During malting, barley storage proteins are partially degraded by protein-ases into amino acids and peptides that are critical for obtaining high-quality malt and therefore high-quality
Trang 2R E V I E W P A P E R
Protein changes during malting and brewing with focus
on haze and foam formation: a review
Elisabeth Steiner•Martina Gastl•Thomas Becker
Received: 17 October 2010 / Revised: 6 December 2010 / Accepted: 13 December 2010 / Published online: 5 January 2011
Ó Springer-Verlag 2010
Abstract Beer is a complex mixture of over 450
con-stituents and, in addition, it contains macromolecules such
as proteins, nucleic acids, polysaccharides, and lipids In
beer, several different protein groups, originating from
barley, barley malt, and yeast, are known to influence beer
quality Some of them play a role in foam formation and
mouthfeel, and others are known to form haze and have to
be precipitated to guarantee haze stability, since turbidity
gives a first visual impression of the quality of beer to the
consumer These proteins are derived from the malt used
and are influenced, modified, and aggregated throughout
the whole malting and brewing process During malting,
barley storage proteins are partially degraded by
protein-ases into amino acids and peptides that are critical for
obtaining high-quality malt and therefore high-quality wort
and beer During mashing, proteins are solubilized and
transferred into the produced wort Throughout wort
boil-ing proteins are glycated and coagulated beboil-ing possible to
separate those coagulated proteins from the wort as hot
trub In fermentation and maturation process, proteins
aggregate as well, because of low pH, and can be
sepa-rated The understanding of beer protein also requires
knowledge about the barley cultivar characteristics on
barley/malt proteins, hordeins, protein Z, and LTP1 This
review summarizes the protein composition and functions
and the changes of malt proteins in beer during the malting
and brewing process Also methods for protein
identifica-tion are described
Keywords Proteins Barley Malt Beer Hazeformation Foam formation
Proteins in barley and maltBarley (Hordeum vulgare L.) is a major food and animalfeed crop It ranks fourth in area of cultivation of cerealcrops in the world Barley is commonly used as rawmaterial for malting and subsequently production of beer,where certain specifications have to be fulfilled Thesespecifications are among others: germinative capacity,protein content, sorting (kernel size), water content, kernelabnormalities, and infestation Malting includes the con-trolled germination of barley in which hydrolytic enzymesare synthesized, and the cell walls, proteins, and starch ofthe endosperm are largely digested, making the grain morefriable [1 3] Proteins in beer are mainly derived from thebarley used The mature barley grain contains a spectrum
of proteins that differ in function, location, structure, andother physical and chemical characteristics Barley seedtissues have different soluble protein contents and distinctproteomes
The three main tissues of the barley seed are the rone layer, embryo, and starchy endosperm that account forabout 9, 4, and 87%, respectively, of the seed dry weight[4, 5] The level of protein in barley is an importantdeterminant in considering the final product quality of beer,for example for cultivar identification or as an indication ofmalting quality parameters [4], and it is influenced by soilconditions, crop rotation, fertilization, and weather condi-tions For malting barley, the balance between carbohy-drates and proteins is important, since high protein contentreduces primarily the amount of available carbohydrates.Proteins present in barley seeds are important quality
aleu-E Steiner ( &) M Gastl T Becker
Lehrstuhl fu¨r Brau- und Getra¨nketechnologie, Technische
Universita¨t Mu¨nchen, Weihenstephaner Steig 20,
85354 Freising, Germany
e-mail: Elisabeth.Steiner@wzw.tum.de
DOI 10.1007/s00217-010-1412-6
Trang 3determinants During malting, barley storage proteins are
partially degraded by proteinases into amino acids and
peptides which are critical for obtaining high-quality malt
and therefore high-quality wort and beer [1,6,7]
Germination provides the necessary hydrolytic enzymes
to modify the grain, which are, in the case of proteins,
endoproteases, and carboxypeptidases These enzymes
degrade storage proteins, especially prolamins (hordeins)
and glutelins [8] and produce free amino acids during
ger-mination by cleavage of reserve proteins in the endosperm
[9] According to Mikola [10], there exist five seine
carb-oxypeptidases in germinating barley, which have
comple-mentary specificities and mostly an acidic pH optimum All
of these carboxypeptidases consist of 2 identical subunits,
each compose of two polypeptide chains, cross-linked by
disulphide bridges [9, 11,12] Barley malt endoproteases
(EC.3.4.21) develop multiple isoforms mainly during grain
germination and pass through kilning almost intact [8,13]
Jones [13–17] surveyed those enzymes and their behavior
during malting and mashing Cysteine proteases (EC
