The resulting extract was then collected in a 60 mL glass vial and evaporated to dryness under N2. Each sample was redissolved in 1 mL of mobile phase (as in the following section), vortex mixed, and transferred to amber glass chromatography vials. TABLE I Contents of Tocopherols and Tocotrienols in Freshly Milled Cereal Grains (Wheat and Rye), as Well as the Red Palm Oil Used in This Studya Tocol Concentration (mg/g) Sample
a-T
a-T3
b-T
b-T3 g-T g-T3 d-T d-T3
Wheat 5.43 2.16 6.71 21.05 ND ND ND Rye 9.36 8.85 4.76 7.31 ND ND ND Palm oil 177.4 149.1 19.53 34.62 ND 491.5 ND a
ND ND ND
Total 35.35 30.28 872.2
Recoveries for wheat and rye were adjusted to a dry matter basis. T = tocopherol; T3 = tocotrienol; and ND = not detected.
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Separation and Detection of Tocopherols and Tocotrienols by HPLC. Chromatographic analysis was undertaken with an HPLC system (model 510) equipped with a fluorescence detector (RF-10A, Waters, Milford, MA, U.S.A.). Data were acquired and processed via a Shimadzu interface CBM-10A communicator bus module connected to an autoinjector (Rowville, VIC, Australia). An injection volume of 25 µL was used, and the sample was run with a 4.6 mm × 25 cm, 5 µm particle size, silica Supelcosil LC-Si normal-phase HPLC column (Supelco/Sigma-Aldrich). An ethyl acetate/acetic acid/n-hexane 1:1:198 (v/v/v) mobile phase was used at a flow rate 1.5 mL/min, and fluorometric detection of all peaks was performed with wavelength excitation of 290 nm and emission of 330 nm, respectively. The total run time was 30 min. All standards, reagents, and solvents used were supplied by SigmaAldrich. Moisture Analysis. The moisture content of samples was measured according to AACC International Approved Method 44-15.02. Empty aluminum moisture dishes were first placed into a preheated oven at 130°C. After 1 h, the dishes were cooled in a desiccator containing active silica gel for a period of 30 min and then weighed. A subsample of the freeze-dried powder (5 g) was then added to each dish, and these samples were dried for 1 h at 130°C. This analysis was undertaken in triplicate in each case. RESULTS To facilitate comparisons and evaluation of retention of the vitamers, samples of freshly milled wheat and rye as well as palm oil were analyzed for all eight forms of vitamin E, and the results are presented in Table I. The total vitamin E content in rye grain was 16% lower than the freshly milled wheat grain. The quantities of vitamin E extracted from freshly milled rye and wheat kernels shown in Table I are similar to those reported by other researchers (Hidalgo et al. 2006; Nystrom et al. 2008). As shown in Table I, palm oil has a significant amount of g-tocotrienol. In a bread formulation containing 2% oil (density of palm oil is 0.9165 g/mL), the addition of 1.83 g of oil to 100 g of meal approximates to 1,596 µg of vitamin E. The content of tocopherols and tocotrienols in 100% wheat bread prepared without and with the addition of oil is shown in Table II. In this experiment, the purpose of producing bread without the addition of oil was to verify the effect of incorporating palm oil on the vitamin E content of whole grain products. The tocopherols present were a-, b-, and g-tocopherol, whereas d-tocopherol was not detected. The tocotrienol content found in the wheat meal bread samples consisted of only b-tocotrienol and ranged from 1.55 to 3.76 µg/g. No a-, g-, or d-tocotrienol was detected. The highest level of total tocols was found in the bread fermented at 37°C for 3 h. The content of tocopherols (a-, b-, and g-tocopherol) in wheat breads with the addition of oil was significantly increased compared with those without oil, ranging from 1.06 to 7.89 µg/g. The principal tocopherol in wheat is a-tocopherol, and no d-tocopherol was detected. The total tocotrienol contents ranged from 0.98 to 23.17 µg/g. The primary wheat bread tocotrienol fraction consisted of b- and g-tocotrienol. As expected, the highest overall levels of tocopherols and tocotrienols were found in bread with the addition of 8% palm oil. The contents of tocopherols and tocotrienols in rye bread, with and without the addition of oil, are presented in Table III. In this series of experiments, when no oil was incorporated the content of tocopherols (a- and b-tocopherol) reflected the naturally occurring vitamin E in the whole rye kernel, particularly the germ. The main tocopherol was a-tocopherol, and no g- and d-tocopherol were detected. The tocotrienol content in the rye bread was formed by b-tocotrienol, with lesser amounts of a- and no g- or d-tocotrienol found. The highest total levels of tocopherols and tocotrienols were found in the bread fermented at 23°C.
