Health safety aspects of foodstuffs intented for phenylketonurics

Po a ina s o® Scien i ic Jou nal o Food Indus y Volume 9 132 No. 1/2015 INTRODUCTION Phenylke onu ia (PKU) is an au osomal ecessi e inhe i ed diso de o phenylalanine me abolism due o a lack o he enzyme phenylalanine hyd oxylase. Mos o ms o PKU and hype phenylalaninemia (HPA) a e caused by mu a ions in a gene on ch omosome 12 o phenylalanine hyd oxylase (PAH) (Bu ne e al., 2008). Cu en ly, mo e han 400 known mu a ions a e iden i ied in he gene o PAH. Lack o o insu icien ac i i y o he phenylalanine hyd oxylase causes accumula ion o phenylalanine (Phe), and phenyl ke one exc e ion in he u ine. Any inc ease in he concen a ion o phenylalanine in he blood esul s in cen al ne ous sys em damage (S no á and Ü ge, 2007). Phenylalanine (Phe) is an essen ial amino acid ha is a p ecu so o melanin, dopamine, and hy oxine. I is an essen ial amino acid and i s na u al occu ence in ood is usually su icien (mean con en 3.5%). In p o ein oods, Phe cons i u es abou 4-5% (Velíšek and Hajšlo á, 2009). The a i icial swee ene aspa ame is also high sou ce o phenylalanine. The ADI o aspa ame o heal hy pe son is 40 mg/kg body weigh (EFSA, 2014). In PKU pa ien s, a es ic ion o oods ich in p o ein (mea , ish, eggs, b ead, dai y p oduc s, nu s and seeds), as well as a oidance o d inks con aining aspa ame help o con ol blood phenylalanine le els. The de eloping e us o women su e ing om PKU is pa icula ly sensi i e o hei mo he ’s phenylalanine le els (EFSA, 2014). The basic ea men s o PKU a e mix u e p epa a ions o aminoacids. Thei use ensu es an adequa e supply o amino acids wi hou phenylalanine, he eby compensa ing educed p o ein in ake in low p o ein die a y egime. Pa ien s wi h phenylke onu ia usually ge hese oods by medical p esc ip ion as mos o hese oods u s can belong o he g oup o oods o special medical pu poses (S no á and Ü ge, 2007), which sa e y is de ined by he Commission Di ec i e 1999/21/EC. Un ea ed child en wi h pe sis en hype phenylalaninemia (PKU) show b ain damage. Symp oms include mic ocephaly, epilepsy, men al disabili y and beha io al p oblems. By he use o neona al sc eening and ea ly die a y in e en ion, child en bo n wi h PKU can li e a ela i ely no mal li e (Bu ne e al., 2008; Mi chell, 2000). The applica ion o die a an ea ly age o li e can signi ican ly educe men al de iciencies associa ed wi h phenylke onu ia. Die a y measu es a e based on he oods con aining a minimum o Phe, while p ese ing he no mal con en o y osine and o he p o eins in ood. This is o en achie ed by subs i u ion o phenylalanine ee p o eins. Die a y es ic ion o na u al p o eins educes and Po a ina s o, ol. 9, 2015, no. 1, p. 132-137 doi:10.5219/455 Recei ed: 9 Ma ch 2015. Accep ed: 6 May 2015. A ailable online: 25 May 2015 a www.po a ina s o.com © 2015 Po a ina s o. All igh s ese ed. ISSN 1337-0960 (online) License: CC BY 3.0 HEALTH SAFETY ASPECTS OF FOODSTUFFS INTENTED FOR PHENYLKETONURICS S anisla a Ma ejo á, Ma ina Fikselo á, F an išek Buňka ABSTRACT Phenylke onu ia is a a e me abolic diso de ha occu s due o he lack o li e enzyme phenylalanine hyd oxylase. Un ea ed i leads o men al e a da ion, delayed de elopmen o speech, mic ocephaly, epilepsy, beha io al p oblems e c. I s ea men consis s o s ic die , e y low in phenylalanine con en . The aim o his s udy was o de e mine he heal h sa e y and sui abili y o 17 selec ed oods labeled