Vitamin B2 (riboflavin) content in cereal products

377 ACTA UNIVERSITATIS AGRICULTURAE ET SILVICULTURAE MENDELIANAE BRUNENSIS Volume LVIII 44 Numbe 5, 2010 VITAMIN B2 (RIBOFLAVIN) CONTENT IN CEREAL PRODUCTS S. Šk o ánko á, P. Siko o á Recei ed: July 8, 2010 Abs ac ŠKROVÁNKOVÁ, S., SIKOROVÁ, P.: Vi amin B2 ( ibo l a in) con en in ce eal p oduc s. Ac a uni . ag ic. e sil ic. Mendel. B un., 2010, LVIII, No. 5, pp. 377–382 Vi amin B2 ( ibofl a in) is a wa e -soluble essen ial i amin. Nowadays an inc eased isk o ibofl a in defi ciency may be seen in people on special die s (diabe es melli us), smoke s o hea y alcohol d inke s. In he Czech die he main sou ces o he i amin in ake a e milk and dai y p oduc s ollowed by ce- eals and mea . Ce eals a e good sou ce o his i amin as i is widely and egula ly consumed in di - e en o ms. Analyses o he i amin B2 con en in diff e en ypes o ce eal p oduc s (fl ou s, b eads, pas ies, b eak as ce eals, cooked pas a) o Czech o igin using HPLC wi h e e sed phase and UV de- ec ion we e done. The i amin B2 con en o chosen ce eal p oduc s dec eased in his p og ession: en iched wholemeal b eak as ce eals ( he bes sou ce o he i amin), en iched whea fl ou s, b eads – ye and wholemeal whea b eads, whole whea and spel fl ou s, whea b ead, cooked whole whea and ye spaghe i, whea and mul ig ain pas ies and fi nally scou ed whea fl ou s. i amin B2, ibofl a in, ce eal p oduc s, HPLC/UV Vi amin B2 – ibofl a in is a wa e -soluble essen ial i amin which is needed in he me abolism o amino acids, a y acids, and saccha ides. I also ac i a es i amin B6 (py idoxine) and olic acid and helps o c ea e niacin. Ribofl a in exe s i s biological unc- ion h ough wo coenzymes – fl a ine adenine dinu- cleo ide (FAD) and fl a ine mononucleo ide (FMN) which pa icipa e in oxida ion- educ ion eac ions in nume ous me abolic pa hways exemplifi ed by he oxida ion o educed NADH, he oxida ion in he ica boxylic acid cycle, and in he –oxida ion o a y acids (Ball, 2006). Humans canno syn hesize his i amin and hus i mus be ob ained om hei die h ough ab- so p ion in he small in es ine. The ecommended daily in ake (RDI) o ibofl a in, ela ed o p o ein and ene ge ic in ake, is om 1.2 o 1.7 mg pe day in he Czech Republic (Velíšek, 2009). Nowadays an inc eased isk o ibofl a in defi - ciency may be seen in people on special die s (dia- be es melli us, pep ic ulce disease, weigh loss), smo- ke s, hea y alcohol d inke s and women using ce ain ypes o bi h con ol. The e o e he solu- ion could be he die en iched wi h some o com- mon i amin sou ces as p oduc s wi h whole ce eals o ibofl a in en iched p oduc s, e.g. b ead and pas- ies. These p oduc s could be en iched by ibofl a- in (E 101) up o 50 % o RDI, ce eal mix u es and pas a e en up o 100 % o RDI. Vi amin o ifi ca ion o oods is e e ed in Regula ion No. 446/2004. This i amin can be also used as a colo an which is e- e ed in Regula ion No. 304/2004. G ea na u al ood sou ces o i amin B2 include: en ails – li e , nu s, yeas , cheese, eggs, milk, lean mea and wholemeal ce eals. In he Czech die he main sou ces o he i amin in ake a e milk and dai y p oduc s ollowed by ce eals and mea (Hlúbik and Opl o á, 2004). Ce eals a e good sou ce o his i amin as i is widely and egula ly consumed in di - e en o ms – fl ou o home culina y pu poses and echnological p oduc ion o ce eal p oduc s, b ead, pas ies, b eak as ce eals, pas as, and biscui s, espe- cially o common ce eals – whea and ye. The aim o his wo k was he e o e o de e mine ibofl a in con en in common ce eal p oduc s o he Czech o igin. Ribofl a in is s able, no des oyed by hea o ox- ida ion, unless exposed o ul a iole adia ion (420–560 nm). So i is a highly pho osensi i e com- pound. Knowledge o i s pho ochemical beha iou 378 S. Šk o ánko á, P. Siko o á in aqueous solu ion o e an app op ia e pH ange is needed o p edic he shel -li e. The choice o he op imum pH is c ucial o liquid p epa a ions. Ahmad e al. (2004) de e mined ha ibofl a in solu- ions a e mos s able o UV and isible adia ions a pH 5–6. Ribofl a in con en could be also dec eased acco ding o cha ac e o oods, echnological and culina y p ocesses (Leško á e al., 2006), e.g. scou - ing and dissolu ion in cooking wa e (Al-Khali a and Dawood, 1993). Vi amins ( i amin B2) in ce eals occu especially in ge m and aleu onic laye , so losses a e connec ed o ce eal scou ing, p ima ily i s le el (Hägg and Kum- pulainen, 1994). Scou ed ce eal p oduc s wi hou b an hen con ain up o en imes less B-complex i- amins han b an p oduc s. Losses o ibofl a in due o baking a e qui e small (up o 10 %). The signifi can losses o his i amin (35–55 %) a e in cooked p o- duc s due o leaching in o wa e (Ay anci and Kaya, 1993; Velíšek, 2009). F eezing doesn’ signifi can ly aff ec ibofl a in con en s in b eads (Hägg and Kum- pulainen, 1994). In oods uff s, he i amin B2 may be p esen ed in ee and phospho yla ed o ms, igh ly bound bu non-co alen ly o p o eins. Fo i amin ex ac ion he e can be used an acid hyd olysis o an enzyma ic ea men ( akadias asa, ypsin, cla asa) (Ndaw e al., 2000). Fo i amin de e mina ion se e al me hods ha e been epo ed – mic obiological assay (Golbach e al., 2007), elec ochemical me hod and High Pe o - mance Liquid Ch oma og aphy (A ella e al., 1996, Tang e al., 2006). The mos widely used me hod in he de e mina ion o i amin B2 is HPLC using e- e se phase wi h fl uo escence (And és-Lacue a e al., 1998; Ca aldi e al., 2003) and UV (Hőlle e al., 2003) de ec ion. MATERIAL AND METHODS Samples 20 ce eal p oduc s a e om he Czech p oduc ion pu chased in ood ma ke s. • fl ou s – whea (b ead, s anda d, pas y) fl ou s • • whole whea and spel fl ou s • • en iched whea (b ead, s anda d, pas y) fl ou s – decla ed amoun o ibofl a in con en o all en- iched fl ou s: 0.640 mg . 100 g−1 • b eads: whea , wholemeal (2 ypes), ye b ead • b eak as ce eals: en iched wholemeal (3 ypes) • pas ies: whea , mul ig ain (2 ypes) pas ies • cooked spaghe i: whole whea and ye spaghe i (cooked in wa e a 90 °C o 10 min). Reagen s • Calib a ion: ibofl a in was ob ained om Supelco (USA) • • s anda d solu ions: 0.25–4 μg . ml−1 • ex ac ion: HCl, ichlo oace ic acid, Ca ez I and Ca ez II we e ob ained om P. Lukeš (Czech Re- public) • HPLC de e mina ion: CH3COONa was pu chased om P. Lukeš (Czech Republic), me hanol o HPLC g ade om Sigma – Ald ich Chemicals (Ge many). Vi amin ex ac ion • 100 ml 0.2 M HCl and 3 ml CCl3COOH • 30 ml o Ca ez I and Ca ez II. Ch oma og aphic condi ions • HPLC (Hewle Packa d 1100, USA) • injec ion olume 20 μl • mobile phase: g adien echnique. CH3COONa : CH3OH 87:13 • column SUPELCOSIL – LC8 (15 cm × 4.6 mm; 5 μm, Supelco, USA) • column empe a u e 30 °C • fl ow a e: 0.8 ml . min−1 • De ec o : UV/VIS DAD G1315A = 270 nm. Ribo l a in Ex ac ion and HPLC De e mina ion By eason o complica ed ma ices based on p o- ein-polysaccha ides, he fi s s ep was isola ion o i amin B2 