In the wholemeal rye bread with the addition of oil, it has been observed that a-, b-, and traces of g-tocopherol contributed to the vitamin content of the final baked product. As observed for the wheat breads, the final bread had no detectable d-tocopherol. The primary rye bread tocotrienol fraction was formed by b- and g-tocotrienol, although a small quantity of d-tocotrienol was detected. The addition of 8% palm oil again resulted in the highest levels of the E vitamers in the final product. In the current study, less vitamin E was detected in the rye breads than the corresponding wheat breads. This may be because of the sourdough fermentation process, as was also reported by Liukkonen et al. (2003). The losses of vitamin E content in bread occur in the doughmaking and fermentation stage. The reduction of vitamin E at various stages in this study is shown in Figures 1 and 2. The U.S. recommended dietary allowance for vitamin E is 15 mg per day. In Table IV, we demonstrate the potential contribution of the baked products from the current study to vitamin E intakes. The values have been estimated on the basis of consuming two servings (two slices), assuming a loaf of bread weighs 680 g and contains approximately 20 slices weighing 34 g each. The sensory characteristics of the loaves of bread produced as a result of the inclusion of palm oil were evaluated on the basis of a comparison of cross-sectional slices, and examples are presented in Figure 3.
The control bread in Figure 3A had the appearance of a normal baked wholemeal bread. The introduction of palm oil into the formulation had a noticeable effect on the crumb color (Fig. 3C and D). The bread shown in Figure 3C appears to be the best loaf of bread, made with the inclusion of 5% palm oil, based upon the greater uniformity of crumb structure and volume compared with other oil untreated and treated loaves. Although a full sensory evaluation of the product was beyond the scope of this study, all breads produced were tested by the authors and colleagues at the Royal Melbourne Institute of Technology and bakery teaching staff at the William Angliss Institute (Melbourne, Australia). There was a slight orange color to the breads with a high (8%) oil content, but no negative comments on taste or texture were received, and the bread looked and felt like a normal loaf. DISCUSSION The quantities of vitamin E extracted from freshly milled rye and wheat kernels confirm that whole grain wheat (containing the wheat germ and thus the wheat germ oil) has a considerable amount of vitamin E. However, because of its high levels of polyunsaturated fatty acids, it is also prone to rancidity and oxidation depending on storage and handling conditions (Enig 2000). Here, as in commercial baking, the whole grain wheat was milled and immediately combined with the other ingredients (water, salt, and yeast) and
TABLE II Contents of Tocopherols and Tocotrienols in Loaves Baked from 100% Wheat Without (Control Experiment) and With the Addition of Oila Tocol Concentration (mg/g) Sample Without the addition of oil 23°C, 3 h 23°C, 7 h 30°C, 5 h (1) 30°C, 5 h (2) 37°C, 3 h 37°C, 7 h With the addition of oil 2%, 23°C, 3 h 2%, 23°C, 7 h 2%, 37°C, 3 h 2%, 37°C, 7 h 5%, 30°C, 5 h (1) 5%, 30°C, 5 h (2) 8%, 23°C, 3 h 8%, 23°C, 7 h 8%, 37°C, 3 h 8%, 37°C, 7 h a
a-T
a-T3
b-T
b-T3
g-T
g-T3
d-T
d-T3
Total
1.42 2.41 1.50 1.35 2.42 1.56
ND ND ND ND ND ND
0.91 1.52 1.01 1.02 2.42 2.68
2.25 3.45 2.61 1.55 3.76 2.45
ND ND ND ND ND ND
ND ND ND ND ND ND
ND ND ND ND ND ND
ND ND ND ND ND ND
4.58 7.38 5.12 3.92 8.60 6.69
2.49 2.72 2.94 2.63 4.83 4.14 6.72 7.11 6.80 7.89
1.02 0.98 1.05 1.01 2.95 2.05 5.53 4.39 5.19 6.38
2.98 3.33 3.57 3.31 3.04 3.77 3.27 4.24 2.98 3.37
7.51 7.78 8.57 7.90 8.04 9.32 8.92 12.65 9.06 10.83
1.27 1.06 1.50 1.20 1.66 1.47 1.67 2.00 1.59 1.97
2.97 3.35 3.42 3.31 9.89 10.72 17.67 23.17 16.80 19.38
ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND ND
18.24 19.22 21.04 19.37 30.41 31.46 43.79 53.56 42.42 49.82
Recoveries were adjusted to a dry matter basis. Samples marked (1) and (2) are center points. T = tocopherol; T3 = tocotrienol; and ND = not detected. TABLE III Contents of Tocopherols and Tocotrienols in Rye Bread Without (Control Experiment) and With the Addition of Oila Tocol Concentration (mg/g)
Sample Without the addition of oil 23°C 23°C 30°C (1) 30°C (2) 37°C 37°C With the addition of oil 2%, 23°C 2%, 37°C 5%, 30°C (1) 5%, 30°C (2) 8%, 23°C 8%, 37°C a
