Plant Sterols in Cereals and Cereal Products

The total plant sterol contents (free sterols and covalently bound structures) of the main cereals cultivated in Finland were determined. Furthermore, sterol contents were determined for different flour and bran fractions in the milling process of wheat and rye, as well as plant sterol contents in various milling and retail bakery products. The sample preparation procedure included acid and alkaline hydrolysis to liberate sterols from their glycosides and esters, respectively. Free sterols were extracted and, after recovery using solid‐phase extraction, derivatized to trimethylsilyl ethers for gas chromatography (GC) analysis. We used GC with a mass spectrometer (MS) for identification. When two cultivars of rye, wheat, barley, and oats grown in the same year were compared, the highest plant sterol content was observed in rye (mean content 95.5 mg/100 g, wb), whereas the total sterol contents (mg/100 g, wb) of wheat, barley, and oats were 69.0, 76.1, and 44.7, respectively. In addition, the 10 rye cultivars and breeding lines compared had total sterol contents of 70.7–85.6 mg/100 g. In the milling process of rye and wheat, the plant sterols fractionated according to the ash content of the corresponding milling product. In all cereal grain and milling product samples, sitosterol was the main sterol. The level of stanols differed in the different milling process samples; it was lower in the most refined rye and wheat flours (≈15%) than in the bran fractions (≈30% in the bran with 4% ash content). Rye bread with whole meal rye flour as the main or only ingredient was a good source of sterols. Sterol content was higher than that of wheat bread, whereas plant sterol content of other bakery products was affected by the type and amount of fat used in baking.     

Fermentation Reduces Free Asparagine in Dough and Acrylamide Content in Bread

Free asparagine is an important precursor for acrylamide in cereal products. The content of free asparagine was determined in 11 milling fractions from wheat and rye. Whole grain wheat flour contained 0.5 g/kg and whole grain rye flour 1.1 g/kg. The lowest content was found in sifted wheat flour (0.2 g/kg). Wheat germ had the highest content (4.9 g/kg). Fermentation (baker's yeast or baker's yeast and sourdough) of doughs made with the different milling fractions was performed to investigate whether the content of free asparagine was reduced by this process. In general, most of the asparagine was utilized after 2 hr of fermentation with yeast. Sourdough fermentation, on the other hand, did not reduce the content of free asparagineas efficiently but had a strong negative impact on asparagine utilization by yeast. This indicates that this type of fermentation may result in breads with higher acrylamide content than in breads fermented with yeast only. The effect of fermentation time on acrylamide formation inyeast‐leavened bread was studied in a model system. Doughs (sifted wheat flour with whole grain wheat flour or rye bran) were fermented for a short (15+15 min) or a long time (180+180 min). Compared with short fermentation time, longer fermentation reduced acrylamide content in bread made with whole grain wheat 87%. For breads made with rye bran, the corresponding reduction was 77%. Hence, extensive fermentation with yeast may be one possible way to reduce acrylamide content in bread.     

Antioxidant activity of alkylresorcinols from rye bran and their protective effects on cell viability of PC-12 AC cells

Alkylresorcinols (ARs) are phenolic lipids that are present in high amounts in the bran layer of different cereals. Rye samples, cultivar Hazlet, and a white rye genotype, RT202, were analyzed for their antioxidant properties and AR content and composition, based on six fractions of the bran, where 1 was the outermost fraction and 6 was the bran fraction closest to the endosperm. Gas chromatography-mass spectrometry (GC-MS) analysis demonstrated that the most commonly found AR homologue in Hazlet rye is C19:0 and that the total amount of ARs decreases from the outermost to innermost fractions. The antioxidant activity using oxygen radical absorbance capacity (ORAC) for both white rye genotype RT202 and Hazlet brans was determined to decrease from the outermost fraction (136.05 μmol TE/g for Hazlet fraction 1 and 186.57 μmol TE/g for white rye genotype RT202 fraction 1) to the innermost fraction (9.84 μmol TE/g for Hazlet fraction 6 and 78.75 μmol TE/g for white rye genotype RT202 fraction 2). A positive relationship was seen with GC-MS results. Treatment of PC-12 AC cells with Hazlet fraction 1 increased mitochondrial biogenesis as determined using mitochondrial fluorescent dyes. In the presence of a prooxidant (AAPH), PC-12 AC cells were better protected from free radical attack when treated with Hazlet fraction 1 than with all other bran fractions. The results suggest that higher AR content in bran fractions confers antioxidant protection against free radical damage.     

