Vitamin B2 (riboflavin) content in cereal products

377 ACTA UNIVERSITATIS AGRICULTURAE ET SILVICULTURAE MENDELIANAE BRUNENSIS Volume LVIII 44 Number 5, 2010 VITAMIN B2 (RIBOFLAVIN) CONTENT IN CEREAL PRODUCTS S. Škrovánková, P. Sikorová Received: July 8, 2010 Abstract ŠKROVÁNKOVÁ, S., SIKOROVÁ, P.: Vitamin B2 (ribofl avin) content in cereal products. Acta univ. agric. et silvic. Mendel. Brun., 2010, LVIII, No. 5, pp. 377–382 Vitamin B2 (ribofl avin) is a water-soluble essential vitamin. Nowadays an increased risk for ribofl avin defi ciency may be seen in people on special diets (diabetes mellitus), smokers or heavy alcohol drinkers. In the Czech diet the main sources of the vitamin intake are milk and dairy products followed by cereals and meat. Cereals are good source of this vitamin as it is widely and regularly consumed in different forms. Analyses of the vitamin B2 content in diff erent types of cereal products (fl ours, breads, pastries, breakfast cereals, cooked pasta) of Czech origin using HPLC with reversed phase and UV detection were done. The vitamin B2 content of chosen cereal products decreased in this progression: enriched wholemeal breakfast cereals (the best source of the vitamin), enriched wheat fl ours, breads – rye and wholemeal wheat breads, whole wheat and spelt fl ours, wheat bread, cooked whole wheat and rye spaghetti, wheat and multigrain pastries and fi nally scoured wheat fl ours. vitamin B2, ribofl avin, cereal products, HPLC/UV Vitamin B2 – ribofl avin is a water-soluble essential vitamin which is needed in the metabolism of amino acids, fatty acids, and saccharides. It also activates vitamin B6 (pyridoxine) and folic acid and helps to create niacin. Ribofl avin exerts its biological function through two coenzymes – fl avine adenine dinucleotide (FAD) and fl avine mononucleotide (FMN) which participate in oxidation-reduction reactions in numerous metabolic pathways exemplifi ed by the oxidation of reduced NADH, the oxidation in the tricarboxylic acid cycle, and in the –oxidation of fatty acids (Ball, 2006). Humans cannot synthesize this vitamin and thus it must be obtained from their diet through absorption in the small intestine. The recommended daily intake (RDI) of ribofl avin, related to protein and energetic intake, is from 1.2 to 1.7 mg per day in the Czech Republic (Velíšek, 2009). Nowadays an increased risk for ribofl avin defi - ciency may be seen in people on special diets (diabetes mellitus, peptic ulcer disease, weight loss), smokers, heavy alcohol drinkers and women using certain types of birth control. Therefore the solution could be the diet enriched with some of common vitamin sources as products with whole cereals or ribofl avin enriched products, e.g. bread and pastries. These products could be enriched by ribofl avin (E 101) up to 50 % of RDI, cereal mixtures and pasta even up to 100 % of RDI. Vitamin fortifi cation of foods is referred in Regulation No. 446/2004. This vitamin can be also used as a colorant which is referred in Regulation No. 304/2004. Great natural food sources of vitamin B2 include: entrails – liver, nuts, yeast, cheese, eggs, milk, lean meat and wholemeal cereals. In the Czech diet the main sources of the vitamin intake are milk and dairy products followed by cereals and meat (Hlúbik and Opltová, 2004). Cereals are good source of this vitamin as it is widely and regularly consumed in different forms – fl our for home culinary purposes and technological production of cereal products, bread, pastries, breakfast cereals, pastas, and biscuits, especially for common cereals – wheat and rye. The aim of this work