Vista arquitectónica del modelo de análisis

1.4.1 BIOSYNTHESIS AND COMPARTMENTALIZATIONIN PLANTSAND GRAINS

Tocopherols and/or tocotrienols have been found in all photosynthetic organisms, and they are always located in membranes of the cell. In higher plants, tocopherols are mainly synthesized in chloroplasts and plastids, and tocotrienols outside chloro-plasts. The chromanol ring is built up from a shikimic acid pathway intermediate homogentisate and the phytyl side chain from an isoprenoid pathway intermediate phytyl pyrophosphate (Figure 1.3). Phytyl transferase combines these two units to form 2-methyl-6-phytyl benzoquinol that is methylated and cyclized by methyl transferases and cyclases. S-adenosylmethionine serves as the methyl donor. To produce δ-tocopherol, 2-methyl-6-phytyl benzoquinol undergoes cyclization, and to produce β- and γ-tocopherols, it is also methylated. The final product of the bio-synthetic pathway, α-tocopherol, is fully methylated. The biosynthesis of toco-trienols follows the same reactions, except geranylgeranyl pyrophosphate is used as the source for the unsaturated side chain. Thus, the availability of homogentizate, S-adenosylmethionine and phytol or geranylgeranol is essential for the synthesis of tocopherols and tocotrienols (e.g., Hess, 1993; Bramley et al., 2000). Many of the biosynthetic reactions are known to regulate the total amount of tocopherols and tocotrienols. The activity of different methyltransferases is important in determining the relative proportions of different vitamers in seeds/grains, which is an interesting finding for plant breeding purposes (see Bramley et al., 2000).

In general, total amounts of tocopherols and tocotrienols in plants are greatest in mature leaves and other light-exposed tissues and smallest in roots and other

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tissues grown under diminished light. In photosynthetic tissues, α-vitamers domi-nate, while in nonphotosynthetic tissues, the ratio of γ- over α-vitamers is higher (Hess, 1993). The vitamin E content of plants varies depending on, e.g., the intensity of sunlight and soil state and other growing conditions (Crawley, 1993).

1.4.2 OILSAND CEREALSAS IMPORTANT SOURCESOF TOCOPHEROLS AND TOCOTRIENOLS

The major sources of vitamin E in the Western diet are fats and oils, cereal and vegetable products. In the Unites States, these three groups of food items contrib-uted to 20.2%, 14.6% and 15.1% (Murphy et al., 1990) and in Finland, to 41%, 18% and 3% (Heinonen and Piironen, 1991) of the total vitamin E intake from the diet, respectively.

1.4.3 OIL GRAINS

The descending order of total tocopherols and tocotrienols in commercial oils is as follows: corn, soybean, palm (800–1100 µg/g) > cottonseed, sunflower, rapeseed FIGURE 1.3 General biosynthetic pathway of tocopherols in chloroplasts and proplasts of plant cells. Biosynthesis of tocotrienols is similar except for reduction of geranylgeranyl pyrophosphate to phytyl pyrophosphate, which is omitted. SAM = S-adenosylmethionine.

[Data modified from Hess (1993).]

Terpenoid pathway

Geranylgeranyl pyrophosphate oxidoreductase

Phytyl pyrophosphate

Shikimic acid pathway

Homogentisic acid

phytyl transferase CO2 + PPi

2-Methyl-6-phytyl benzoquinol methyl transferase cyclase SAM

2,3-Dimethyl-6-phytyl δ-Tocopherol benzoquinol

methyl transferase cyclase SAM

γ -Tocopherol β-Tocopherol

methyl transferase methyl transferase SAM SAM

α -Tocopherol

Tocopherols and Tocotrienols from Oil and Cereal Grains 11

(550–800 µg/g) > peanut, olive (150–350 µg/g) > coconut, palm kernel (≤50 µg/g) (Tan, 1989). The contents in some breeding lines significantly exceed those men-tioned above. Most oils contain only tocopherols, while palm, palm kernel and rice bran oil contain significant amounts of tocotrienols (Eitenmiller, 1997). The richest source of tocopherols, wheat germ oil, is mainly consumed as a tocopherol supple-ment and not as ordinary edible oil (Table 1.2).