a-T
a-T3
b-T
b-T3
g-T
g-T3
d-T
d-T3
Total
1.42 1.38 1.50 1.36 1.06 1.45
0.53 0.39 0.46 0.54 0.44 0.40
0.38 0.26 0.18 0.21 0.21 0.25
1.15 1.49 1.29 1.15 1.20 1.34
ND ND ND ND ND ND
ND ND ND ND ND ND
ND ND ND ND ND ND
ND ND ND ND ND ND
3.48 3.52 3.43 3.26 2.90 3.44
6.20 5.70 7.55 7.70 8.39 9.29
0.42 ND ND ND 0.45 ND
1.51 1.32 1.37 1.56 1.37 1.50
2.12 1.88 2.39 3.12 2.59 2.78
0.77 0.73 1.32 1.56 0.96 0.96
2.49 2.04 5.91 6.27 9.34 9.93
ND ND ND ND ND ND
0.40 ND ND ND 0.49 ND
13.92 11.66 18.53 20.19 23.60 24.46
Recoveries were adjusted to a dry matter basis. Samples marked (1) and (2) are center points. T = tocopherol; T3 = tocotrienol; and ND = not detected. Vol. 92, No. 3, 2015
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mixed into a dough, but this was found to result in a substantial loss of vitamin E. This may be because the dough-making process (without the addition of palm oil) resulted in a substantial incorporation of oxygen in the dough, which may have facilitated lipoxygenase-catalyzed oxidation of unesterified, polyunsaturated fatty acids; indeed, the destruction of vitamin E in this way has been reported previously (Wennermark and J¨agerstad 1992) but was not investigated in this study. Whole grain cereals naturally have relatively high levels of enzyme activity. When the germ is incorporated during the milling process, these enzymes may have a negative effect on breadmaking qualities. A reduced enzyme activity in refined (white) flour has been reported in literature, and aging for refined flour is recommended to enhance baking performance. In contrast, however,
Fig. 1. Vitamin E content in wheat bread sample production (5% palm oil fermented at 30°C for 5 h) including after mixing, fermentation, final proof, and baking. Error bars show the standard error of the mean of three replicates.
wholemeal flour must be used fresh (unaged) to avoid changes to, or degradation of, significant components including glutenforming proteins and essential nutrients, preventing oxidation of lipids and any associated nutrients, particularly vitamin E (Dai Suter, personal communication). It was found in the current work that both temperature and fermentation time had minimal effects on total tocol quantity in the final wheat meal bread, and the increase of vitamin E content in the bread is mainly attributable to the addition of palm oil. In the wheat bread variety without oil, the decline of vitamin E from flour to bread ranged from 76 to 89%, whereas the wheat bread with an addition of 2% palm oil had a reduction of only 48%. The total reduction of vitamin E content decreased with the content of palm oil, so that at 8% palm oil the total of vitamin E content was increased by 52% compared with wheat meal with no oil. Most typical rye breads made from wholemeal are prepared by using the sourdough process in which the main ingredients (flour, water, and starter culture) are mixed and fermented for about 24 h, and the red palm oil in this study was added after the fermentation stage. The first step involved 35% of the total rye meal without the addition of yeast. Yeast, when added to a wheat dough formulation, consumes oxygen (which may account for the increased vitamin E in the final baked bread). However, the absence of yeast in the rye bread formulation gave a reduction of vitamin E compared with the wholemeal wheat bulk fermentation, and previous studies have reported a reduction of up to 60% of vitamin E activity in rye sourdough processes (Wennermark and J¨agerstad 1992). The content of vitamin E in rye bread without the addition of red palm oil was also diminished by 88–90% compared with the raw meal. However, with the addition of 2% palm oil, the loss was reduced to 61%. Furthermore, reduction was even lower at 8% palm oil, with a deterioration of only 18% of the vitamin E content. Palm oil contains similar amounts of unsaturated and saturated fatty acids, making it a very stable ingredient that is less prone to oxidize. It is well known to have high levels of b-carotene (the precursor of vitamin A), as well as several other carotenes and vitamin E. This causes the commercial-grade palm oil to appear a reddish color because of the high carotene content (Enig 2000). b-Carotene is insoluble in water but soluble in fats and oils as a result of the conjugated, double-bond structure of the molecule. Once exposed to air it will not only oxidize but also decompose.