as „low in p o ein“ espec i ely „ oods low in phenylalanine“. Analyses we e ocused on hei phenylalanine con en and aminoacid ep esen a ion. The o he aim was o compa e phenylalanine measu ed wi h he con en decla ed by he p oduce s on hei label. By p oduc o igin, he la ges amoun o oods u s o igina ed om Poland (59%), ollowed by Ge many (23%) and equal ep esen a ion (6%) had Slo akia, Hunga y and Sweden. Au oma ic analyze AAA 400, using ion exchange ch oma og aphy, analyzed he samples. The lowes de e mined phenylalanine con en was 1 mg.100g-1 (in ubbe candy) and he highes 299 mg.100g-1 (in pa é). Powde ed egg al e na i e was moni o ed as he second highes sou ce o phenylalanine. Phenylalanine con en in he moni o ed oods anged om 1 o 299.6 mg. 100 g-1 espec i ely, and inc eased in he ollowing o de : Gum candies (1 mg.100g-1) < Che y jelly (1.4 mg. 100g-1) < Flou (5.8 mg.100g-1) < Flou (5.9 mg.100g-1) < Spaghe i (8.7 mg.100g-1) < Pas a (9.9 mg.100g-1) < Wa le (14.2 mg.100g-1) < Sal y s icks (23.8 mg.100g-1) < Chocola e (24.1 mg.100g-1) < B ead (26 mg.100g-1) < B eadc umbs (27.1 mg.100g-1) < S icks wi h sal (30.3 mg.100g-1) < Ice Co ne s (30.8 mg.100g-1) < Walnu cookie (37.2 mg.100g-1) < Ins an soup wi h noodles (46.1 mg.100g-1) < Powde ed egg al e na i e (58 mg.100g-1) < Pa e (299.6 mg.100g-1). Only se en p oduc s o 17 obse ed showed low phenylalanine con en , less han 20 mg.100g-1. Keywo ds: phenylke onu ia; phenylalanine; low-p o ein p oduc ; acid hyd olysis; ch oma og aphy Po a ina s o® Scien i ic Jou nal o Food Indus y Volume 9 133 No. 1/2015 main ains he le el o Phe in he blood (Macdonald e al. 2009; Pous ie and Wildgoose, 2010; S ačina, 2008). The aim o his wo k was o analyze selec ed amino acids (aspa ic acid, h eonine, se ine, glu amic acid, p oline, glycine, alanine, aline, isoleucine, leucine, y osine, phenylalanine, his idine, lysine, a ginine) in se en een ood samples, decla ed by he p oduce as "Low-p o ein oods” espec i ely “Foods low in phenylalanine con en " by au oma ic analyze AAA 400 and consequen ly o e alua e hei sa e y and sui abili y o a pa icula nu i ional use. To compa e he con en o phenylalanine decla ed on he label wi h con en o phenylalanine measu ed. MATERIAL AND METHODOLOGY As ma e ial used we e he oods u s o e ed o phenylke onu ics ob ained om in e ne sale and labeled as ood sui able o phenylke onu ics as „low in p o ein“ espec i ely „ oods low in phenylalanine“. O e iew o he ma e ial used is gi en in Table 1. By o igin o p oduc s, mos o hem o igina ed om Poland (59%), ollowed by Ge many (23%) and equal ep esen a ion (6%) had Slo akia, Hunga y and Sweden. Fi een amino acids (aspa ic acid, h eonine, se ine, glu amic acid, p oline, glycine, alanine, aline, isoleucine, leucine, phenylalanine, y osine, his idine, lysine and a ginine) we e de e mined in obse ed samples using ion- exchange ch oma og aphy. The o al amoun o amino Table 1 O e iew o he ma e ial es ed. No. P oduc P oduce Main componen s 1. Pa e P oduce A, Ge many bacon, po k, li e , onion, wa e , sal , s abilize E 250, spice, emulsi ie 2. B eadc umbs P oduce 1, Poland glu en ee whea s a ch, co n s a ch, ege able oil, glucose, suga , yeas , sal , gua gum, E 464, ib e, aising agen s, E 500, E 575 3. Spaghe i Slo akia co n s a ch, modi ied co n s a ch, emulsi ie E 471, dye cu cuma (E 100) 4. Ins an soup wi h noodles P oduce 1, Poland mal odex in, glu en ee pas a, co n s a ch, wa