by acid hyd olysis. Tha is essen ial o de- na u e he p o eins and o elease he i amin om his associa ion wi h he p o eins and when he ood- s uff s con ain a lo o s a ch, o con e i in o solu- ble o m (Ndaw e al., 2000). Ex ac ion was done wi h 0.2 M hyd ochlo ic acid in a wa e ba h a 95 °C o 1 hou wi h addi ion o 80 % ichlo oace ic acid in 50 minu e. Mo eo e he p o eins we e subsequen ly p ecipi a ed by so- lu ions Ca ez I and Ca ez II ollowed by fi l a ion. All samples we e kep away om ligh because o i s low pho os abili y. The sui able condi ions o he de e mina ion by HPLC echnique we e es ablished pa ly acco ding o Buňka e al. (2003). Mobile phase composed o so- dium ace a e, pH adjus ed o 4.5 as ibofl a in solu- ions a e mos s able o UV and isible adia ions a pH abou 5–6, and me hanol, in he a io 87:13 ( / ) wi h g adien echnique ( ill 15 min., hen 0:100) o 30 min. and fl ow a e 0.8 ml . min−1. Fo ibofl a in de ec ion UV/VIS DAD de ec o was used a he wa eleng h o 270 nm wi h Chem- S a ion-Ins umen 1 p og amme o he con en calcula ion. Me hod alida ion Confi ma o y analysis o alida ion pa ame e s we e expe imen ally e ifi ed and confi med, as p e- iously published by Buňka e al. (2003). Calib a ion cu e (y = ax + b): y = 79.572x − 2.243 Vi amin B2 ( ibofl a in) con en in ce eal p oduc s 379 Co ela ion coeffi cien : = 0.9995 Reco e y: 90.1 ± 2.3 % – 91.4 ± 3.1 % (known amoun o i amin B2 was added o ce eal samples) In e and in a-day epea abili y: SD < 3 %. RESULTS AND DISCUSSION Table I. p esen s he con en o ibofl a in in 20 ce eal p oduc s – fl ou s, b eads, pas ies, b eak as ce eals and cooked spaghe i. The e is also shown con ibu ion o 100 g ce eal p oduc o RDI (Recom- mended Daily In ake in he Czech Republic is 1.5 mg pe day). The i amin B2 con en o chosen Czech ce eal p oduc s dec eased in his p og ession: en iched wholemeal b eak as ce eals, en iched whea fl ou s, b eads – ye and wholemeal whea b eads, whole whea and spel fl ou s, whea b ead, cooked whole whea and ye spaghe i, whea and mul ig ain pas- ies and fi nally scou ed whea fl ou s. F om he chosen p oduc s he bes ones as o i- bofl a in con en we e wholemeal b eak as ce eals (0.553–2.779 mg . 100 g−1) en iched wi h i amins. This ange ep esen s 36.9–185.3 % con ibu ion o RDI o ibofl a in. The esul s a e in ag eemen wi h he li e a u e (Hägg and Kumpulainen, 1993). B an ce eals also con ain mo e i amin B2 han scou ed ones as i was p e iously e e ed (Hägg and Kum- pulainen, 1994). These p oduc s don’ need any u he culina y ea men so hey a e eally good sou ce o his i amin. Also all en iched whea fl ou s – b ead (Fig. 1A), pas y and s anda d (0.525–0. 8 00 mg . 100 g−1; con i- bu ion o 35.0–53.3 % o RDI) and whole spel and whea fl ou s (0.204 and 0.265 mg . 100 g−1) seem as good sou ces. The i amin concen a ion in scou ed fl ou s was e y low (up o 0.087 mg . 100 g−1 in whea b ead fl ou (Fig. 1B)), as expec ed. The esul s co e- spond o he p oduce alues and a e also in ag ee- men wi h he li e a u e (Ba i oulie e al., 2005; Hägg and Kumpulainen, 1994; Ndaw e al., 2000). The a- ia bi li y o he con en in fl ou s could be explained by milling p ocedu e, gene ic a iabili y and g ow- ing loca ion. So milling and he ype o he fl ou (b ead, s anda d, pas y) a e e y impo an o he esponsibili y in he diff e ences o he i amin esul s. And fl ou s be o e consump ion need an- o he echnological o culina y ea men so i could lead o ano he loss o i amin due o ha p epa a- ion. Ribofl a in con en in b ead p oduc s was highe han in fl ou s. The concen a ion inc eased du - ing b ead making, sugges ing a po en ial syn hesis o con ibu ion by yeas as e e ed Ba i oulie e al. (2005) and Ba i oulie e al. (2006). Lowe i amin concen a ion was ound in com- mon whea b ead (0.208 mg . 