Alkylresorcinol content and homologue composition in durum wheat (Triticum durum) kernels and pasta products

The total alkylresorcinol (AR) content and relative homologue composition of 21 durum wheat (Triticum durum) kernel samples, as well as 5 pasta products and the corresponding flour mixtures, were determined. Durum wheat contained on average 455 microg/g ARs, and the average relative homologue composition was C17:0 (0.4%), C19:0 (14%), C21:0 (58%), C23:0 (21%), and C25:0 (6.5%). The homologue composition was found to be relatively consistent among samples, with durum wheat being different from common wheat by having a higher proportion of the longer homologues. No differences in content or homologue composition were observed in pasta products compared to flour ingredients, showing that alkylresorcinols are stable during pasta processing. The ratio of the homologues C17:0 to C21:0 was < or =0.02 for whole grain durum wheat products, which is different from those of common wheat (0.1) and rye (0.9).     

Alkylresorcinols as markers of whole grain wheat and rye in cereal products

The alkylresorcinol (AR) content of Swedish wheat grain samples, as well as of cereal ingredients and cereal foods containing wheat and rye, was determined. The average total AR content in Swedish wheat was 412 microg/g (ranging between 227 and 639 microg/g), which is lower than that in Swedish rye analyzed in a previous study. The relative composition of AR homologues was consistent for wheat samples and differed markedly from that of rye. Notably, the ratio of the homologues C17:0/C21:0 was approximately 0.1 in wheat and approximately 1.0 in rye, indicating that it can be used to distinguish between those two cereals. The AR content in cereal foods commonly consumed in Sweden varied widely, from nondetectable levels in white wheat flour and products not containing the outer parts of wheat and/or rye to >900 microg/g in some whole grain rye products. AR content in cereal foods was calculated from their recipes using average AR values for the cereal ingredients determined in this study. As there was a good correlation between calculated and analyzed AR levels in cereal foods (R2 = 0.91), it is possible to estimate the proportion of whole grain wheat and/or rye in a given cereal product on the basis of AR content and C17:0/C21:0 ratio. ARs appear to be good markers of whole grain wheat and rye in foods, and their analysis may be an objective way to identify foods rich in whole grain wheat and/or rye or brans thereof.     

Oxalate content of cereals and cereal products

Detailed knowledge of food oxalate content is of essential importance for dietary treatment of recurrent calcium oxalate urolithiasis. Dietary oxalate can contribute considerably to the amount of urinary oxalate excretion. Because cereal foods play an important role in daily nutrition, the soluble and total oxalate contents of various types of cereal grains, milling products, bread, pastries, and pasta were analyzed using an HPLC-enzyme-reactor method. A high total oxalate content (>50 mg/100 g) was found in whole grain wheat species Triticum durum (76.6 mg/100 g), Triticum sativum (71.2 mg/100 g), and Triticum aestivum (53.3 mg/100 g). Total oxalate content was comparably high in whole grain products of T. aestivum, that is, wheat flakes and flour, as well as in whole grain products of T. durum, that is, couscous, bulgur, and pasta. The highest oxalate content was demonstrated for wheat bran (457.4 mg/100 g). The higher oxalate content in whole grain than in refined grain cereals suggests that oxalic acid is primarily located in the outer layers of cereal grains. Cereals and cereal products contribute to the daily oxalate intake to a considerable extent. Vegetarian diets may contain high amounts of oxalate when whole grain wheat and wheat products are ingested. Recommendations for prevention of recurrence of calcium oxalate stone disease have to take into account the oxalate content of these foodstuffs.     

Steryl phenolic acid esters in cereals and their milling fractions

The steryl ferulate contents of rye and wheat grains and their milling fractions were analyzed using a reversed-phase high-performance liquid chromatographic (HPLC) method. HPLC-mass spectrometry was used for identification. In addition, steryl ferulates of some selected milling byproducts were determined. The total steryl ferulate contents of rye and wheat grains were 6.0 and 6.3 mg/100 g, respectively. Uneven distribution of steryl ferulates in the grains led to considerable differences in the milling products; their steryl ferulate contents ranged from trace amounts in flours with low ash content to 20 and 34 mg/100 g in rye and wheat brans, respectively. Campestanyl ferulate and sitostanyl ferulate were the main components, followed by campesteryl ferulate and sitosteryl ferulate, whereas sitosterol was the main component in total sterols. Among the other samples, a byproduct of rice milling (pearling dust) was the best source of steryl ferulates, its total steryl ferulate content being 119 mg/100 g, whereas no measurable amounts of steryl ferulates were measured in oat bran or pearling dust of barley. The results indicated that rye and wheat and especially their bran fractions are comparable to corn as steryl ferulate sources.     