e , sal , mono and diglyce ides o a y acids, E 464, E 575, be aca o ene, sal , monosodium glu ama e, ege able oil, ca o , suga , onion, a oma, cele y, leek, black peppe , pa sley s alk, cu cuma, ci ic acid 5. S icks wi h sal P oduce 1, Poland co n s a ch, sal , whea s a ch glu en ee, ege able oil, suga , yeas , gua gum, E 464, sal , ib e, E 500, E 503, mono and diglyce ides o a y acids, leci hin, E 575 6. Walnu cookie P oduce 2, Poland co n s a ch, glu en ee whea s a ch, ege able oil, suga , walnu s, peanu s, oil, pec in, gua gum, pec in, glucose, bu e essence, mono and diglyce ides o a y acids 7. Powde ed egg al e na i e P oduce B, Ge many co n s a ch, mono and diglyce ides o a y acids, palm oil, xan han, ice lou , pea p o ein 8. Sal y s icks P oduce 2, Poland ma ga ine, co n s a ch, glu en ee whea s a ch, wa e , suga , po a o s a ch, yolk, gua gum, yeas , po a o sy up, whea glu en ee cellulose, sal , E 503, mono and diglyce ides o a y acids, leci hin, anillin 9. Chocola e P oduce C, Ge many cocoa bu e , suga , bu e , cocoa ma e , powde ed whey, anilla 10. Pas a Hunga y co n s a ch, wa e , a y acids, E 471 11. Wa le P oduce 2, Poland glu en ee whea s a ch, co n s a ch, gua gum, bamboo ib e, glucose, apple pec in, mono and diglyce ides o a y acids 12. Che y jelly P oduce 2, Poland suga , glucose sy up, wa e , aga , ci ic acid, che y a oma, dye E 124 13. Ice Co ne s P oduce 2, Poland co n s a ch, glu en ee whea s a ch, po a o s a ch, suga , yolk, gua gum 14. B ead P oduce 1, Poland glu en ee whea s a ch, wa e , ege able a , suga , glucose, yeas , sal , ca ob, gua gum, E 464, ib e, E 500, E 575 15. Gum candies P oduce C, Ge many po a o and apioca s a ch, ci ic acid, malic acid, galac ic acid, calcium ci a e, a ome, ui concen a es, ege able ex ac s (blackbe ies, ca o , peppe , cu cuma, spi ulina, ne le, spinach), na u al dye : chlo ophyll, ege able oil, beeswax, ca nauba wax 16. Flou P oduce 1, Poland glu en ee whea s a ch, glucose, co n s a ch, gua gum, E 464, ege able ib e, E 500, E 575 17. Flou Sweden whea s a ch (max. 0.2 g glu en/100 g), oligo uc ose, gua gum, i amins, e um Po a ina s o® Scien i ic Jou nal o Food Indus y Volume 9 134 No. 1/2015 acids and phenylalanine con en o ood p oduc s we e moni o ed a e 23-hou acid hyd olysis using HCl (6 mol.dm-3) and 115 ±2 °C. A e he hyd olysis, he es ubes we e cooled down o 20°C. Hyd ochlo ic acid was e apo a ed and he opy esidue was dilu ed in loading bu e in a 25 cm-3 olume ic lask. The mix u e was il e ed h ough 0.22 m il e and loaded in o an analyse . Amino Acid Analyse AAA400 (Ingos, P ague, Czech Republic) was equipped wi h a column (370×3.7 mm illed wi h a ion exchange Os ion LG ANG – Ingos, P ague, Czech Republic), pos -column ninhyd ine de i a iza ion and spec opho ome ic de ec ion (440 nm o p oline and 570 nm o o he amino acids). Me hod was pe o med acco ding o he Buňka e al. (2009). S a is ical analysis o esul s The esul s we e e alua ed by calcula ing he mean, coe icien o a ia ion and s anda d de ia ion using he s a is ical so wa e S a is ica 8.0. RESULTS AND DISCUSSION T ea men o phenylke onu ia should be a combina ion o se e al die s ha educe he amoun o phenylalanine: glu en- ee die , lac ose- ee and low p o eins die which is he mos impo an . The p inciple o he die is o educe he amoun o phenylalanine in he body and o c ea e a balance be ween i s income and he po en ial use (Komá ko á and Pazdí ko á, 