Soybean oil is the most important source of vitamin E in the Western world. It contains, on average, 1000 µg/g of tocopherols (Eitenmiller, 1997; Kamal-Eldin and Andersson, 1997). The total tocopherol contents of 14 breeding lines with commodity-type fatty acid composition were 1406–2195 µg/g, while those of the major isomers γ-, δ- and α-tocopherols were 850–1171, 254–477 and 44–158 µg/g, respectively (Dolde et al., 1999).

Sunflower oil is a rich source of vitamin E. It contains, on average, 400–700 µg/g total tocopherols (Van Niekerk and Burger, 1985; Eitenmiller, 1997; Kamal-Eldin and Andersson, 1997). Total tocopherol concentration of 66 experimental breeding lines with commodity-type fatty acid compositions was 982 ± 27 µg/g, of which >99%

was α-tocopherol. α-Tocopherol was present in some lines as a minor component.

The tocopherol compositions of breeding lines with adjusted fatty acid compositions were similar (Dolde et al., 1999).

Palm oil is an exception among oil crops, because more than 85% of its vitamin E content consists of γ-tocotrienol, α-tocotrienol and α-tocopherol (Tan, 1989).

TABLE 1.2

Tocopherol (T) and Tocotrienol (T3) Contents of Refined Vegetable Oils (µg/g FW)

Oil α-T α-T3 β-T β-T3 γ-T γ-T3 δ-T δ-T3 Reference

Soybean 95 — 13 — 689 — 239 — 1

120 — 10 — 610 — 260 — 2

179 — 28 4 604 — 371 — 3

Corn 257 15 9 — 752 20 32 — 1

120 — 10 — 400 — 20 — 2

272 54 — 11 566 62 25 — 3

Cottonseed 403 — — 9 383 — 4 — 3

Sunflower 622 — 23 — 27 — — — 1

610 — 10 — 30 — 10 — 2

564 — 24 — 4 — — — 3

Safflower 449 — 12 — 26 — 6 — 1

Rapeseed (LEAR) 189 — — — 486 — 12 — 1

200 — — — 430 — 10 — 2

Linseed 5 — — — 573 — 7 — 1

Wheat germ 1507 36 312 — 527 18 — — 1

Olive oil 119 — — — 13 — — — 1

90 — — 4 5 — — — 3

References: 1. Syväoja et al., J. Am. Oil Chem. Soc. 63: 328–329 (1986); 2. Warner and Mounts, J. Am.

Oil Chem. Soc. 67: 827–831 (1990); and 3. Van Niekerk and Burger, J. Am. Oil Chem. Soc. 62: 531–538 (1985).

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Other tocotrienols (i.e., β-, δ-) are also present (Syväoja et al., 1986; Ong and Choo, 1997). Total vitamin E content in crude palm oil is between 600–1200 µg/g, while contents in palm oil fractions may be significantly smaller. Palm kernel oil has only

<100 µg/g vitamers (Eitenmiller, 1997; Ong and Choo, 1997). Tocotrienols and tocopherols are concentrated up to eightfold in the palm fatty acid distillate compared with the crude palm oil, which enables extraction of vitamin E compounds from this by-product.

Oils from genetically modified rapeseed/canola with a wide range of fatty acid compositions contained 478–677 µg/g of total tocopherols, of which >50% was γ- and about 30% was α-tocopherol. There was no relationship between the fatty acid and tocopherol compositions (Abidi et al., 1999). Genetically modified canola oils had similar tocopherol compositions with a range of 504–687 µg/g of total tocopherols (Dolde et al., 1999).

A study on flaxseed showed that this seed contains an average of 93 µg/g tocopherols, of which >96% was γ-vitamer (Oomah et al., 1997). The level of tocopherols was cultivar specific and was regulated by environmental conditions.