Alkylresorcinols in selected Polish rye and wheat cereals and whole-grain cereal products

The alkylresorcinol content and homologue composition in selected Polish rye and wheat cultivars and selected whole-grain cereal products were determined in this study. Cereal grains and whole-grain cereal products were extracted with acetone, whereas bread types were extracted with hot 1-propanol. The average alkylresorcinol content in tested rye (approximately 1100 mg/kg DM) and wheat (approximately 800 mg/kg DM) grains harvested in Poland was within the range previously reported in Swedish and Finnish samples. The total alkylresorcinol content in tested cereal products available on the Polish market varied from very low levels in barley grain-based foods up to 3000 mg/kg DM in wheat bran. The total alkylresorcinol content in 14 bread samples extracted with hot 1-propanol varied from approximately 100 mg/kg DM in whole bread made with honey up to approximately 650 mg/kg DM in whole-rye bread. Calculated ratios of C17:0 to C21:0 homologues, a useful parameter previously used to distinguish between rye and wheat cereals and their derived products, was about 1.2-1.4 in rye products, about 0.2 in wheat products, and varied between 0.2 and 0.6 in cereal-derived products containing a mixture of whole rye and/or wheat. The data set obtained were subsequently compared using cluster and principal component analysis, which allowed the tested cereal products to be classified into two major groups consisting of whole-rye or whole-wheat products, respectively. On the basis of that approach, mixed cereal products containing rye and wheat bran or whole rye and wheat flour were grouped between those two well-defined clusters. Our work not only provides a detailed examination of alkylresorcinols in selected Polish rye and wheat cultivars and selected whole-grain cereal products, but also demonstrates that this type of analysis accompanied by the use of proper statistical algorithms offers an objective way to evaluate the quality of whole-grain rye and/or wheat and their derived products.     

Contents of phenolic acids, alkyl- and alkenylresorcinols, and avenanthramides in commercial grain products

The contents of free and total phenolic acids and alk(en)ylresorcinols were analyzed in commercial products of eight grains: oat (Avena sativa), wheat (Triticum spp.), rye (Secale cerale), barley (Hordeum vulgare), buckwheat (Fagopyrum esculentum), millet (Panicum miliaceum), rice (Oryza sativa), and corn (Zea mays). Avenanthramides were determined in three oat products. Free phenolic acids, alk(en)ylresorcinols, and avenanthramides were extracted with methanolic acetic acid, 100% methanol, and 80% methanol, respectively, and quantified by HPLC. The contents of total phenolic acids were quantified by HPLC analysis after alkaline and acid hydrolyses. The highest contents of total phenolic acids were in brans of wheat (4527 mg/kg) and rye (4190 mg/kg) and in whole-grain flours of these grains (1342 and 1366 mg/kg, respectively). In other products, the contents varied from 111 mg/kg (white wheat bread) to 765 mg/kg (whole-grain rye bread). Common phenolic acids found in the grain products were ferulic acid (most abundant), ferulic acid dehydrodimers, sinapic acid, and p-coumaric acid. The grain products were found to contain either none or only low amounts of free phenolic acids. The content of avenanthramides in oat flakes (26-27 mg/kg) was about double that found in oat bran (13 mg/kg). The highest contents of alk(en)ylresorcinols were observed in brans of rye (4108 mg/kg) and wheat (3225 mg/kg). In addition, whole-grain rye products (rye bread, rye flour, and whole-wheat flour) contained considerable levels of alk(en)ylresorcinols (524, 927, and 759 mg/kg, respectively).     