was therefore to determine ribofl avin content in common cereal products of the Czech origin. Ribofl avin is stable, not destroyed by heat or oxidation, unless exposed to ultraviolet radiation (420–560 nm). So it is a highly photosensitive compound. Knowledge of its photochemical behaviour 378 S. Škrovánková, P. Sikorová in aqueous solution over an appropriate pH range is needed to predict the shelf-life. The choice of the optimum pH is crucial for liquid preparations. Ahmad et al. (2004) determined that ribofl avin solutions are most stable to UV and visible radiations at pH 5–6. Ribofl avin content could be also decreased according to character of foods, technological and culinary processes (Lešková et al., 2006), e.g. scouring and dissolution in cooking water (Al-Khalifa and Dawood, 1993). Vitamins (vitamin B2) in cereals occur especially in germ and aleuronic layer, so losses are connected to cereal scouring, primarily its level (Hägg and Kumpulainen, 1994). Scoured cereal products without bran then contain up to ten times less B-complex vitamins than bran products. Losses of ribofl avin due to baking are quite small (up to 10 %). The signifi cant losses of this vitamin (35–55 %) are in cooked products due to leaching into water (Ayranci and Kaya, 1993; Velíšek, 2009). Freezing doesn’t signifi cantly aff ect ribofl avin contents in breads (Hägg and Kumpulainen, 1994). In foodstuff s, the vitamin B2 may be presented in free and phosphorylated forms, tightly bound but non-covalently to proteins. For vitamin extraction there can be used an acid hydrolysis or an enzymatic treatment (takadiastasa, trypsin, clarasa) (Ndaw et al., 2000). For vitamin determination several methods have been reported – microbiological assay (Golbach et al., 2007), electrochemical method and High Performance Liquid Chromatography (Arella et al., 1996, Tang et al., 2006). The most widely used method in the determination of vitamin B2 is HPLC using reverse phase with fl uorescence (Andrés-Lacueva et al., 1998; Cataldi et al., 2003) and UV (Hőller et al., 2003) detection. MATERIAL AND METHODS Samples 20 cereal products are from the Czech production purchased in food markets. • fl ours – wheat (bread, standard, pastry) fl ours • • whole wheat and spelt fl ours • • enriched wheat (bread, standard, pastry) fl ours – declared amount of ribofl avin content of all enriched fl ours: 0.640 mg . 100 g−1 • breads: wheat, wholemeal (2 types), rye bread • breakfast cereals: enriched wholemeal (3 types) • pastries: wheat, multigrain (2 types) pastries • cooked spaghetti: whole wheat and rye spaghetti (cooked in water at 90 °C for 10 min). Reagents • Calibration: ribofl avin was obtained from Supelco (USA) • • standard solutions: 0.25–4 μg . ml−1 • extraction: HCl, trichloroacetic acid, Carrez I and Carrez II were obtained from P. Lukeš (Czech Republic) • HPLC determination: CH3COONa was purchased from P. Lukeš (Czech Republic), methanol of HPLC grade from Sigma – Aldrich Chemicals (Germany). Vitamin extraction • 100 ml 0.2 M HCl and 3 ml CCl3COOH • 30 ml of Carrez I and Carrez II. Chromatographic conditions • HPLC (Hewlett Packard 1100, USA) • injection volume 20 μl • mobile phase: gradient technique. CH3COONa : CH3OH 87:13 • column SUPELCOSIL – LC8 (15 cm × 4.6 mm; 5 μm, Supelco, USA) • column temperature 30 °C • fl ow rate: 0.8 ml . min−1 • Detector: UV/VIS DAD G1315A = 270 nm. Ribofl avin Extraction and HPLC Determination By reason of complicated matrices based on protein-polysaccharides, the fi rst step was isolation of vitamin B2 by acid hydrolysis. That is essential to denature the proteins and to release