100 g−1) han in whole- meal and ye b eads (0.322–0.561 mg . 100 g−1; con i- bu ion o 21.5–37.4 % o RDI). This could be ela ed o he fl ou scou ing le el as he i amin is con- ained especially in he b an. I efl ec s a mo e d as- I: Ribo l a in con en in ce eal p oduc s (meanSE) [mg.100 g−1] and con ibu ion o 100 g p oduc o RDI [%] Ce eal p oduc s A e age ibofl a in con en Con ibu ion o 100 g p oduc o RDI Flou : En iched whea b ead 0.800 ± 0.004 53.3 En iched whea pas y 0.584 ± 0.006 38.9 En iched whea s anda d 0.525 ± 0.005 35.0 Whole whea 0.265 ± 0.005 17.7 Whole spel 0.204 ± 0.004 13.6 Whea b ead 0.087 ± 0.006 5.8 Whea pas y and s anda d nd. - B ead: Rye 0.561 ± 0.010 37.4 Wholemeal 1 0.504 ± 0.013 33.6 Wholemeal 2 0.322 ± 0.013 21.5 Whea 0.208 ± 0.008 13.9 Pas ies: Whea 0.094 ± 0.007 6.3 Mul ig ain 1 0.081 ± 0.007 5.4 Mul ig ain 2 nd. - Wholemeal b eak as ce eals: 1 2.779 ± 0.007 185.3 2 1.246 ± 0.010 83.1 3 0.553 ± 0.012 36.9 Cooked spaghe i: Whole whea 0.179 ± 0.007 11.9 Whole ye 0.223 ± 0.010 14.9 nd. – no de ec ed 380 S. Šk o ánko á, P. Siko o á ic milling eff ec wi h efi ned fl ou han wi h un e- fi ned, whole fl ou (Hägg and Kumpulainen, 1994). Low concen a ions o ibofl a in in cooked spa- ghe i (0.179–0.223 mg . 100 g−1) o Czech o igin, hough wholemeal p oduc , a e due o i amin leaching in o wa e which co espond o esul s o Ay anci and Kaya (1993). Acco ding o hem he pe - cen age o ibofl a in loss o maca oni du ing cook- ing a 90 °C was 18.3 %, (10 min cooking wi h wa e ) and 53.3 % (20 min cooking).As expec ed, he lo- wes i amin B2 le els we e ound in pas ies (up o 0.094 mg . 100 g−1) made om scou ed fl ou s. So, common whea o mul ig ain pas ies a e no e y good sou ce o his i amin in he Czech Republic. Ribofl a in is wa e -soluble i amin which leaches in o wa e , e.g. du ing cooking wi h wa e . O he losses could happen du ing s o age e.g. UV adia ion impac . In some pape s he e ha e been p o ed ha ibofl a in losses depend on echnological condi ion (Ba i oulie e al., 2005; Ba i oulie e al., 2006; Hägg and Kumpulainen, 1994; P odano e al., 2004). 1: Ch oma og aphic analysis o i amin B2 in en iched whea b ead lou (A) and whea b ead lou (B) by HPLC/UV SUMMARY Ribofl a in is a wa e -soluble essen ial i amin which is needed in he me abolism o amino acids, a y acids, and saccha ides (RDI=1.5 mg pe day). Nowadays an inc eased isk o ibofl a in defi ciency may be seen in people on special die s (e.g. diabe es melli us). In he Czech die he main sou ces o he i a- min in ake a e milk and dai y p oduc s ollowed by ce eals and mea . Ce eals a e good sou ce o his i amin as i is widely and egula ly consumed in diff e en o ms. The aim o his wo k was he e o e o de e mine ibofl a in con en in common ce eal p oduc s o he Czech o igin. Vi amin B2 ( ibofl a in) con en in ce eal p oduc s 381 One o widely used me hod in he de e mina ion o i amin B2 is HPLC using e e se phase wi h UV de ec ion. The i amin B2 con en o chosen Czech ce eal p oduc s, de e mined by his me hod, de- c eased in his p og ession: en iched wholemeal b eak as ce eals (0.553–2.779 mg . 