The hulls contained about 26% of total tocopherols and relatively more α- and δ-tocopherols than the embryo (Oomah and Mazza, 1998). Flaxseeds also contain an analogous compound, plastochromanol-8, consisting of a γ-chromanol and eight isoprene units in the side chain (Olejnik et al., 1997). The level of plastochromanol-8 in seeds was 17–74 µg/g and that of total tocopherols was 74–184 µg/g, of which 98–100% was γ-tocopherol (Velasco and Goffman, 2000). Tocopherol contents in linseed oil range from 400 to 600 µg/g (Syväoja et al., 1986; Kamal-Eldin and Andersson, 1997; Oomah and Mazza, 1998; Schöne et al., 1998).

For olive oil, total tocopherol level is ca 100–150 µg/g and α-tocopherol is the dominant (>90%) tocopherol present (Van Niekerk and Burger, 1985; Kamal-Eldin and Andersson, 1997). α-Tocopherol values of virgin olive oils from Greece ranged between 98 and 370 µg/g, while the sum of other vitamers was <50 µg/g. Small-sized Greek olives had slightly higher contents of α-tocopherol than medium-sized olives, at 239 and 198 µg/g, respectively (Psomiadou et al., 2000).

1.4.4 CEREAL GRAINS

Cereal grains are mainly recognized for their proteins and carbohydrates and for being valuable sources of energy and other nutrients, but they also contain substantial amounts of various lipids. Lipids are located in the cereal grain in membranes and spherosomes. They are unevenly distributed in grain fractions. Nonstarch lipid con-tents of cereal grains are for oat groats 5–9%, maize kernels 3.9–5.8%, barley 3.3–4.6%, rye 2.0–3.5%, millet 4.0–5.5% and rice 0.8–3.1% (Morrison, 1978; Youngs, 1986; Chung and Ohm, 2000). Cereals are considered only moderate sources of vitamin E, providing 6–23 mg of α-tocopherol equivalents/kg (Bramley et al., 2000).

Because cereals contain great amounts of tocotrienols (Table 1.3) with very low vitamin E activity, they are more valuable as sources of tocotrienols and tocopherols than vitamin E.

Wheat grains contain total tocopherol and tocotrienol of >40 µg/g (e.g., Barnes, 1983; White and Xing, 1997), where β-tocotrienol (>50%) and α-tocopherol

Tocopherols and Tocotrienols from Oil and Cereal Grains 13

(ca 20%) are the major E-vitamers, and α-tocotrienol and β-tocopherol are the minor E-vitamers (Table 1.3). Wheat germ is an especially rich source of α- and β-tocopherols and oil produced from it provides more vitamin E activity than most other oils (Piironen et al., 1986; Syväoja et al., 1986; Balz et al., 1992). In wheat, as in other cereals, tocopherols are concentrated in the germ, and tocotrienols are concentrated in the bran and endosperm (Table 1.4).

Barley grains contain all eight tocopherols and tocotrienols (Table 1.3), but there is a large variation in the total contents of tocopherols and tocotrienols. The major vitamers are α-tocotrienol (contributing to >50%), α-tocopherol, β-tocotrienol and γ-tocotrienol, and they are generally correlated positively with each other (Peterson and Qureshi, 1993). The total vitamer content in whole-grain barley of 30 genotypes TABLE 1.3

Tocopherol (T) and Tocotrienol (T3) Contents of Cereal Grains (µg/g FW)

Cereal α-T α-T3 β-T β-T3 γ-T γ-T3 δ-T δ-T3 Total

T+T3 Reference

Wheat 10 4 5 21 — — <1 — 40.4 1

12 4 6 26 — — — — 47.8 2

Rye 10 14 3 11 — — — — 38.0 1

10 12 2 7 <0.2 — — — 32.4 2

Barley 3 16 <1 6 1 6 <1 — 32.0 1

4 20 <0.2 4 1 8 — 0.6 36.9 2

10 33 0.7 7 0.7 5 0.7 0.9 58.5 3

Oats 9 25 0.6 3 — <0.2 — — 38.0 2

8 15 0.9 0.9 0.9 — — 0.2 25.9 3

Rice, brown 6 4 1 <0.1 1 7 <1 — 19.0 1

8 4 0.7 <0.2 4 10 0.5 0.7 27.0 2

References: 1. Piironen et al., Cereal Chem. 63: 78–81 (1986); 2. Balz et al., Fat Sci. Technol. 94: 209–213 (1992); and 3. Peterson and Qureshi, Cereal Chem. 70: 157–162 (1993).