3.4.22) are clearly important players in the hydrolysis of
barley proteins during malting and mashing However, it
seems likely that they do not play as predominant a role as
was attributed to them in the past [15, 16,18–22] It has
been found out that metalloproteases (EC 3.4.24) play a
very significant role in solubilizing proteins, especially
during mashing at pH 5.8–6.0 [23] All current evidence
suggests that the serine proteases (EC 3.4.21) play little or
no direct role in the solubilization of barley storage proteins
[23,24], even though they comprise one of the most active
enzyme forms present in malt [22] While none of the barley
aspartic proteases (EC 3.4.23), that have been purified and
characterized, seem to be involved in hydrolyzing the seed
storage proteins, it is likely that other members of this group
do participate Jones [17] investigated endoproteases in
malt and wort and discovered that they were inactivated at
temperatures above 60°C Jones et al [6] examined the
influence of the kilning process toward the endoproteolytic
activity These enzymes were affected by heating at 68 and
85°C, during the final stages of kilning, but these changes
did not influence the overall proteolytic activity
Other proteins are involved in protein folding, such as
protein disulfide isomerase (EC 5.3.4.1), which catalyzes
the formation of protein disulfide bridges Due to their
heat-sensitivity, proteinases are inactivated when the
tem-perature rises above 72°C [25–30] They are almost totally
inactive within 16 min [1,7,13]
Summarizing the most important factors for the protein
composition, as origin in finished beer are barley cultivar
and the level of protein modification during malting, which
is judged by malt modification which is conventionally
measured in the brewing industry as the Kolbach index
To get an overview of the main proteins in malt and beer,the most studied proteins are described in the next para-graphs Proteins can be classified pursuant to their solubil-ity Osborne [33–37] took advantage of this fact anddeveloped a procedure to separate the proteins Proteins aredivided into water-soluble (albumins), salt-soluble (globu-lins), alcohol-soluble (prolamins), and alkali-soluble(glutelins) fractions [34–36,38,39] Osborne fractionation
is a relatively simple, fast, and sensitive extraction–analysisprocedure for the routine quantitation of all protein types incereals in relative and absolute quantities, including theoptimization of protein extraction and of quantitativeanalysis by RP-HPLC High-performance liquid chroma-tography (or high-pressure liquid chromatography, HPLC)
is a chromatographic technique that can separate a mixture
of compounds and is used in biochemistry and analyticalchemistry to identify, quantify, and purify the individualcomponents of the mixture
Not only Osborne fractionation and HPLC but alsoseveral other methods exist to separate and identify pro-teins in barley, malt, wort, and beer To get an overviewover the applications of the described methods in thereview, a description follows in the next paragraphs.Several authors [5, 39–60] characterized barley andbarley malt proteins with help of 2D-PAGE Other authors[25, 26,29,30, 32, 41,61–65] used 2D-PAGE and massspectrometry to fingerprint the protein composition in beerand to evaluate protein composition with regard to foamstability and haze formation Klose [39] followed proteinchanges during malting with the help of a Lab-on-a-Chiptechnique and validated the results with 2D-PAGE Iimure
et al [64] invented a protein map for the use in beer qualitycontrol This beer proteome map provides a strong detec-tion platform for the behaviors of beer quality–relatedproteins, like foam stability and haze formation Thenucleotide and amino acid sequences defined by the proteinidentification in the beer proteome map may have advan-tages for barley breeding and process control for beerbrewing The nucleotide sequences also give access toDNA markers in barley breeding by detecting sequencepolymorphisms
Hejgaard et al [66–73] worked with phoresis and could identify several malt and beer proteins.Shewry et al [54,74–78] determined several methods forinvestigation of proteins in barley, malt, and beer mainlywith different electrophoresis methods Asano et al [62,
immunoelectro-63] worked with size-exclusion chromatography, noelectrophoresis and SDS–PAGE Mills et al [79] madeimmunological studies of hydrophobic proteins in beerwith main focus and foam proteins He discovered that themost hydrophilic protein group contained the majority ofthe proteinaceous material but it also comprised polypep-
Trang 4immu-Vaag et al [28] established a quantitative ELISA
method to identify a 17 kDa Protein and Ishibashi et al
[80] used an ELISA technique to quantify the range of
foam-active protein found in malts produced in different
geographic regions, and using different barley cultivars
Van Nierop et al [30] used an ELISA technique to follow
LTP1 content during the brewing process
Osman et al [18–20] investigated the activity of
endo-proteases in barley, malt, and mash Hence, protein
