Content of phytic acid in selected sorts of legumes

217 ACTA UNIVERSITATIS AGRICULTURAE ET SILVICULTURAE MENDELIANAE BRUNENSIS SBORNÍK MENDELOVY UNIVERZITY V BRNĚ Volume LVIII 26 Numbe 1, 2010 CONTENT OF PHYTIC ACID IN SELECTED SORTS OF LEGUMES P. Voj íško á, S. K áčma , I. Hoza Recei ed: Oc obe 7, 2009 Abs ac VOJTÍŠKOVÁ, P., KRÁČMAR, S., HOZA, I.: Con en o phy ic acid in selec ed so s o legumes. Ac a uni . ag- ic. e sil ic. Mendel. B un., 2010, LVIII, No. 1, pp. 217–222 The aim o his s udy was o de e mine he con en o phy ic acid (phy a e) in soybeans (Glycine max), yellow shelled peas (Pisum sa i um) and len il (Lens esculen a). D y seeds we e g ounded o a fi ne pow- de . The mois u e o samples was de e mined acco ding o he Offi cial Jou nal o he Eu opean Union (2009). The mois u e o he samples was 8.42% in soybeans, 11.19% in yellow shelled peas and 10.07% in len il. The con en o phy ic acid was de e mined by he modifi ed Hol 's me hod (1955) using he spec opho ome e . S anda d cu e was measu ed using he Na phy a e s anda d solu ion (0.2 mM). The phy a e con en in G. max a ied om 1.28 o 1.86% in d y ma e and om 1.17 o 1.70 g pe 100 g o he sample. The con en o phy a e in P. sa i um anged om 0.49 o 0.86% and om 0.43 o 0.77 g pe 100 g o he sample. The phy a e con en in L. esculen a a ied om 0.45 o 1.39% in d y ma e and om 0.40 o 1.25 g pe 100 g o he sample. These ob ained alues could be infl uenced by many ac o s, e.g. clima ic condi ions, loca ion, a ie y, e c. phy ic acid, phy a e, Glycine max, Pisum sa i um, Lens esculen a Legumes a e d y edible seeds o some plan s om he amily Fabaceae, as beans, lupine, peas and len il a e. The nu i ional po en ial o he seeds om his g oup o plan s is based on hei high le el o p o- eins. Howe e , he legumes also con ain an inu i- ional ac o s, such as p o einase inhibi o s, lec in, afi nose oligosaccha ides, saponins, polyphenols and phy a e (Sandbe g, 2002). Phy ic acid (myo-inosi ol 1,2,3,4,5,6-he xa kis- pho spha e, IP6) ep esen s a majo an inu ien in ood and eed. In seeds, i is s o ed as mixed sal s o ca ions, mainly K and Mg, oge he wi h mino amoun s o Ca, Fe, Zn and Mn (Raboy, 1997). Inosi ol phospha es consis o an inosi ol ing and a leas one phospha e g oup. B eaking he name in o i s sepa a e pa s desc ibes he exac s uc u e and appea ance: he p efi x “myo” e e s o he con o - ma ion o he hyd oxyl g oups on he inosi ol ing. The e a e nine s e eoisome s o inosi ol, o which se en a e meso s uc u es and wo o m a chi al pai . They a e (1) cis-, (2) epi-, (3) allo-, (4) neo-, (5) myo-, (6) muco-, (7) 1L-chi o-, (9) 1D-chi o-, and (9) scyllo-inosi- ol (Loewus and Loewus, 1980). The myo-inosi ol is common in plan s (Cosg o e, 1980). The nine pos- sible confi gu a ions o he inosi ol ing ha e been anno a ed in a numbe o ways, bu he adop ed no- mencla u e is acco ding o he se o ules sugges ed by Pos e nak (1965). The con o ma ion myo-inosi ol hus has one plane o symme y, going di ec ly om he mos le o he mos igh a om. The D/L-p efi xes speci y he numbe ing di ec ion o ca bons in he inosi ol ing, whe e he D anno a es coun e clockwise and L clockwise coun ing, espec i ely. In gene al che- mis y, numbe ing o he a oms should always ol- low he lowes possible ou e. Con usions ega ding myo-inosi ols and enzymes ela ed o hem ha e led he In e na ional Union o Biochemis y (IUPAC- IUB, 1989) o ecommend ha he a oms in he myo- inosi ol ing should always be numbe ed acco ding o he D confi gu a ion. Myo-inosi ol is he majo nu i ionally ele an o m o inosi ol, and al hough some o he o he s e eoisome s a e also ound in na u e. Myo-inosi- ol (1,2,3,4,5,6) hexakisphospha e has six g oups o phospha es a ached o he