the vitamin from his association with the proteins and when the foodstuff s contain a lot of starch, to convert it into soluble form (Ndaw et al., 2000). Extraction was done with 0.2 M hydrochloric acid in a water bath at 95 °C for 1 hour with addition of 80 % trichloroacetic acid in 50 minute. Moreover the proteins were subsequently precipitated by solutions Carez I and Carez II followed by fi ltration. All samples were kept away from light because of its low photostability. The suitable conditions for the determination by HPLC technique were established partly according to Buňka et al. (2003). Mobile phase composed of sodium acetate, pH adjusted to 4.5 as ribofl avin solutions are most stable to UV and visible radiations at pH about 5–6, and methanol, in the ratio 87:13 (v/v) with gradient technique (till 15 min., then 0:100) for 30 min. and fl ow rate 0.8 ml . min−1. For ribofl avin detection UV/VIS DAD detector was used at the wavelength of 270 nm with ChemStation-Instrument 1 programme for the content calculation. Method validation Confi rmatory analysis of validation parameters were experimentally verifi ed and confi rmed, as previously published by Buňka et al. (2003). Calibration curve (y = ax + b): y = 79.572x − 2.243 Vitamin B2 (ribofl avin) content in cereal products 379 Correlation coeffi cient: r = 0.9995 Recovery: 90.1 ± 2.3 % – 91.4 ± 3.1 % (known amount of vitamin B2 was added to cereal samples) Inter and intra-day repeatability: SD < 3 %. RESULTS AND DISCUSSION Table I. presents the content of ribofl avin in 20 cereal products – fl ours, breads, pastries, breakfast cereals and cooked spaghetti. There is also shown contribution of 100 g cereal product to RDI (Recommended Daily Intake in the Czech Republic is 1.5 mg per day). The vitamin B2 content of chosen Czech cereal products decreased in this progression: enriched wholemeal breakfast cereals, enriched wheat fl ours, breads – rye and wholemeal wheat breads, whole wheat and spelt fl ours, wheat bread, cooked whole wheat and rye spaghetti, wheat and multigrain pastries and fi nally scoured wheat fl ours. From the chosen products the best ones as for ribofl avin content were wholemeal breakfast ce reals (0.553–2.779 mg . 100 g−1) enriched with vitamins. This range represents 36.9–185.3 % contribution to RDI for ribofl avin. The results are in agreement with the literature (Hägg and Kumpulainen, 1993). Bran cereals also contain more vitamin B2 than scoured ones as it was previously referred (Hägg and Kumpulainen, 1994). These products don’t need any further culinary treatment so they are really good source of this vitamin. Also all enriched wheat fl ours – bread (Fig. 1A), pastry and standard (0.525–0. 8 00 mg . 100 g−1; contribution of 35.0–53.3 % to RDI) and whole spelt and wheat fl ours (0.204 and 0.265 mg . 100 g−1) seem as good sources. The vitamin concentration in scoured fl ours was very low (up to 0.087 mg . 100 g−1 in wheat bread fl our (Fig. 1B)), as expected. The results correspond to the producer values and are also in agreement with the literature (Batifoulier et al., 2005; Hägg and Kumpulainen, 1994; Ndaw et al., 2000). The varia bi li ty of the content in fl ours could be explained by milling procedure, genetic variability and growing location. So milling and the type of the fl our (bread, standard, pastry) are very important for the responsibility in the diff erences of the vitamin results. And fl ours before consumption need another technological or culinary treatment so it could lead to another loss of vitamin due to that preparation. Ribofl avin content in bread products was higher than in fl ours. The concentration increased during bread making, suggesting a potential synthesis or contribution by yeast as referred Batifoulier et al. (2005) and Batifoulier et al. (2006). Lower vitamin concentration was found in common wheat bread (0.208 mg . 