Rapid assessmen o selec ed ee amino acids du ing Edam cheese ipening by nea
in a ed spec oscopy
Jiří Mlček1, K ě osla a Šus o á2, O aka Rop3, Tünde Ju íko á4, Pe Humpolíček5
,
Š e an Balla4
1Tomas Ba a Uni e si y, Facul y o Technology, Depa men o Food Technology, Zlín, Czech Republic
2Mendel Uni e si y, Facul y o Ag onomy, Depa men o Food Technology, B no, Czech Republic
3College o Business and Ho el Managemen , Depa men o Gas onomy, B no, Czech Republic
4Cons an ine he Philosophe Uni e si y, Facul y o Cen al Eu opean S udies, Ins i u e o Na u al
and In o ma ics Sciences, Ni a, Slo akia
5Tomas Ba a Uni e si y, Cen e o Polyme Sys ems, Polyme Cen e, Zlín, Czech Republic
Recei ed July 2, 2012
Accep ed Ma ch 19, 2013
Abs ac
The s udy ocuses on apid de e mina ion o ee amino acids p oduced du ing he ipening
o cheese, by using nea in a ed spec oscopy. Analyses o 96 samples o Edam cheese (30%
and 45% o a in d y ma e ) we e pe o med a mon hly in e als up o he ipening age o 6
mon hs. In o al, 19 amino acids we e analysed wi h in a ed spec ome e using wo di e en
me hods, ei he in he egime o e lec ance in he in eg a ing sphe e o he appa a us o using a
ib e op ic appa a us wi h he ib e op ic p obe. Re e ence da a based on high-pe o mance liquid
ch oma og aphy we e used o calib a ion o he spec opho ome e . Calib a ion models we e
de eloped using a pa ial leas squa e algo i hm and es ed by means o c oss- alida ion. When
measu ed wi h he in eg a ing sphe e and wi h he p obe, he alues o co ela ion coe icien s
anged om 0.835 o 0.993 and om 0.739 o 0.995, espec i ely. Pai ed - es did no show
signi ican di e ences be ween he e e ence and p edic ed alues (P < 0.05). The esul s o his
new calib a ion me hod showed he possibili y o nea in a ed echnology o as de e mina ion
o ee amino acids, which occu du ing he ipening o Edam cheese. The con en o ee amino
acids allow us o p epa e Edam cheese quickly and e icien ly o sale o o p epa e he ma e ial
o p ocessed cheese.
NIR, ipeness o cheese, me hod o pa ial leas squa es, p o eolysis
The key ac o o ageing in cheese is i s chemical composi ion. The wa e con en is
one o he mos impo an ac o s in egula ion o cu d hyd olysis and mic obial g ow h
in manu ac u ed cheese (Janš o á e al. 2010). Ne e heless, changes in and analysis
o majo compounds p esen ed in young cheese a e no good indica o s o he cou se o
ipening. This is because hese cheese componen s a e s able and mo e o less unchanging
(He ing e al. 2008). On he o he hand, howe e , changes in con en s o pep ides and
ee amino acids a e much be e indica o s o cheese ageing. Cheese acqui es i s ypical
as e, smell, consis ency and appea ance only in he cou se o ageing, due o e men a ion
p ocesses ha change all h ee basic milk componen s, i.e. lac ose, p o eins and a .
P o eolysis is he mos complex and in he majo i y o cheese ypes also one o he mos
impo an biochemical p ocesses ha ake place in he cou se o ageing. The concen a ion
o amino acids, he inal p oduc s o p o eolysis, is dependen on he ype o cheese and
s age o cheese ageing. In Edam cheese, he ollowing amino acids a e he mos abundan :
glu amic acid, leucine, a ginine, lysine, phenylalanine and se ine. The ype and quan i y o
ee amino acids p esen in cheese in luences i s as e and indica es he deg ee o ageing
and i s p og ess. The composi ion and spec um o ee amino acids change depends on
he ype and age o cheese. Du ing cheese ageing, he concen a ions o amino acids a e
inc easing wi h he excep ion o a ginine because i s concen a ion dec eases in he inal
s age o ageing (McSweeney 2004). F ee amino acids hemsel es can subs an ially
ACTA VET. BRNO 2013, 82: 191–196; doi:10.2754/a b201382020191
Add ess o co espondence:
Ing. Jiří Mlček, Ph.D.