Alkylresorcinols in wheat varieties in the HEALTHGRAIN Diversity Screen

The contents of alkylresorcinols (AR) were analyzed in 131 winter wheats, 20 spring wheats, 10 durum wheats, 5 spelt wheats, and 10 early cultivated forms of wheat (5 diploid einkorn and 5 tetraploid emmer), which are part of the HEALTHGRAIN diversity screen. AR were analyzed by gas chromatography (GC), which provides both total contents and relative homologue compositions, as well as with a Fast Blue colorimetric method that provides only total contents but which is fast and easily screens a large number of samples. There was considerable variation in the total AR content analyzed with GC: winter wheat (220-652 microg/g of dm), spring wheat (254-537 microg/g of dm), durum wheat (194-531 microg/g of dm), spelt (490-741 microg/g of dm), einkorn (545-654 microg/g of dm), and emmer wheat (531-714 microg/g of dm). The relative AR homologue composition was different for different types of wheat, with a C17:0 to C21:0 ratio of 0.1 for winter, spring, and spelt wheats, 0.04 for einkorn and emmer wheat, and 0.01 for durum wheat. The total AR content analyzed with the Fast Blue method was lower than that analyzed with GC but there was a good correlation between the two methods (R(2) = 0.76).     

Fructan Content of Rye and Rye Products

The fructan content of Finnish rye grains (13 samples, seven cultivars, harvested in 1998‐2000) varied at 4.6–6.6 g/100 g (db). Commercial whole grain rye flour and rye flakes had fructan content of 4 g/100 g, light refined rye flour had fructan content of 3 g/100 g, and rye bran had fructan content of 7 g/100 g. Fructan content as high as 23 g/100 g was detected in the water‐extractable concentrate of rye bran. Finnish soft rye bread and rye crisp bread contained 2–3 g of fructan/100 g. According to the suggested new definition of dietary fiber, fructans are also classified as dietary fiber. This means that the dietary fiber content of some cereal foods such as rye products may be increased by as much as 20% due to the presence of fructans in the grain.     

Bran Characteristics and Bread‐Baking Quality of Whole Grain Wheat Flour

Variations in physical and compositional bran characteristics among different sources and classes of wheat and their association with bread‐baking quality of whole grain wheat flour (WWF) were investigated with bran obtained from Quadrumat milling of 12 U.S. wheat varieties and Bühler milling of six Korean wheat varieties. Bran was characterized for composition including protein, fat, ash, dietary fiber, phenolics, and phytate. U.S. soft and club wheat brans were lower in insoluble dietary fiber (IDF) and phytate content (40.7–44.7% and 10.3–17.1 mg of phytate/g of bran, respectively) compared with U.S. hard wheat bran (46.0–51.3% and 16.5–22.2 mg of phytate/g of bran, respectively). Bran of various wheat varieties was blended with a hard red spring wheat flour at a ratio of 1:4 to prepare WWFs for determination of dough properties and bread‐baking quality. WWFs with U.S. hard wheat bran generally exhibited higher dough water absorption and longer dough mixing time, and they produced smaller loaf volume of bread than WWFs of U.S. soft and club wheat bran. WWFs of two U.S. hard wheat varieties (ID3735 and Scarlet) produced much smaller loaves of bread (<573 mL) than those of other U.S. hard wheat varieties (>625 mL). IDF content, phytate content, and water retention capacity of bran exhibited significant relationships with loaf volume of WWF bread, whereas no relationship was observed between protein content of bran and loaf volume of bread. It appears that U.S. soft and club wheat bran, probably owing to relatively low IDF and phytate contents, has smaller negative effects on mixing properties of WWF dough and loaf volume of bread than U.S. hard wheat bran.     

Tocopherol and Tocotrienol Content in Commercial Wheat Mill Streams

The content of tocopherols and tocotrienols, collectively known as vitamin E (tocols), was determined in fractions of roller‐milled wheat grains. The results showed that vitamin E components are present in all major flour fractions of wheat, but that the vitamin E content and composition differed significantly between fractions. The total content of vitamin E, calculated as alpha‐tocopherol equivalents, changed from 16.1 mg α‐TE/g in wheat grain to 12.2 mg α‐TE/g in roller‐milled wheat flour. The germ fraction had the highest content of tocopherols, and the content of α‐tocopherol (195.2 μg/g) was 16 times higher (on average) than in any other fraction. The content of tocotrienols was distributed more uniform in the wheat grain with the highest content in the bran fractions, and the content of β‐tocotrienol was higher than the content of α‐tocopherol in all milling fractions except the wheat germ. The content of β‐tocotrienol was 24.1 μg/g in wheat grain, 25.3–31.0 μg/g in the bran fractions, and 14.3–21.9 μg/g in the fractions of endosperm. Overall, germ and fine bran fractions represent good sources of vitamin E and might be used in breadmaking.     