2010; D ořák, 2009). Phenylalanine con en in he moni o ed oods in ou wo k was ep esen ed in a ying amoun s, i s con en anged om 1 o 299.6 mg. 100 g-1 espec i ely, and inc eased in he ollowing o de : Gum candies (1 mg.100g-1) < Che y jelly (1.4 mg.100g-1) < Flou no.16 (5.8 mg.100g-1) < Flou no. 17 (5.9 mg.100g-1) < Spaghe i (8.7 mg.100g-1) < Pas a (9.9 mg.100g-1) < Wa le (14.2 mg.100g-1) < Sal y s icks (23.8 mg.100g-1) < Chocola e (24.1 mg.100g-1) < B ead (26 mg.100g-1) < B eadc umbs (27.1 mg.100g-1) < S icks wi h sal (30.3 mg.100g-1) < Ice Co ne s (30.8 mg.100g-1) < Walnu cookie (37.2 mg.100g-1) < Ins an soup wi h noodles (46.1 mg.100g-1) < Powde ed egg al e na i e (58 mg.100g-1) < Pa e (299.6 mg.100g-1). Following legisla ion, i seems ha na ional o Eu opean legisla ion con ain no speci ic equi emen s o „low p o ein oods“, o oods low in phenylalanine, so in pu pose o e alua e ou samples we used as he s anda d gi en in he Czech legisla ion (Vyhláška Minis e s a zd a o nic í České epubliky č. 54/2004 Sb), acco ding o which ood wi h no phenylalanine con en is de ined as ood made by special echnological p ocess so ha he phenylalanine con en should no exceed 20 mg pe 100 g o 100 cm-3 in he ood in ended o consump ion. Unde his legisla ion, sui able oods o phenylke onu ics could be classi ied as ollows: Gum candies (1 mg.100g-1) < Che y jelly (1.4 mg. 100g-1) < Flou no. 16 (5.8 mg.100g-1) < Flou no.17 (5.9 mg.100g-1) < Spaghe i (8.7 mg.100g-1) < Pas a (9.9 mg.100g-1) < Wa le (14.2 mg.100g-1). Table 2 Aminoacid con en (mg.100g-1) de e mined in sample o pa e. Aminoacids Con en o aminoacids [mg.100g-1] S anda d de ia ion CV (%) Aspa ic acid 571.11 0.14 2 Th eonine 201.60 0.04 2 Se ine 156.48 0.04 3 Glu amic acid 916.62 0.55 6 P oline 546.50 0.27 5 Glycine 762.99 0.12 2 Alanine 471.35 0.02 0 Valine 371.65 0.33 9 Isoleucine 269.77 0.15 5 Leucine 522.17 0.19 4 Ty osine 121.15 0.03 3 Phenylalanine 299.59 0.13 4 His idine 205.64 0.03 2 Lysine 435.07 0.01 0 A ginine 387.21 0.24 6 To al 6238.90 Po a ina s o® Scien i ic Jou nal o Food Indus y Volume 9 135 No. 1/2015 Table 3 Aminoacid con en (mg.100g-1) de e mined in sample o powde ed egg al e na i e. Aminoacids Con en o aminoacids [mg.100g-1] S anda d de ia ion CV (%) Aspa ic acid 125.69 0.05 4 Th eonine 33.60 0.02 7 Se ín 55.26 0.03 6 Glu amic acid 163.12 0.08 5 P oline 39.72 0.01 2 Glycine 46.35 0.02 5 Alanine 58.73 0.02 3 Valine 38.43 0.00 0 Isoleucine 29.31 0.02 7 Leucine 87.81 0.04 5 Ty osine 28.76 0.01 5 Phenylalanine 58.00 0.01 1 His idine 32.71 0.01 4 Lysine 60.59 0.01 1 A ginine 87.63 0.02 3 To al 945.70 Figu e 1 Di e ences be ween phenylalanine con en measu ed and labeled by p oduce s (mg.100g-1). Samples: 1.Pa e, 2. B eadc umbs, 3. Spaghe i, 4. Ins an soup wi h noodles, 5. S icks wi h sal , 6. Walnu cookie, 7. Powde ed egg al e na i e, 8. Sal y s icks, 9. Chocola e, 10. Pas a, 11. Wa le, 12. Che y jelly, 13. Ice Co ne s, 14. B ead, 15. Gum candies, 16. Flou , 17. Flou . 