TABLE 1.4

Distribution of Tocopherols (T) and Tocotrienols (T3) in Milling Fractions of Swedish Winter Wheat (µg/g dry matter)

Milling Fraction α-T α-T3 β-T β-T3

Wholemeal 12 5 5 30

White flour 6 2 2 21

Bran 4 20 2 94

Germ 130 5 50 25

Data modified from Jägerstad and Håkansson (1988) and Wennermark and Jägerstad (1992).

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grown in three locations in the United States ranged from 42 to 80 µg/g with an average of 58.5 µg/g and suggested that the variation was mainly between genotypes and to a lesser extent between locations (Peterson and Qureshi, 1993). The tocopherol and tocotrienol contents of these 30 genotypes as well as that of two hull-less barley cultivars, 71.0 ± 9.5 µg/g (Wang et al., 1993), were greater than the average level of 30 µg/g reported earlier (Barnes, 1983; Piironen et al., 1986; Balz et al., 1992).

Within a barley grain, the germ contains high levels of α-tocopherol, while the hull and endosperm contain all vitamers and are especially rich in tocotrienols (Barnes, 1983; White and Xing, 1997). Unlike most cereals, the barley contains significant amounts of tocotrienol, namely, the β-vitamer, in the germ (Peterson, 1994). α-Tocotrienol seems to accumulate in the starchy endosperm (Wang et al., 1993). When studying the distribution of tocopherols and tocotrienols in hull-less barley fractions, pearling flour had the greatest total concentration of 205 µg/g, which was almost three times as much as in the whole grain. Since the yield of pearling flour is ca 20% of the total grain weight, it is a potential ingredient for health-promoting food products (Wang et al., 1993; Peterson, 1994).

Oat grains and dehulled grains (called groats) contain on average 2–11% of lipids, most of which are located in endosperm and bran, as reviewed by Zhou et al.

(1999). The major E-vitamers of oat groats are α-tocotrienol and α-tocopherol, contributing to 57% and 31% of the total, respectively (Peterson and Qureshi, 1993).

β-, γ- and δ-Vitamers occur only as minor components (Table 1.3). Oat groats of 12 genotypes contained 25.9 µg/g of tocopherols and tocotrienols with a range of 19–30 µg/g (Peterson and Qureshi, 1993), which is at a similar level to that found in other studies (Barnes, 1983; Piironen et al., 1986; Peterson and Wood, 1997).

There were significant differences in the total vitamin contents between the 12 genotypes and the three growing locations (Peterson and Qureshi, 1993). Within the oat grain, the germ was richest in total E-vitamers, because the contents in the hulls, germ and endosperm were 3, 130 and 40 µg/g, respectively (Peterson, 1995).

In 25 high-oil oat selections with 6.9–18.1% of lipids, the total tocopherol and tocotrienol contents ranged from 25 to 67 µg/g with a significant positive correlation to lipids, especially for tocotrienols (Peterson and Wood, 1997). In these high-oil oats, more lipids including tocotrienols were distributed in the endosperm than in control oats, while the germs were similar. Analysis of hand-dissected fractions indicates that tocotrienols, mainly α-tocotrienol, were located in the endosperm, while tocopherols, mainly α-tocopherol and γ-tocopherol, were present in the germ (Peterson, 1995; Peterson and Wood, 1997).

In rye grain and its products, α-tocopherol and α- and β-tocotrienols are the three dominant vitamers (Table 1.3). The total amount of tocopherols and toco-trienols in whole grain lies in the range of 30–50 µg/g (e.g., Barnes, 1983; White and Xing, 1997).