Fig. 2. Vitamin E content in rye bread production, including inoculation, fermentation (at 30°C, 5% palm oil), mixing, final proof, and baking. Error bars show the standard error of the mean of three replicates. TABLE IV Vitamin E Contents of Final (Baked) Breads Sample Wheat breads 0% 2% 5% 8% Rye breads 0% 2% 5% 8% 330
Per 100 g (mg)
Per 64 g Serving (%)
488 1,947 3,093 4,740
2.2 8.8 14.0 21.4
334 1,279 1,936 2,403
1.6 5.8 8.8 10.8
CEREAL CHEMISTRY
Fig. 3. Images of wheat bread produced with A, 0%; B, 2%; C, 5%; and D, 8% red palm oil.
For example, Heinonen et al. (1997) reported a synergistic effect of a-tocopherol and b-carotene on oxidation of 10% oil-in-water emulsion of rapeseed oil. Natural secondary antioxidants including carotenoids often possess excellent singlet oxygenquenching properties (Decker 1998). This synergistic effect of a-tocopherol and b-carotene could be the reason for the increasing amount of vitamin E recovered in final baked loaves, and further research is warranted to elucidate this. Nevertheless, the current results demonstrate that, environmental considerations aside, palm oil could be a useful ingredient in wholemeal and rye flour based baked goods to increase the health benefits of the final products. In addition, it has been demonstrated that vitamin E incorporated into baked products in the form of palm oil has survived the baking temperatures exceeding 90°C (Simonne and Eitenmiller 1998) within the loaves. CONCLUSIONS Our results show that compared with the control loaves baked in this study, the inclusion of palm oil increased the quantity of tocopherols and tocotrienols found in both wholemeal wheat and rye. It is concluded that palm oil was effective in increasing the vitamin E content of whole grain breads. A further advantage was that a wider range of E vitamers was retained during breadmaking. Therefore, the use of palm oil from sustainable plantations shows promise as a method of increasing the health benefits of baked goods via an increased nutrient content in the final product. Although the use of 8% palm oil causes change in color and a slight decrease in final loaf volume, if a dose of 5% palm oil is used, rather than the 2% vegetable oils (e.g., canola or sunflower) used in commercial baking processes, the health benefits are improved but quality of the final product (in terms of color, taste, volume, and shelf life) is not affected. ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support provided through a Grain Research Scholarship (awarded to Oliver Buddrick) from the Grains Research and Development Corporation, Canberra, Australia. They also thank David Hogan of Laucke Flour Mill of Bridgewater, Victoria, for the supply of whole grain wheat and rye and the Malaysian Palm Oil Board for supplying the red palm oil used in this work. The authors declare that they have no conflict of interest. LITERATURE CITED AACC International. Approved Methods of Analysis, 11th Ed. Method 44-15.02. Moisture—Air-oven methods. Approved October 30, 1975. http://dx.doi.org/10.1094/AACCIntMethod-44-15.02. Available online only. AACC International: St. Paul, MN. Agnoletti, D., Zhang, Y., Czernichow, S., Galan, P., Hercberg, S., Safar, M. E., and Blacher, J. 2011. Fortification of vitamin B12 to flour and the metabolic response. Pages 437-449 in: Flour and Breads and Their Fortification in Health and Disease Prevention. V. R. Preedy, R. Watson, and V. Patel, eds. Academic Press: San Diego, CA. Akhtar, S., and Ashgar, A. 2011. Mineral fortification of whole wheat flour: An overview. Pages 263-271 in: Flour and Breads and Their Fortification in Health and Disease Prevention. V. R. Preedy, R. Watson, and V. Patel, eds. Academic Press: San Diego, CA. Al-Saqer, J. M., Sidhu, J. S., Al-Hooti, S. N., Al-Amiri, H. A., Al-Othman, A., Al-Haji, L., Ahmed, N., Mansour, I. B., and Minal, J. 2004. Developing functional foods using red palm olein. IV. Tocopherols and tocotrienols. Food Chem. 85:579-583. Azzi, A. 2007. Molecular mechanism of alpha-tocopherol action. J. Free Radic. Biol. Med. 43:16-21. Buddrick, O., Jones, O., Morrison, P. D., and Small, D. M. 2013. Heptane as a less toxic option than hexane for the separation of vitamin E from food products using normal phase HPLC. RSC Adv. 3:24063-24068.
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[Received July 24, 2014. Accepted December 9, 2014.] Vol. 92, No. 3, 2015
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