Phenolic Content and Antioxidant Activity of Pearled Wheat and Roller‐Milled Fractions

Wheat contains phenolic compounds concentrated mainly in bran tissues. This study examined the distribution of phenolics and antioxidant activities in wheat fractions derived from pearling and roller milling. Debranning (pearling) of wheat before milling is becoming increasingly accepted by the milling industry as a means of improving wheat rollermilling performance, making it of interest to determine the concentration of ferulic acid at various degrees of pearling. Eight cultivar samples were used, including five genotypes representing four commercial Canadian wheat classes with different intrinsic qualities. Wheat was pearled incrementally to obtain five fractions, each representing an amount of product equivalent to 5% of initial sample weight. Wheat was also roller milled without debranning. Total phenolic content of fractions was determined using the modified Folin‐Ciocalteau method for all pearling fractions, and for bran, shorts, bran flour, and first middlings flour from roller milling. Antioxidant activity was determined on phenolic extracts by a method involving the use of the free radical 2,2‐diphenyl‐l‐picrylhydrazyl (DPPH). Total phenolics were concentrated in fractions from the first and second pearlings (>4,000 mg/kg). Wheat fractions from the third and fourth pearlings still contained high phenolic content (>3,000 mg/kg). A similar trend was observed in antioxidant activity of the milled fractions with ≈4,000 mg/kg in bran and shorts, ≈3,000 mg/kg in bran flour, and <1,000 mg/kg in first middlings flour. Total phenolic content and antioxidant activity were highly correlated (R² = 0.94). There were no significant differences between red and white wheat samples. A strong influence of environment (growing location) was indicated. Pearling represents an effective technique to obtain wheat bran fractions enriched in phenolics and antioxidants, thereby maximizing health benefits associated with wheat‐based products.     

Effects of Fermentation and Baking of Whole Wheat and Whole Rye Sourdough Breads on Cereal Alkylresorcinols

Alkylresorcinol (AR) content was determined in multiple-stage whole wheat and whole rye flour sours, as well as in whole wheat and whole rye flour doughs and breads. AR content decreased considerably during fermentation and baking. AR content was reduced by 20 and 46%, respectively, at the end of sourdough starter fermentation of whole wheat and whole rye flour sours. AR content, which was 512 and 210 μg/g in whole rye and whole wheat flour doughs, respectively, was 30 and 0 μg/g, respectively, after baking of breads. Synthetic AR added at different levels to doughs was also greatly reduced during fermentation and baking.     

Distribution of Peroxidases in Durum Wheat (Triticum durum)

A biochemical study of the main durum wheat milling fractions (bran, embryo, and semolina) showed that peroxidases (POD) were present in multiple forms in the kernel and appeared to be tissue specific: one form for the embryo, one for the endosperm, one for the subaleuronic layer, and one for the outer layers. Large varietal differences were found regarding both the composition and the POD activity. POD activity, detected by diaminobenzidine, was found mainly in the cell wall of the subaleurone layer and inside some specific, differentiated cells of the embryo. Immuno‐localization with antibodies of durum wheat POD showed the presence of POD in several layers of the pericarp (epidermis) and the seed coat (testa), in the embryo, and also in the endosperm. In this latter tissue, the staining intensity decreased gradually from the outer layers toward the center of the kernel. The localization of POD in durum wheat kernel suggests specific functions for different forms.     

Effect of Single‐Pass and Multipass Milling Systems on Whole Wheat Durum Flour and Whole Wheat Pasta Quality

Single‐pass and multipass milling systems were evaluated for the quality of whole wheat durum flour (WWF) and the subsequent whole wheat (WW) spaghetti they produced. The multipass system used a roller mill with two purifiers to produce semolina and bran/germ and shorts (bran fraction). The single‐pass system used an ultracentrifugal mill with two configurations (fine grind, 15,000 rpm with 250 μm mill screen aperture; and coarse grind, 12,000 rpm with 1,000 μm mill screen aperture) to direct grind durum wheat grain into WWF or to regrind the bran fraction, which was blended with semolina to produce a reconstituted WWF. Particle size, starch damage, and pasting properties were similar for direct finely ground WWF and multipass reconstituted durum flour/fine bran blend and for direct coarsely ground WWF and multipass reconstituted semolina/coarse bran blend. The semolina/fine bran blend had low starch damage and had desirable pasting properties for pasta cooking. WW spaghetti was better when made with WWF produced using the multipass than single‐pass milling system. Mechanical strength was greatest with spaghetti made from the semolina/fine bran or durum flour/fine bran blends. The semolina/fine bran and semolina/coarse bran blends made spaghetti with high cooked firmness and low cooking loss.     