Depa men o Food Technology
Facul y o Technology, Tomas Ba a Uni e si y in Zlin
nam. T.G. Masa yka 275, 762 72 Zlin, Czech Republic
Phone: +420 576 033 030
Fax: +420 576 031 111
E-mail: [email p o ec ed]
h p://ac a e . u.cz/
con ibu e o he as e o cheese because hey a e one o i s senso y componen s (Lane e
al. 1997). Good knowledge o he s age o cheese ageing is he e o e an impo an ac o
o i s u he de elopmen and o he p oduc ion o p ocessed cheese (Bunka e al. 2004).
The aim o he s udy was o e alua e he possibili y o using nea in a ed spec ome y
o de e mina ion o ee amino acids which a ise du ing he ipening o Edam cheese as
an impo an indica o o he p oduc ion o p ocessed cheese. The esul s ob ained by
measu emen s pe o med in he in eg a ing sphe e and by p obe we e compa ed.
Ma e ials and Me hods
Analyses we e pe o med on Edam cheese samples o igina ing om wo dai y ac o ies (ma ked as A and B)
in he Czech Republic. Each ac o y supplied ou di e en p oduc s. Two o hem con ained 45% and 30% o
a in d y ma e (FDM), espec i ely, and wo we e so-called s a ing cul u es (Lac ococcus lac is subsp. lac is,
Lac ococcus lac is subsp. c emo is and Lac ococcus lac is subsp. c emo is, Lac ococcus lac is subsp. lac is,
Leuconos oc mesen e oides subsp. c emo is, Lac ococcus lac is subsp. diace ylac is, S ep ococcus he mophilus,
Lac obacillus hel e icus). Each o he samplings in ol ed h ee samples. Analyses o hese samples we e
pe o med a mon hly in e als up o he ipening age o 6 mon hs. The cen al pa (co e) and ind o cheese we e
e alua ed sepa a ely and he e o e he o al numbe o cheese samples was 96.
E e y mon h he cheese samples we e analysed o he p esence o ollowing 19 aminoacids: soluble y osine,
yp ophan, a ginine, o ni hine, lysine, his idine, glu amine, se ine, aspa agine, glycine, h eonine, alanine, p oline,
aspa ic acid, aline, glu amic acid, leucine, isoleucine and phenylalanine wi h he aim o calib a e he Fou ie
ans o m in a ed (FTIR) appa a us o indi idual componen s which change in dependence on he deg ee o
cheese ipeness. Concen a ions o ee amino acids we e es ima ed in he liquid ch oma og aph (HPLC) Agilen
1100 Se ies wi h he mass de ec o Agilen MSD 1456 VL (Agilen Technologies, USA) based on he me hod
o A do and Polych onidau (1999). All eagen s including s anda ds we e p o ided in he EZ: aas ™ amino
acid analysis sample es ing ki by Phenomenex Inc. (To ance, CA, USA). Da a ob ained om HPLC we e used
o calib a ion o he nea in a ed spec opho ome e . Edam cheeses we e analysed wi h he Nicole An a is nea
in a ed spec ome e ollowing he p ocedu e acco ding o Mlček e al. (2011). Two di e en me hods we e
used: in eg a ing sphe e (IS) and ib e op ic appa a us wi h a ib e op ic p obe. Calib a ion models we e c ea ed
wi h he TQ Analys e sion 6.2.1.509 (The mo Elec on Co po a ion, Madison, USA); s a is ical me hods used
we e simila as desc ibed by Mlček e al. (2006).
Resul s
Al oge he 96 samples o Edam cheese we e used o e alua e he amino acids. The
esul s ob ained by measu emen s pe o med in he in eg a ing sphe e and using he p obe
we e compa ed. All de eloped calib a ion models showed high co ela ion coe icien s
(Table 1). The highes co ela ion coe icien s we e ound in models o phenylalanine
(0.993) and yp ophan (0.992) in samples o Edam cheese measu ed in he in eg a ing
sphe e and o phenylalanine (0.995) and o ni hine (0.993) measu ed by he p obe. When
using he in eg a ing sphe e and p obe, he lowes alues o co ela ion coe icien s we e
ound in he calib a ion models o glycine (0.835) and p oline (0.739), espec i ely. In
gene al, i can be concluded ha he e we e no ma ked di e ences in alues o co ela ion
coe icien s when using bo h a o emen ioned me hods.