inosi ol ing. Using 218 P. Voj íško á, S. K áčma , I. Hoza he p efi x ”hexakis” ins ead o “hexa” indica es ha he phospha es a e no in e nally connec ed and he compound is consequen ly a polyden a e li- gand, which is a chela o ha can bind o mo e han one coo dina ion si e o he me al a om. Each o he phospha e g oups is es e ifi ed o he inosi- ol ing and oge he hey can bind up o 12 p o ons in o al. The acidi y o he p o ons a ies om e y s ong acids o e y weak al hough ionic s eng h o he solu ion and empe a u e infl uence hese alues (To es e al., 2005; He e al., 2006). Ce eals and legumes con ain signifi can amoun s o phospho us in he o m o phy ic acid (Tables I and II). I se es se e al physiological unc ions and also signifi can ly infl uences he unc ional and nu- i ional p ope ies o ce eals and legumes and hei de i ed oods by complexing wi h p o eins and es- sen ial mine als. The e ms phy ic acid, phy a e and phy in e e o ee acid, sal and calcium/magne- sium sal , espec i ely. In he li e a u e, he name phy ic acid has been used in e changeably wi h he e m phy a e, which is a sal (Reddy e al., 1989). Phy ic acid accumula es du ing seed de elopmen un il he seeds each ma u i y and accoun s o 60–90% o o al phospho us con en in ce eals, le- gu mes, nu s and oil seeds (Lo e al., 2000). The p i- ma y unc ions o phy ic acid in seeds a e s o age o phospha es as ene gy sou ce and an ioxidan o he ge mina ing seed (Raboy, 2003). Legume seeds a e he iches and cheapes al e - na i e sou ces o p o ein among all oods o plan o igin (U bano e al., 2000). The chela ing eff ec o he phospha e g oups causes phy ic acid o bind eadily o mine al ca ions, especially o Cu2+ and Zn2+ which appea o ha e a high affi ni y o inosi- ol phospha es. The o de o he abili y o he mi- ne al ca ions o o m complexes in i o wi h inosi- ol phospha es has been ound o be Cu2+>Zn2+>Cd2+ o all InsP3-InsP6 a pH 3-7, bu binding s eng h is weake o he lowe inosi ol phospha es (Pe sson e al., 1998). Recen fi nding shows ha phy ic acid is s o ed in i o in complexes no only wi h hese mi ne- als, bu o a much la ge ex en s wi h Mg, Ca and K (Bohn e al., 2007). In p esence o excess phy ic acid, o ma ion o soluble complexes be ween phy ic acid and me al ion displaying 1 : 1 s oichiome ies p edomina es. Howe e , when me al ions a e in excess, an insolu- ble solid called phy a e is o med (To es e al., 2005). Phy a e as he mine al bound sal o phy ic acid is also an impo an mine al ese e in seeds, and i is s o ed in p o ein s o age acuoles in he aleu one cell laye o he emb yo o he seed (S o cksdieck e al., 2007). MATERIALS AND METHODS Legume sampling The con en o phy ic acid in selec ed so s o le- gu mes was de e mined. Legumes o his s udy we e ob ained om he Food Resea ch Ins i u e in B a i- sla a, Slo akia. D y seeds we e g ounded in a mill o a fi ne powde and ho oughly mixed. They we e s o ed a labo a o y empe a u e. Analysis Fi s , he mois u e o samples was de e mined (O - fi cial Jou nal o he Eu opean Union, 2009). The de e mina ion o phy a e was ealized by modifi ed Hol 's me hod (Hol , 1955). T iplica e 1.0 g o samples was ex ac ed wi h 40 ml 0.5M HNO3 o 3 hou s wi h con inuous shaking. The ex ac was fi l a ed h ough he fi l e FILTRAK, No. 390, ∅ 12.5 cm. Nex , 0.2–1.0 ml o fi l a e was dilu ed wi h dis- illed wa e o a fi nal olume o 1.4 ml in polyp o- pylene es - ubes; a e ha 1 ml o a e ic ammo- nium sulpha e solu ion (con aining 50 μg o Fe) was added, es - ubes we e s oppe ed and mixed. Then, hey we e pu in o a boiling wa e ba h (Memme , Ge many) o 20 minu es and a e ha cooled o a oom empe a u e. Fi e millili es o amyl alcohol and 0.1 ml o am- monium hiocyana e solu ion (100 g . l−1) was sub- sequen ly added o each es - ube. They we e s op- pe ed and immedia ely mixed by in e sion and shaking. A e cen i uga ion o a sho ime a low speed, he in ensi y o he colou in he amyl laye was de e mined a 465 nm using a spec opho ome- e (Bioch om Lib a S6, Camb idge, England) agains an amyl alcohol “blank” exac ly 15 minu es a e ad- di ion o NH4CNS (Da ies and Reid, 1979). S anda d cu e was de e mined he same way us- ing Na phy a e s anda d solu ion (0.2mM; Sigma Ald ich, USA) ins ead o he fi l a e. The equa ion om he s anda d cu e was used o he calcula ion I: Con en o phy ic acid in d y ma e o ce eals and legumes [%] (Reddy, 1982) Ce eal / Legume Amoun o phy ic acid Whea 0.62–1.35 Rye 0.97 Ba ley 0.97–1.16 Co n (Maize) 0.89–0.99 Oa 0.79–1.01 Soya 1.00–1.47 Peas 1.20 Beans 0.55–0.75 Len il 0.51 II: Con en o phy ic acid in some legume seeds [g pe 100 g] (Híd égi and Lász i y, 2002) Legume Amoun o phy ic acid Soybeans 1.20–1.75 Peas 0.72–1.23 Common bean 0.55–1.69 Con en o phy ic acid in selec ed so s o legumes 219 o he amoun o phy a e in G. max, P. sa i um and L. esculen a (Fig. 1). All esul s we e e alua ed using he a ia ion s a- is ics (ANOVA). Co ela ion ma ices and eg es- sion unc ions we e calcula ed acco ding o Snede- co and Coch an (1967) using he s a is ical package Unis a , . 5.5. RESULTS AND DISCUSSION The con en o phy ic acid in selec ed so s o le- gu mes, Glycine max, Pisum sa i um and Lens esculen a, was s udied. Fi s o all, he mois u e o he samples was de e mined. As can be seen om Table III, he con en o mois u e in G. max was 8.42%, in P. sa i um 11.19% and in L. esculen a 10.07%. Consequen ly, he legume samples we e analysed using he modifi ed Hol 's me hod. Values in Table IV show ha he amoun o phy a e in d y ma e o G. max a ies om 1.28 o 1.86%. These alues a e sligh ly highe han hose epo ed by Reddy e al. (1982; Table I) who p esen ed a ange o 1.00–1.47% o phy a e con en in d y ma e o soy- beans. Híd égi and Lász i y (2002; Table II) assigned he con en o phy a e in soybeans in he ange o 1.20–1.75 g pe 100 g o he sample. The quan ifi ca- ion o phy a e in G. max (Table V) a ies om 1.17 o 1.70 g pe 100 g o he sample. As can be seen, hese alues a e almos he same as hose epo ed alues a e. The con en o phy a e in d y ma e o P. sa i um is lowe han in epo s by Reddy (1982) and Híd égi and Lász i y (2002). Reddy (1982; Table I) assigned he alue o 1.20% o phy a e in d y ma e o yel- low shelled peas, and Híd égi and Lász i y (2002; Ta- ble II) p esen ed he phy a e con en in he ange o 0.72–1.23 g pe 100 g o he sample. The quan ifi ca- ion o phy a e in P. sa i um a ies om 0.49 o 0.86% in d y ma e o he sample (Table IV) and om 0.43 o 0.77 g pe 100 g o he sample (Table V). These de- e mined alues a e almos by hal lowe han hose epo ed by Reddy (1982) and Híd égi and Lász i y (2002). The con en o phy a e in d y ma e o L. escu- len a (Table IV) is in he ange o 0.45–1.39%. Reddy (1982; Tab. I) assigned he alue o 0.51% in d y ma - e o len il. The con en o phy a e is highe han in epo s by Reddy (1982). The amoun o phy a e in L. esculen a a ied om 0.40 o 1.25 g pe 100 g o he sample. Cu en da a ob ained by he de e mina ion o phy a e con en in G. max, P. sa i um and L. esculen a using he modifi ed Hol ´s me hod can diff e om y = –11196x + 2.0265 R2 = 0.9895 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0.00E+00 2.00E-05 4.00E-05 6.00E-05 8.00E-05 1.00E-04 1.20E-04 1.40E-04 Phy a e con en (g) Abso bance (nm) 1: S anda d cu e o phy a e de e mina ion III: Mois u e and d y ma e in legume samples (mean ± S.E.) [%] Sample A e age mois u e A e age d y ma e Glycine max 8.42 ± 0.042 91.58 ± 0.042 Pisum sa i um 11.19 ± 0.064 