degra-dation during malting and brewing is very important for the
later beer quality (mouthfeel, foam, and haze stability) It
was suggested that estimation of the levels of degraded
hordein (the estimation of the levels of hordein degraded
during malting truly reflects the changes in proteins during
malting and can measure the difference in barley varieties
related to proteins and their degrading enzymes) during
malting is a sensitive indicator of the total proteolytic action
of proteinases as well as the degradability of the reserve
proteins And therefore, it is possible to predict several beer
quality parameters according the total activity of all
pro-teinases and the protein modification during malting
To obtain good results, those separation and
identifica-tion methods can be combined Van Nierop et al [30], for
example, used ELISA, 2D-PAGE, RP-HPLC, electrospray
mass spectrometry (ESMS), and circular dichroism (CD)
spectrophotometry to follow the changes of LTP1 before
and after boiling
Since there exist various methods to separate and
identify proteins in this review, an overview over existent
proteins in barley, malt, wort, and beer is provided
according to only one method, which is Osborne
fraction-ation These fractions are described more closely in the
next sections
Barley glutelin
About 30% of barley protein is glutelin that dissolves only
in diluted alkali [54] Glutelin is localized almost entirely
in the starchy endosperm (Fig.1), is not broken down later
on, and passes unchanged into the spent grains [81,82].Glutelin is the least well-understood grain protein frac-tion This is partly because the poor solubility of thecomponents has necessitated the use of extreme extractionconditions and powerful solvents which often cause dena-turation and even degradation (e.g., by the use of alkali) ofthe proteins, rendering electrophoretic analysis difficult.Also, because glutelin is the last fraction to be extracted, it
is frequently affected by previous treatments and inated with residual proteins from other fractions, notablyprolamins, which are incompletely extracted by classicalOsborne procedures [83] It has not been possible to pre-pare an undenatured glutelin fraction totally free of con-taminating hordein [3]
contam-Barley prolaminThe prolamin in barley is called hordein and it constitutesabout 37% of the barley protein It dissolves in 80% alcoholand part of it passes into spent grains Hordein is a low-lysine, high-proline, and high-glutamine alcohol-solubleprotein family found in barley endosperm (Fig.1) It is themajor nitrogenous fraction of barley endosperm composing35–55% of the total nitrogen in the mature grain [1,84–86].Hordeins are accumulated relatively late in grain develop-ment, first being observed about 22 days after anthesis(when the grain weighs about 33% of its final dry weight)and increasing in amount until maximum dry weight isreached [87] The major storage proteins in most cerealgrains are alcohol-soluble prolamins These are not singlecomponents, but form a polymorphic series of polypeptides
of considerable complexity [88] Hordein is synthesized onthe rough endoplasmic reticulum during later stages of grainfilling and deposited within vacuoles in protein bodies [89,
90] Silva et al [91] ascertained that the exposure ofhordeins to a proteolytic process during germination redu-ces its content and originates in less hydrophobic peptides.Fig 1 Shematic longitudinal
section of a barley grain [ 81 ]
Trang 5Some malt water–soluble proteins result from the hordein
proteolysis Hordeins are the most abundant proteins in
barley endosperm characterized by their solubility in
alco-hol These storage proteins form a matrix around the starch
granules, and it is suggested that their degradation during
malting directly affects the availability of starch to
amylo-lytic attack during mashing [92]
Shewry [75,77] divided the hordeins according to their
size and amino acid composition in four different fractions
(A-D), dependent on their size and amino acid
composi-tion A-hordeins (15–25 kDa) seem to be no genuine
storage proteins as they contain protease inhibitors and
a-amylases B-hordeins (32–45 kDa) are rich in sulfur
content and are, with 80%, the biggest hordein fraction
B-hordeins have a general structure, with an assumed
sig-nal peptide of 19 aminoacid residues, a central repetitive
domain rich in proline and glutamine residues, and a
C-terminal domain containing most of the cysteine residues
are encoded by a single structural locus, Hor2, located on
the short arm 1 of chromosome 1H(5), 7–8 cM distal to the
Hor1 locus which codes for the C-hordeins C-hordeins
(49–72 kDa) are low in sulfur content, and D-hordeins
([100 kDa) are the largest storage proteins and are
enco-ded by the Hor3 locus located on the long arm of
chro-mosome 1H(5) [85,87,93,94]
Cereal prolamins are not single proteins but complex
polymorphic mixtures of polypeptides [54] During
malt-ing, disulfide bonds are reduced and B- and D-hordeins are
broken down by proteolysis Well-modified malt contains
less than half the amount of hordeins present in the original