025 50 75 100 125 150 175 200 225 250 275 300 325 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Con en o phenylalanine (mg.100g-1) Sample PHE decla ed PHE measu ed Po a ina s o® Scien i ic Jou nal o Food Indus y Volume 9 136 No. 1/2015 Con a y, as sample wi h he highes con en o phenylalanine (299.6 mg.100g-1) was de e mined pa e p oduced in Ge many. The highes amino acid con en was also de ec ed (6238.90 mg.100g-1) in his sample p obabaly due o mea p esen , and he highes con en o glu amic acid (916.62 mg.100g-1) among amino acids was de e mined as well. Powde ed egg al e na i e (sample no.7) was moni o ed as he second highes sou ce o phenylalanine. I consis s o co ns a ch, mono and diglyce ides o a y acids, palm oil, xan han, ice lou and pea p o ein. Glu amic acid also p e ailed in his sample (Table 3). The only p oduc o Slo ak o igin was he sample no. 3, spaghe i. To al sum o amino acids 127.91 mg.100g-1 was obse ed, which can be assumed oge he wi h he obse ed phenylalanine con en (8.75 mg.100g-1) as low con en and he e o e his p oduc can be conside ed as sui able o consump ion by phenylke onu ics. As he mos sui able o enylke onu ics wi h he lowes con en o phenylalanine we e obse ed gum candies and che y jelly (less han 2 mg. 100g-1). To al aminoacid con en de ec ed in hese wo p oduc s was also he lowes , a gum candies 12.04 mg.100g-1 and che y jelly 14.81 mg.100g-1. Sample no. 8 was he s icks o Polish o igin. They a e compa able wi h he sample no. 5, which a e also s icks wi h sal wi h a simila composi ion bu made om o he p oduce . Phenylalanine con en de e mined was 30.0 esp. 23.8 mg.100g-1. Compa able could be also wo samples o lou s o igina ing om Poland and Sweden, wi h de ec ed phenylalanine con en a lou no.16 (5.8 mg.100g-1), and lou no.17 (5.9 mg.100g-1). To al aminoacid con en we e obse ed as simila (85.08 mg.100g-1 s. 86.65 mg.100g-1) as well. As i was s a ed p e iously, oods o phenylke onu ics a e o en included in he ca ego y o oods o special medical pu poses ha means a ca ego y o oods o pa icula nu i ional uses specially p ocessed o o mula ed and in ended o he die a y managemen o pa ien s and o be used unde medical supe ision. They a e in ended o he exclusi e o pa ial eeding o pa ien s wi h a limi ed, impai ed o dis u bed capaci y o ake, diges , abso b, me abolise o exc e e o dina y oods u s o ce ain nu ien s con ained he ein o me aboli es, o wi h o he medically-de e mined nu ien equi emen s, whose die a y managemen canno be achie ed only by modi ica ion o he no mal die , by o he oods o pa icula nu i ional uses, o by a combina ion o he wo (Commission Di ec i e 1999/21/EC). The labelling o oods u s in ended o pa icula nu i ional uses shall include by he Di ec i e 2009/39/EC he pa icula elemen s o he quali a i e and quan i a i e composi ion o he special manu ac u ing p ocess which gi es he p oduc i s pa icula nu i ional cha ac e is ics. Compa ing he esul s in phenylalanine con en measu ed and he con en o phenylalanine labeled by p oduce s, we obse ed se e al di e ences shown in he Fig. 1. Sample no. 2 Figu e 2 To al aminoacid con en (mg.100g-1) de e mined in selec ed p oduc s. Samples: 1.Pa e, 2. B eadc umbs, 3. Spaghe i, 4. Ins an soup wi h noodles, 5. S icks wi h sal , 6. Walnu cookie, 7. Powde ed egg al e na i e, 8. Sal y s icks, 9. Chocola e, 10. Pas a, 11. Wa le, 12. Che y jelly, 13. Ice Co ne s, 14. B ead, 15. Gum candies, 16. Flou , 17. Flou . 6238.9 443.13 127.91 2907.39 541.36 678.89 945.7 376.39 385.36 142.54 270.29 14.81 543.08 452.62 12.04 86.65 85.08 0 1000 2000 3000 4000 5000 6000 7000 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Sample To al aminoacid con en (mg.100g-1) Po a ina s o® Scien i ic Jou nal o Food Indus y Volume 9 137 No. 1/2015 ep esen ed, as b eadc umbs and sample no. 6 we e he p oduc s wi h he lowes di e ence in obse ed and decla ed alues o phenylalanine in he packaging. Con a y, highe di e ences in hese wo amoun s we e de ec ed in samples 1, 4, 7, 8. To al aminoacid con en de e mined in selec ed p oduc s is shown in he Figu e 2. I s con en in selec ed p oduc s anged om 12.04 (gum candies) o 6238.90 mg.100g-1 (in pa e) and inc eased in he o de : Gum candies (12.04 mg.100g-1) < Che y jelly (14.81 mg.100g-1) < Flou (85.08 mg.100g-1) < Flou (86.65 mg.100g-1) < Spaghe i (127. 