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
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