100 g−1) than in wholemeal and rye breads (0.322–0.561 mg . 100 g−1; contribution of 21.5–37.4 % to RDI). This could be related to the fl our scouring level as the vitamin is contained especially in the bran. It refl ects a more drasI: Ribofl avin content in cereal products (meanSE) [mg.100 g−1] and contribution of 100 g product to RDI [%] Cereal products Average ribofl avin content Contribution of 100 g product to RDI Flour: Enriched wheat bread 0.800 ± 0.004 53.3 Enriched wheat pastry 0.584 ± 0.006 38.9 Enriched wheat standard 0.525 ± 0.005 35.0 Whole wheat 0.265 ± 0.005 17.7 Whole spelt 0.204 ± 0.004 13.6 Wheat bread 0.087 ± 0.006 5.8 Wheat pastry and standard nd. - Bread: Rye 0.561 ± 0.010 37.4 Wholemeal 1 0.504 ± 0.013 33.6 Wholemeal 2 0.322 ± 0.013 21.5 Wheat 0.208 ± 0.008 13.9 Pastries: Wheat 0.094 ± 0.007 6.3 Multigrain 1 0.081 ± 0.007 5.4 Multigrain 2 nd. - Wholemeal breakfast cereals: 1 2.779 ± 0.007 185.3 2 1.246 ± 0.010 83.1 3 0.553 ± 0.012 36.9 Cooked spaghetti: Whole wheat 0.179 ± 0.007 11.9 Whole rye 0.223 ± 0.010 14.9 nd. – not detected 380 S. Škrovánková, P. Sikorová tic milling eff ect with refi ned fl our than with unrefi ned, whole fl our (Hägg and Kumpulainen, 1994). Low concentrations of ribofl avin in cooked spaghetti (0.179–0.223 mg . 100 g−1) of Czech origin, though wholemeal product, are due to vitamin leaching into water which correspond to results of Ayranci and Kaya (1993). According to them the percentage of ribofl avin loss for macaroni during cooking at 90 °C was 18.3 %, (10 min cooking with water) and 53.3 % (20 min cooking).As expected, the lowest vitamin B2 levels were found in pastries (up to 0.094 mg . 100 g−1) made from scoured fl ours. So, common wheat or multigrain pastries are not very good source of this vitamin in the Czech Republic. Ribofl avin is water-soluble vitamin which leaches into water, e.g. during cooking with water. Other losses could happen during storage e.g. UV radiation impact. In some papers there have been proved that ribofl avin losses depend on technological condition (Batifoulier et al., 2005; Batifoulier et al., 2006; Hägg and Kumpulainen, 1994; Prodanov et al., 2004). 1: Chromatographic analysis of vitamin B2 in enriched wheat bread flour (A) and wheat bread flour (B) by HPLC/UV SUMMARY Ribofl avin is a water-soluble essential vitamin which is needed in the metabolism of amino acids, fatty acids, and saccharides (RDI=1.5 mg per day). Nowadays an increased risk for ribofl avin defi ciency may be seen in people on special diets (e.g. diabetes mellitus). In the Czech diet the main sources of the vitamin intake are milk and dairy products followed by cereals and meat. Cereals are good source of this vitamin as it is widely and regularly consumed in diff erent forms. The aim of this work was therefore to determine ribofl avin content in common cereal products of the Czech origin. Vitamin B2 (ribofl avin) content in cereal products 381 One of widely used method in the determination of vitamin B2 is HPLC using reverse phase with UV detection. The vitamin B2 content of chosen Czech cereal products, determined by this method, decreased in this progression: enriched wholemeal breakfast cereals (0.553–2.779 mg . 100 g−1; contribution of 36.9–185.3 % to RDI), which are really good source of the vitamin as they don’t need any further culinary treatment; then enriched fl ours (0.525–0. 