100 g−1; con ibu- ion o 36.9–185.3 % o RDI), which a e eally good sou ce o he i amin as hey don’ need any u he culina y ea men ; hen en iched fl ou s (0.525–0. 8 00 mg . 100 g−1), b eads – wholemeal wi h highe con en as i amin (0,322–0,561 mg . 100 g−1) which occu s especially in g ain ge m and aleu onic laye ; ollowing by whea b ead (0.208 mg . 100 g−1). In b eads, in compa ison o fl ou s, could he ibofl a in con en inc eased du ing b ead making (syn hesis o by yeas con ibu ion); nex whea fl ou s – due o he ce eal scou ing he concen a ion was e y low (up o 0.087 mg . 100 g−1; con ibu ion o 5.8 % o RDI). Low concen a ions o ibofl a in in cooked spaghe i (0.179–0.223 mg . 100 g−1, con ibu ion o 11.9–14.9 % o RDI) a e due o i amin leaching in o wa e . The lowes i amin B2 le els we e ound in pas ies (up o 0.094 mg . 100 g−1), so common whea o mul ig ain pas ies om scou ed fl ou s a e no e y good sou ce o his i amin. SOUHRN Obsah i aminu B2 ( ibofl a inu) ce eálních p oduk ech Ribofl a in je e odě ozpus ný i amin, k e ý je zapo řebí p o me abolismus aminokyselin, uků a sacha idů (DDD = 1,5 mg na den). V dnešní době mají něk eří lidé (např. nemocní s diabe es melli us) ě ší po řebu přísunu i aminu B2. Zd oje oho o i aminu u lidí České epublice pochází přede- ším z mléka a mléčných ý obků, ce eálií a masa. K nej ýznamnějším os linným zd ojům ěch o i aminů pa ří obilo iny k ůli jejich běžné a pomě ně ozsáhlé konzumaci ůzné o mě. Cílem é o p áce p o o bylo s ano ení obsahu ibofl a inu běžných ce eálních ý obcích českého pů odu. Jednou z nej íce ozšířených me od p o jeho s ano ení je HPLC s e e zní ází a UV de ekcí. Obsah i aminu B2 e yb aných ce eálních p oduk ech, s ano ený ýše u edenou me odou, klesal om o pořadí: obohacené celoz nné snídaňo é ce eálie (0,553–2,779 mg . 100 g−1; kde 100 g ý obku zabez- pečí 36,9–185,3 % DDD ibofl a inu), k e é jsou edy nejlepším zd ojem z hodnocených p oduk ů bez nu nos i dalších kuliná ních úp a . Následují i aminem obohacené mouky (0,525–0,800 mg . 100 g−1), dále chleba – celoz nný s yšším obsahem i aminu (0,322–0,561 mg . 100 g−1), k e ý se nachází přede- ším klíčku a aleu ono é s ě, a pšeničný chleba (0,208 mg . 100 g−1). Chleba po o nání s odpo- ídajícími moukami může mí yšší obsah i aminu B2 z dů odu ná ůs u p ůběhu pečení (syn- éza nebo přída ek d oždí). Bílé pšeničné mouky měly pomě ně nízký obsah ibofl a inu s ohledem na ymílání ěch o mouk (do 0,087 mg . 100 g−1; 5,8 % DDD). Nízké obsahy ibofl a inu e ařených špa- ge ách (0,179–0, 223 mg . 100 g−1, 11,9–14,9 % DDD) sou isí s yluho áním do odného p os ředí. Nej- nižší hodno y obsahů i aminu B2 byly zjiš ěny peči u (do 0,094 mg . 100 g−1), edy běžné ymílané bílé a ícez nné peči o není ýhodným zd ojem oho o i aminu. i amin B2, ibofl a in, ce eální p oduk y, HPLC/UV REFERENCES AHMAD, I., FASIHULLAH, Q., NOOR, A., ANSARI, I. A., MANZAR ALI, Q. N., 2004: Pho olysis o i- bofl a in in aqueous solu ion: a kine ic s udy. In e - na ional Jou nal o Pha maceu ics, 280, 1–2: 199–208. ISSN 0378-5173. AL-KHALIFA, A. S., DAWOOD, A. A., 1993: Eff ec s o cooking me hods on hiamine and ibofl a in con en s o chicken mea , Food Chemis y, 48, 1: 69– 74. ISSN 0308-8146. ANDRÉS-LACUEVA, C., MATTIVI, F., TONON, D., 1998: De e mina ion o ibofl a in, fl a in mononu- cleo ide and fl a in–adenine dinucleo ide in wine and o he be e ages by high-pe o mance liq- uid ch oma og aphy wi h fl uo escence de ec ion. 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