Co ela ion coe icien s o alida ion anged om 0.733 o 0.983 and om 0.558 o 0.960
when using he in eg a ing sphe e and p obe, espec i ely. Simila ly as wi h calib a ion
esul s also in case o he co ela ion coe icien o alida ion he bes esul s we e ob ained
o phenylalanine (R = 0.983 and 0.960 when measu ing in he in eg a ing sphe e and
using he p obe, espec i ely). The wo s esul s we e eco ded o glycine (R = 0.733)
and p oline (R = 0.558) when measu ing wi h he in eg a ing sphe e and using he p obe,
espec i ely.
Table 2 indica es ha in case o he p obe, be e s anda d de ia ions o calib a ion
alues we e ob ained o en amino acids (o ni hine, lysine, his idine, glu amine, glycine,
h eonine, alanine, aspa ic acid, leucine, phenylalanine) whe eas be e s anda d de ia ions
o alida ion alues we e ob ained only o i e amino acids (his idine, y osine, glu amine,
glycine and alanine). Excep o he amino acids men ioned abo e, o he amino acids
192
showed be e esul s ob ained by measu ing in he in eg a ing sphe e. This means ha
he in eg a ing sphe e can be ecommended o he es ima ion o amino acids in cheese by
means o NIR spec oscopy because lowe e o alues a e ob ained.
Fo all amino acids he alues o calib a ion coe icien s o a ia ion (CCV) we e highe
han 5% and he alues o p edic ion coe icien o a ia ion (PCV) exceeded 10%. The
inc eased a iabili y esul ed om complex dependences and also om a mino p esence
o amino acids in cheese. The lowes alues o CCV we e eco ded o phenylalanine when
measu ing bo h in he in eg a ing sphe e and using he p obe (CCV = 6.48% and CCV =
5.84%, espec i ely). As a as he alue o PCV was de ec ed, he lowes alues we e
eco ded also in case o phenylalanine (PCV = 10.12% and PCV = 15.81%, espec i ely).
Discussion
NIR spec oscopy shows good abili y o se alues o a s, wa e con en and p o eins in
na u al and p ocessed cheese and he ob ained esul s co ela e well wi h o he e e ence
me hods (Rod iguez-O e o e al. 1995; McQueen e al. 1995; Adamopoulos e al.
2001; Cu da e al. 2001; McKenna 2001). These me hods a e o in e es especially when
moni o ing he p ocess o cheese ageing because hey enable analysis o changes in hei
composi ion. The composi ion o young cheeses is a key ac o o hei u he ageing
and he mois u e con en is one o he mos impo an ac o s o he egula ion o cu d
hyd olysis and o cheese mic obial g ow h.
193
Table 1. Values o ee amino acids in Edam cheese, es ablished by e e ence me hods (n = 96), calib a ion and
alida ion esul s o ee amino acids in Edam cheese es ablished by in eg a ing sphe e
In eg a ing sphe e x ± SD (nmol/g) Calib a ion Valida ion
R SEC (nmol/g) CCV (%) R SEP (nmol/g) PCV (%)
A ginine 10.57 ± 7.68 ns 0.970 1.87 17.69 0.837 4.22 39.92
O ni hine 230.06 ± 146.96 ns 0.977 31.6 13.74 0.966 38.1 16.56
Lysine 258.89 ± 149.34 ns 0.958 42.7 16.49 0.930 55 21.25