88.81 ± 0.064 Lens esculen a 10.07 ± 0.019 89.93 ± 0.019 IV: Con en o phy a e in d y ma e o selec ed legumes ( alue ± S.E.) [%] Sample Minimum A e age Maximum Glycine max 1.28 ± 0.033 1.60 ± 0.033 1.86 ± 0.033 Pisum sa i um 0.49 ± 0.022 0.62 ± 0.022 0.86 ± 0.022 Lens esculen a 0.45 ± 0.046 0.85 ± 0.046 1.39 ± 0.046 V: Con en o phy a e in selec ed legumes ( alue ± S.E.) [g pe 100g o he sample] Sample Minimum A e age Maximum Glycine max 1.17 ± 0.030 1.46 ± 0.030 1.70 ± 0.030 Pisum sa i um 0.43 ± 0.020 0.55 ± 0.020 0.77 ± 0.020 Lens esculen a 0.40 ± 0.042 0.55 ± 0.042 1.25 ± 0.042 220 P. Voj íško á, S. K áčma , I. Hoza hose ob ained p e iously because o many ac o s, e.g. clima ic condi ions, loca ion, ype o soil, diff e - en a ie ies o legumes, diff e en c op pe iod, using diff e en , modifi ed me hod o de e mina ion, chem- icals om diff e en p oduce s, e c. No many au ho s deal wi h phy ic acid con en in legumes. Mo e o hem s udied he con en o phy a e in ce eals and ce eal b an, o example in whea (Egli e al., 2003), mille (Talamond e al., 1998), ba ley (Ha land and Mo is, 1995), whea , ye, oa (Reddy and Sa he, 2002) and so ghum (Deshpande, 2002). Con en o phy ic acid, and also phy a e is e y im- po an because o i s chela ing p ope ies. Ca ions, as Ca2+, Zn2+, Fe3+, Cu2+ and o he s, can be bound by he phy a e anion and o m indiges ible compounds. The capaci y o binding mine als makes phy ic acid an an inu i ional ac o . On he o he hand, phy ic acid has also posi i e p ope ies. I can ac as an an i- oxidan . Phy ic acid can also pa icipa e in lowe ing o choles e ol and iglyce ide le el in blood, kid- ney s one o ma ion, and maybe in cance he apy. As Golian (2009) b ings ou , good nu i ion can lead o an imp essi e ange o benefi s, o example im- p o ed heal h and g ea e esilience o shocks in- duced by social, economic and na u al causes. Soybeans, peas and len il a e ich in p o eins, sac- cha ides, i amins and o he compounds; phy a e is one o hem. The e o e, his s udy was ca ied ou o de e mine he con en o phy a e in G. max, P. sa i um and L. esculen a. SOUHRN Obsah kyseliny y o é e yb aných d uzích luš ěnin V p áci byl sledo án obsah kyseliny y o é ( y á u) e yb aných d uzích luš ěnin, sójo ých bobů (Glycine max), žlu ého loupaného h achu (Pisum sa i um) a čočky (Lens esculen a). Zkoumané zo ky byly homogenizo ány. Vlhkos zo ků byla s ano ena podle Nařízení komise (ES) č. 152/2009 u edeného Úředním ěs níku E opské Unie. Vlhkos sójo ých bobů byla 8,42 %, žlu ého loupaného h achu 11,19 % a čočky 10,07 %. Obsah kyseliny y o é byl s ano en modifi ko anou me odou podle Hol a jako množs í y á u. S anda dní kři ka byla p oměřena pomocí 0,2mM s anda dního oz oku y á u sodného ozmezí koncen ací 40 až 200 nmol. Obsah y á u sójo ých bobech (G. max) kolísal mezi 1,28 až 1,86 % sušině a mezi 1,17 až 1,70 g e 100 g zo ku, e žlu ém loupaném h achu (P. sa i um) ozmezí od 0,49 do 0,86 % sušině a od 0,43 do 0,77 g e 100 g zo ku a u čočky (L. esculen a) ozmezí 0,45–1,39 % sušině a 0,40–1,25 g e 100 g zo ku. kyselina y o á, y á , Glycine max, Pisum sa i um, Lens esculen a This wo k was kindly suppo ed by a p ojec o he Minis y o Educa ion, You h and Spo s o he Czech Republic (g an No. MSM 70883 52101). REFERENCES BOHN, L., JOSEFSEN, L., MEYER, A. S., RASMUS- SEN, S. 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Jou nal o Physiology and Biochemis y, 56, 3: 283–294. ISSN 1138-7548. Add ess Ing. Pe a Voj íško á, p o . Ing. S anisla K áčma , D Sc., p o . Ing. Ignác Hoza, CSc., Uni e zi a Tomáše Ba i e Zlíně, Fakul a echnologická, Ús a biochemie a analýzy po a in, nám. T. G. Masa yka 275, 762 72 Zlín, Česká epublika, e-mail: oj isko a@ .u b.cz 222