8 00 mg . 100 g−1), breads – wholemeal with higher content as vitamin (0,322–0,561 mg . 100 g−1) which occurs especially in grain germ and aleuronic layer; following by wheat bread (0.208 mg . 100 g−1). In breads, in comparison to fl ours, could the ribofl avin content increased during bread making (synthesis or by yeast contribution); next wheat fl ours – due to the cereal scouring the concentration was very low (up to 0.087 mg . 100 g−1; contribution of 5.8 % to RDI). Low concentrations of ribofl avin in cooked spaghetti (0.179–0.223 mg . 100 g−1, contribution of 11.9–14.9 % to RDI) are due to vitamin leaching into water. The lowest vitamin B2 levels were found in pastries (up to 0.094 mg . 100 g−1), so common wheat or multigrain pastries from scoured fl ours are not very good source of this vitamin. SOUHRN Obsah vitaminu B2 (ribofl avinu) v cereálních produktech Ribofl avin je ve vodě rozpustný vitamin, který je zapotřebí pro metabolismus aminokyselin, tuků a sacharidů (DDD = 1,5 mg na den). V dnešní době mají někteří lidé (např. nemocní s diabetes mellitus) větší potřebu přísunu vitaminu B2. Zdroje tohoto vitaminu u lidí v České republice pochází především z mléka a mléčných výrobků, cereálií a masa. K nejvýznamnějším rostlinným zdrojům těchto vitaminů patří obiloviny kvůli jejich běžné a poměrně rozsáhlé konzumaci v různé formě. Cílem této práce proto bylo stanovení obsahu ribofl avinu v běžných cereálních výrobcích českého původu. Jednou z nejvíce rozšířených metod pro jeho stanovení je HPLC s reverzní fází a UV detekcí. Obsah vitaminu B2 ve vybraných cereálních produktech, stanovený výše uvedenou metodou, klesal v tomto pořadí: obohacené celozrnné snídaňové cereálie (0,553–2,779 mg . 100 g−1; kde 100 g výrobku zabezpečí 36,9–185,3 % DDD ribofl avinu), které jsou tedy nejlepším zdrojem z hodnocených produktů bez nutnosti dalších kulinárních úprav. Následují vitaminem obohacené mouky (0,525–0,800 mg . 100 g−1), dále chleba – celozrnný s vyšším obsahem vitaminu (0,322–0,561 mg . 100 g−1), který se nachází především v klíčku a aleuronové vrstvě, a pšeničný chleba (0,208 mg . 100 g−1). Chleba v porovnání s odpovídajícími moukami může mít vyšší obsah vitaminu B2 z důvodu nárůstu v průběhu pečení (syntéza nebo přídavek droždí). Bílé pšeničné mouky měly poměrně nízký obsah ribofl avinu s ohledem na vymílání těchto mouk (do 0,087 mg . 100 g−1; 5,8 % DDD). Nízké obsahy ribofl avinu ve vařených špagetách (0,179–0, 223 mg . 100 g−1, 11,9–14,9 % DDD) souvisí s vyluhováním do vodného prostředí. Nejnižší hodnoty obsahů vitaminu B2 byly zjištěny v pečivu (do 0,094 mg . 100 g−1), tedy běžné vymílané bílé a vícezrnné pečivo není výhodným zdrojem tohoto vitaminu. vitamin B2, ribofl avin, cereální produkty, HPLC/UV REFERENCES AHMAD, I., FASIHULLAH, Q., NOOR, A., ANSARI, I. A., MANZAR ALI, Q. N., 2004: Photolysis of ribofl avin in aqueous solution: a kinetic study. International Journal of Pharmaceutics, 280, 1–2: 199–208. ISSN 0378-5173. AL-KHALIFA, A. S., DAWOOD, A. A., 1993: Eff ects of cooking methods on thiamine and ribofl avin contents of chicken meat, Food Chemistry, 48, 1: 69– 74. ISSN 0308-8146. ANDRÉS-LACUEVA, C., MATTIVI, F., TONON, D., 1998: Determination of ribofl avin, fl avin mononucleotide and fl avin–adenine dinucleotide in wine and other beverages by high-performance liquid chromatography with fl uorescence detection. 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Pavlína Sikorová, Ústav biochemie a analýzy potravin, Fakulta technologická, Univerzita T. Bati ve Zlíně, nám. T. G. Masaryka 275, 762 72 Zlín, Česká republika, e-mail: skrovankova@ .utb.cz