His idine 66.40 ± 46.23 ns 0.929 17.1 25.75 0.874 22.6 34.03
T yp ophane 17.43 ± 9.03 ns 0.992 11.6 6.65 0.949 28.6 16.41
Ty osine 95.85 ± 45.43 ns 0.949 80.9 9.60 0.884 213.0 22.22
Glu amine 166.61 ± 118.54 ns 0.983 22.1 13.27 0.915 47.9 28.75
Se ine 162.23 ± 90.15 ns 0.912 36.9 22.75 0.881 42.7 26.32
Aspa agine 282.07 ± 177.73 ns 0.890 81 28.72 0.849 94.1 33.36
Glycine 126.75 ± 85.32 ns 0.835 47 37.08 0.733 58.6 46.23
Th eonine 110.59 ± 71.19 ns 0.941 24 21.70 0.918 28.2 25.50
Alanine 238.52 ± 160.57 ns 0.848 85.2 35.72 0.788 99.4 41.67
P oline 200.80 ± 179.42 ns 0.935 63.8 31.77 0.821 102 50.80
Aspa ic acid 46.75 ± 29.45 ns 0.975 6.5 13.9 0.961 8.16 17.46
Valine 292.14 ± 232.48 ns 0.967 59.5 20.37 0.940 79.5 27.21
Glu amic acid 610.20 ± 453.28 ns 0.959 129 21.14 0.919 179 29.34
Leucine 497.40 ± 380.19 ns 0.973 88.2 17.73 0.953 115 23.12
Isoleucine 92.57 ± 85.88 ns 0.969 21.2 22.90 0.940 29.3 31.65
Phenylalanine 274.69 ± 150.39 ns 0.993 17.8 6.48 0.983 27.8 10.12
x - a e age alues, SD - s anda d de ia ion, R - co ela ion coe icien , SEC - s anda d e o o calib a ion, CCV
- calib a ion coe icien o a ia ion, SEP - s anda d e o o p edic ion, PCV - p edic ion coe icien o a ia ion,
ns - non-signi ican di e ence (P < 0.05)
In he cou se o cheese ageing, he cu d is hyd olysed by enzymes o igina ing om
enne , by na i e enzymes o milk, and by mic oo ganisms p esen in he cheese ma ix.
Casein is deg aded o pep ides and hese a e u he deg aded o indi idual amino acids
ha can become p ecu so s o a bad as e, which is caused by a ious acids, alcohols,
aldehydes, and ke ones (McSweeney e al. 2000). Types and amoun s o ee amino
acids p esen in cheese in luence i s as e and indica e o cha ac e ise he s age o ageing.
The spec um o ee amino acids in cheese changes in dependence on i s ype and age.
The au ho s ound ha he e we e di e ences in he con en o ee amino acids no only
be ween indi idual ypes bu also be ween indi idual ba ches o cheese; hese di e ences
we e caused by a ying con en s o d y ma e , sal , amoun s o added lac ic acid bac e ia,
and o so-called non-s a e lac ic acid bac e ia (NSLAB) which occu in cheese unde so
a unexplained condi ions (Øs lie e al. 2004; Øs lie e al. 2005).
The deg ada ion o some speci ic ee amino acids is impo an o he de elopmen
o senso y subs ances con ained in cheese (Y on e al. 2001). Me hionine is deg aded
o me hane hiol, which is he cause o unpleasan sulphu ous s ench o cheese. The
deg ada ion o b anched-chain amino acids (leucine, aline and isoleucine) and o
a oma ic amino acids (phenylalanine, y osine a yp ophan) esul s in o ma ion o a ious
aldehydes and alcohols (i.e. ue a oma ic cheese componen s) and o a ious ca boxylic
acids (i.e. p ecu so s o a oma ic compounds) (Y on e al. 2001). Recen ly i was ound
ha also deg ada ion o se ine (Se ) esul ed in p oduc ion o s ong a oma ic compounds
(Liu e al. 2003).