Proteolytic activities in dormant rye (Secale cereale L.) grain.

Endoproteolytic, exoproteolytic, carboxypeptidase, aminopeptidase, and N-alpha-benzoyl-arginine-p-nitroanilide hydrolyzing activities were detected in 0.05 M sodium acetate buffer (pH 5.0) extracts of whole meal of the rye (Secale cereale L.) varieties Amando, Halo, and Humbolt. The proteolytic enzymes of Humbolt, the variety with the highest proteolytic activity, optimally hydrolyzed hemoglobin around pH 3.5 and 40-45 degrees C. In the different milling fractions of Humbolt, azocasein and hemoglobin hydrolytic activities were especially found in the bran and shorts. Proteolytic enzymes in the bran extract were concentrated in the 35-60% ammonium sulfate precipitate. Pepstatin A, an inhibitor of aspartic proteases, reduced approximately 88 and approximately 75% of the hemoglobin and azocasein hydrolyzing activities of this precipitate, respectively. Phenylmethanesulfonyl fluoride, an inhibitor of serine proteases, inhibited approximately 33% of both cited activities. Both rye and wheat storage proteins were degraded by Humbolt rye whole meal enzyme extract and the above-mentioned ammonium sulfate rye bran fraction in vitro. With the latter fraction digestion was more pronounced.     

Partial Purification of a Water-Extractable Rye (Secale cereale) Protein Capable of Improving the Quality of Wheat Bread

Rye water-soluble extracts contain a protein fraction that, when added at low concentrations to a straight-dough breadmaking recipe, significantly increased bread volume. Enrichment of the active component is possible by anion-exchange fractionation with diethylaminoethyl-cellulose (DEAE), by ammonium sulfate precipitation, or by using rye bran or shorts milling fractions as the starting material. The active material was not bound to DEAE-cellulose. With ammonium sulfate precipitation, the fractions obtained at 30, 40, and 50% saturation were active in straightdough baking experiments. Iso-electric focusing revealed that fractions active in breadmaking invariably contained alkaline protein fractions (pI > 7.5). Inactivation of enzyme material by boiling the water-soluble extract from rye destroyed all breadmaking activity. The activity of the bread improver was additive to that of potassium bromate but not to that of ascorbic acid. It was not counteracted by catalase, showing that it does not work by a mechanism involving the production of hydrogen peroxide. The extract was not able to overcome the detrimental effect on bread quality resulting from mixing dough in a nitrogen atmosphere.     

Content of phenolic acids and ferulic acid dehydrodimers in 17 rye (Secale cereale L.) varieties

The contents of pnenolic acids and ferulic acid dehydrodimers were quantified by HPLC analysis after alkaline hydrolysis in kernels of 17 rye (Secale cereale L.) varieties grown in one location in Denmark during 1997 and 1998. Significant variations (P < 0.05) with regard to the concentration of the analyzed components were observed among the different rye varieties and also between different harvest years. However, the content of phenolic acids in the analyzed rye varieties was narrow compared to cereals such as wheat and barley. The concentration of ferulic acid, the most abundant phenolic acid ranged from 900 to 1170 microgram g(-1) dry matter. The content in sinapic acid ranged from 70 to 140 microgram g(-1) dry matter, p-coumaric acid ranged from 40 to 70 microgram g(-1) dry matter, and caffeic, p-hydroxybenzoic, protocatechuic, and vanillic acids were all detected in concentrations less than 20 microgram g(-1) dry matter. The most abundant ferulic acid dehydrodimer 8-O-4 -DiFA was quantified in concentrations from 130 to 200 microgram g(-1) dry matter followed by 8,5 -DiFA benzofuran form (50-100 microgram g(-1) dry matter), 5,5 -DiFA (40-70 microgram g(-1) dry matter), and 8,5 -DiFA (20-40 microgram g(-1) dry matter).