Two other cereal grains; namely, corn and rice, are important in terms of being utilized as sources of tocopherols and tocotrienols. In corn, there is a large variation, 28–102 µg/g, in the total amount and profile of tocopherols and tocotrienols (White and Xing, 1997). In general, γ-tocopherol is the predominant vitamer contributing up to 80% of the total amount in most cultivars, while α-tocopherol levels are greater or equal to those of γ-tocopherol in some varieties and genotypes (Weber, 1984;

Tocopherols and Tocotrienols from Oil and Cereal Grains 15

Kurilich and Juvik, 1999). The germ and tip-cap contained 63–91% of total toco-pherols and tocotrienols of hand-dissected corn kernels, while the endosperm con-tained 9–37% of them. As with other cereals, tocotrienols (25–35% of total) are concentrated in the endosperm and are almost lacking in the germ, which is rich in α- and γ-tocopherols (Weber, 1987).

In rice grain, γ-tocotrienol, γ-tocopherol and α-tocopherol are the major vitamers (White and Xing, 1997), although some studies report α-tocotrienol levels higher than those of γ-vitamers (Table 1.3). Comparison of tocopherol and tocotrienol results from different studies is difficult, because rice samples may have been processed to different extents, being rough (unshelled), brown (hull was removed) or milled (bran was removed and sometimes polished) (Moldenhauer et al., 1998).

A coproduct from milling, rice bran, is an exceptionally rich source of tocotrienols, and rice bran oil is produced from it. There is evident variation in the tocopherol and tocotrienol compositions of rice bran oils, which can be explained both by differences in rice varieties and/or by processing. For example, total vitamer contents of five brands varied from 88 to 1610 µg/g, and the proportion of tocotrienols varied from 51 to 82% (Rogers et al., 1993).

Tocopherol and tocotrienol profiles of buckwheat and millet are different compared to other cereal grains. In both cereals, γ-tocopherol is the dominant vitamer, and tocotrienols occur in minor amounts. Buckwheat contains 54–62 µg/g and millet 22–32 µg/g of vitamers, of which ca 90% and 70% is γ-tocopherol (Piironen et al., 1986; Balz et al., 1992). Grain amaranth of several cultivars and growing environments showed a large variation in the content of total E-vitamers, 5–29 µg/g, and tocotrienols, mainly β- and γ-, representing 39–74% of this total (Lehmann et al., 1994).

1.4.5 EFFECTS OF GROWING CONDITIONS

Tocopherol and tocotrienol contents depend to a large extent on the geographical and climatic conditions, the maturity of seeds during harvesting and the variety of the plant (Kamal-Eldin and Andersson, 1997; White and Xing, 1997). The temperature is the most effective environmental factor controlling tocopherol and tocotrienol content. In general, the levels are higher in oilseeds and cereal grains grown at high temperatures than at low temperatures, indicating that the biosynthesis is promoted by increased temperature (Marquard, 1990; White and Xing, 1997). However, an experiment performed by Dolde et al. (1999) showed the opposite for soybeans, in which tocopherol contents were higher at low rather than high temperatures. Despite the variation due to growing temperature, the tocopherol composition is mainly determined by the crop (Dolde et al., 1999).

Tocopherol and tocotrienol contents and compositions may be modified by traditional and modern plant breeding techniques (Marquard, 1990). Genetic modifi-cation has been used to change the profile and content of canola tocopherols (Abidi et al., 1999). In general, plant breeding of grains is focused on the fatty acid composition to produce tailor-made oils with special characteristics such as good stability or high linolenic content. Only recently, the importance of tocopherols and other minor lipid components has been recognized. It is suggested that genetic modifications for altered tocopherol and tocotrienol contents and for altered fatty

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acid composition of oil grains can be done independently in many oil grains, while there may be some relationship in soybean (Dolde et al., 1999).

1.5 PRODUCTION OF TOCOPHEROL AND