9 mg.100g-1) < Pas a (142.54 mg.100g-1) < Wa le (270.29 mg.100g-1) < Sal y s icks (376.39 mg.100g-1) < Chocola e (385.36 mg.100g-1) < B eadc umbs (443.13 mg.100g-1) < B ead (452.62 mg.100g-1) < S icks wi h sal (541.36 mg.100g-1) < Ice Co ne s (543.08 mg.100g-1) < Walnu cookie (678.89 mg.100g-1) < Powde ed egg al e na i e (945.70 mg.100g-1) < Ins an soup wi h noodles (2907.39 mg.100g-1) < Pa e (6238.90 mg.100g-1). CONCLUSION In his wo k, amino acids ep esen a ion, ocused on phenylalanine con en in selec ed samples in en ed o phenylke onu ics was obse ed. We can conclude ha may be due o he lack o legisla ion equi emen s, mos o obse ed oods was high in con en o phenylalanine. Only se en p oduc s o 17 obse ed showed lowe phenylalanine con en , less han 20 mg.100g-1. The ob ained esul s also sugges ha ce ain oods a he con en o phenylalanine di e om hose lis ed on he p oduc label. REFERENCES Di ec i e 2009/39/EC o he Eu opean Pa lamen and o he Council o 6 May 2009 on oods u s in ended o pa icula nu i ional uses, OJ L 124, 20.5.2009, p. 21-29. Buňka, F., Kříž, O., Veličko á, A., Buňko á, L., K áčma , S. 2009. E ec o acid hyd olysis ime on amino acid de e mina ion in casein and p ocessed cheeses wi h di e en a con en . Jou nal o Food Composi ion and Analysis, ol. 22. no 3. p. 224-232. h p://dx.doi.o g/10.1016/j.j ca.2008.10.023 Bu ne , J. R., Mamo e, C. D. S., Williams, R. A. 2008. Phenylke onu ia: An Inbo n E o o Phenylalanine Me abolism. Clin. Biochem. Re iew. ol. 29, no.1, p. 31-41. PMid:18566668 Commission Di ec i e 1999/21/EC o 25 Ma ch 1999 on die a y oods o special medical pu poses, OJ L 91, 7.4.1999, p. 29-36. D ořák, J. 2009. Fenylke onu ie PKU. 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S ačina, Š., B e šnajd o á, A., Holcá o á, I. e al. 2008. Klinická die ologie (Clinical Die e ics). 1s ed. P aha: G ada Publishing a.s., 384 p. ISBN 978-80-247-2256-6. Velíšek, J., Hajšlo á, J. 2009. Chemie po a in. II. (Food Chemis y. II.) 3 d ed Ha ličku B od: OSSIS. 644 p. ISBN 978-80-86659-16-9. Dec ee o he Czech Minis y o Heal h on 30 Janua y 2004 no. 54/2004 Coll. on oods u s in ended o pa icula nu i ional uses and how hey a e used. Acknowledgmen : This s udy was suppo ed by a p ojec o he in e nal g an s o Tomas Ba a Uni e si y in Zlin, Czech Republic no IGA/FT/2015/004 unded om he esou ces o speci ic uni e si y esea ch. Con ac add ess: S anisla a Ma ejo á, Slo ak Uni e si y o Ag icul u e in Ni a, Facul y o Bio echnology and Food Sciences, Depa men o Hygiene and Food Sa e y, T . A. Hlinku 2, 949 76 Ni a, Slo akia, E-mail: s.[email p o ec ed]m. Ma ina Fikselo á, Slo ak Uni e si y o Ag icul u e in Ni a, Facul y o Bio echnology and Food Sciences, Depa men o Hygiene and Food Sa e y, T . A. Hlinku 2, 949 76 Ni a, Slo akia, E-mail: ma ina. ikselo [email protected]. F an išek Buňka, Tomas Ba a Uni e si y in Zlín, Facul y o Technology, Depa men o Food Technology, nám. T. G. Masa yka 5555, 760 01 Zlín Czech Republic E-mail: b[email p o ec ed].