194
Table 2. Values o ee amino acids in Edam cheese, es ablished by e e ence me hods (n = 96), calib a ion and
alida ion esul s o ee amino acids in Edam cheese, es ablished by p obe
P obe x ± SD (nmol/g) Calib a ion Valida ion
R SEC (nmol/g) CCV (%) R SEP (nmol/g) PCV (%)
A ginine 10.7 ± 7.57 ns 0.925 2.87 27.15 0.821 4.35 41.15
O ni hine 242.64 ± 152.7 ns 0.993 18 7.42 0.946 49.5 20.40
Lysine 285.73 ± 186.02 ns 0.976 40.2 14.07 0.927 70.1 24.53
His idine 71.56 ± 53.91 ns 0.970 13.1 18.31 0.911 22.2 31.02
T yp ophane 16.89 ± 7.96 ns 0.951 24.7 14.62 0.819 46 27.23
Ty osine 93.74 ± 44.04 ns 0.957 128.0 13.66 0.929 163.0 17.39
Glu amine 171.09 ± 118.48 ns 0.991 16.1 9.41 0.931 43.4 25.37
Se ine 160.39 ± 86.18 ns 0.900 37.6 23.44 0.844 46.3 28.87
Aspa agine 316.46 ± 234.94 ns 0.937 82.3 26.01 0.901 102 32.23
Glycine 155.73 ± 119.56 ns 0.940 40.7 26.14 0.888 55 35.32
Th eonine 117.98 ± 76.06 ns 0.986 12.8 10.85 0.889 35.2 29.84
Alanine 264.84 ± 193.46 ns 0.912 79.3 29.94 0.873 94.8 35.80
P oline 188.34 ± 144.10 ns 0.739 97 51.50 0.558 122 64.78
Aspa ic acid 51.68 ± 29.00 ns 0.981 5.62 10.88 0.934 10.4 20.12
Valine 306.08 ± 244.40 ns 0.946 79.4 25.94 0.877 118 38.55
Glu amic acid 656.52 ± 471.24 ns 0.955 140 21.32 0.915 191 29.09
Leucine 577.19 ± 416.78 ns 0.979 84.5 14.64 0.935 148 25.64
Isoleucine 121.70 ± 120.81 ns 0.975 26.7 21.94 0.916 48.7 40.02
Phenylalanine 282.67 ± 160.03 ns 0.995 16.5 5.84 0.960 44.7 15.81
x - a e age alues, SD - s anda d de ia ion, R - co ela ion coe icien , SEC - s anda d e o o calib a ion, CCV
- calib a ion coe icien o a ia ion, SEP - s anda d e o o p edic ion, PCV - p edic ion coe icien o a ia ion,
ns
- non-signi ican di e ence (P < 0.05)
Good knowledge o he s age o ipeness o na u al cheese is impo an in o ma ion o
he subsequen manu ac u ing o p ocessed cheese (Fo man 1996; Fox 2004).
Based on he highes alues o co ela ion coe icien s o bo h calib a ion and
alida ion expe imen s, he model de eloped o he es ima ion o phenylalanine using
bo h a o emen ioned me hods can be ecommended as he mos eliable. Es ima ion
o p oline, alanine, aspa agine and glycine a e he leas eliable because hey showed
no only he highes di e ences be ween coe icien s o co ela ion bu also he highes
a iabili y.
Pa ame ic - es co obo a ed ha he e alua ed e e ence and ins umen al me hods did
no gi e di e en esul s ( s a < k i ).
Fo all models used o he es ima ion o ee amino acids, NIR spec oscopy seems
o be sui able o o ien a ion pu poses because only in he model o phenylalanine he
PCV alue measu ed in he in eg a ion sphe e was a he le el o 10% (PCV = 10.12%);
whe eas using he p obe he CCV alue was equal o 5.84%. Fo he measu emen o his
amino acid we can e alua e his model as eliable. Howe e , compa ing bo h me hods o
measu emen , measu ing wi h he in eg a ing sphe e seems o be mo e sui able.
Al hough he es ima ion o ee amino acids by means o NIR spec oscopy ga e, as
expec ed, a li le wo se esul s (mainly due o he ac ha compa ed o he basic componen s
o cheese, he accu acy o es ima ion o some less equen compounds was lowe ), i is
possible o ecommend he use o NIR spec oscopy o an o ien a ion es and es ima ion
o hese subs ances mainly due o i s speed. I could con ibu e no only o he e alua ion
o he deg ee o ipeness o Edam cheese bu also when selec ing op imum aw ma e ial
o making p ocessed cheeses. Compa ed o adi ional me hods o de e mina ion o he
chemical composi ion and quali y o cheese, he NIRS me hod enables a as , simple and
simul aneous e alua ion o mo e componen s a once and p ope ies o cheese wi hou he
need o any chemicals. I is also a non-des uc i e me hod sui able o ood e alua ion e en
in in e -ope a ional analysis.
Acknowledgemen
This s udy was suppo ed by p ojec s NAZV KUS QJ1230044 which we e inanced by he Minis y
o Ag icul u e o he Czech Republic.
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