barley D-hordeins are degraded more rapidly than their
B-type counterparts, and the latter are more rapidly degraded
than C-hordeins [3,95]
Barley albumins and globulins
Many researchers extract a combined salt-soluble protein
fraction, because water extracts contain globulins as well as
albumins The two classes of proteins may be separated by
dialysis, but there is considerable overlap between the two
[83] Albumins and globulins consist mainly of metabolic
proteins, at least in the cereal grains [96] and are found in
the embryo and the aleurone layer, respectively [81, 82]
Whereas prolamins are degraded during germination,
al-bumins and other soluble proteins increased during the
germination process [92]
Globulins
The globulin fraction of barley is called edestin It dissolves
in dilute salt solutions and hence also in the mash It forms
about 15% of the barley protein Edestin forms 4 components
not completely precipitate even on prolonged boiling and cangive rise to haze in beer Enzymes and enzyme-related pro-teins are mainly albumins and globulins [42]
AlbuminsThe albumin of barley is called leucosin It dissolves inpure water and constitutes about 11% of the protein inbarley During boiling, it is completely precipitated.a-Amylase, protein Z, and lipid transfer proteins are barleyalbumins and are important for the beer quality attributes:foam stability and haze formation [97] Albumins can befurther divided into protein Z and lipid transfer proteins asfunctional proteins
Protein ZProtein Z belongs to a family of barley serpins and consists
of at least four antigenically identical molecular forms withisoelectric points in the range 5.55—5.80 (in beer:5.1–5.4), but same molecular mass near 40 kDa [1,55,67,
68, 98] Protein Z is hydrophobe and exists in free andbound forms in barley, like a-amylase, and there also existheterodimers Protein Z contains 2 cysteine and 20 lysineresidues per monomer molecule and is relatively rich inleucine and other hydrophobic residues Protein Z accountsfor 5% of the albumin fraction and more than 7% in somehigh-lysine barleys [67, 99] The content of protein Z inbarley grains depends on the level of nitrogen fertilization[67, 100] Protein Z makes up to 20–170 mg/L of beerprotein [79] In mature seeds, protein Z is present in thiolbound forms, which are released during germination [101].The function of the protein is at present unknown but it isknown that it is deposited specifically in the endospermresponding to nitrogen fertilizer, similar to the hordeinstorage proteins The synthesis is regulated during graindevelopment at the transcriptional level in dependence ofthe supply of nitrogen [98,100,102,103] It is stated thatupregulation of transcript levels could be effectuatedwithin hours, if ammonium nitrate was supplied throughthe peduncle, and equally rapid reduced when the supplywas stopped [103] Finnie et al [49] investigated the pro-teome of grain filling and seed maturation in barley Theyidentified a group of proteins that increased gradually both
in intensity and abundance, during the entire examinationperiod of development and were identified as serpins AlsoSorensen [55] and Giese [98] could detect the expression ofprotein Z4 (a subform of protein Z) only during germina-tion Protein Z4 has an expression profile similar tob-amylase and seed storage proteins (hordeins)
Three distinct serpin sequences from barley could befound in the databases SWISSPROT and TREMBL: pro-
Trang 6Z forms are thought to have a role as storage proteins in
plants, due to their high ‘‘Lys’’ content and the fact that
serpin gene expression is regulated by the ‘‘high-Lys’’
alleles lys1 and lys3a [49,104]
Hejgaard et al [68] suggest that the precursors of protein Z
originate from chromosomes 4 and 7, and thus, they are named
protein Z4 and protein Z7 Rasmussen and co-workers [105]
were able to estimate the size of protein Z mRNA at 1.800 b
This is sufficient to code for the 46.000 or 44.000 MW
pre-cursor peptides found in vitro translations plus leave 400–500
b for the 50 and 30non-coding regions Doll [106] and
Ras-mussen [107] suggest that protein Z could be a candidate for
modulation of the barley seed protein composition to balance
the nutritional quality of the grain Giese and Hejgaard [98]
found out that during germination, protein Z becomes the
dominant protein in the salt-soluble fraction in developing
barley The proteins in barley malt are known to be glycated
by D-glucose, which is a product of starch degradation during
malting [108] Bobalova et al [109] investigated in their
research the glycation of protein Z and found out that protein Z
glycation is detectable from the second day of malting The
role of protein Z in beer is described more detailed in the
sections foam and haze formation
Lipid transfer protein
Lipid transfer proteins (LTPs) are ubiquitous plant
lipid-binding proteins that were originally identified by their
ability to catalyze the transfer of lipids between
mem-branes LTPs are abundant soluble proteins of the aleurone
layers from barley endosperm The compact structure of
the barley LTP1 comprises four helices stabilized by four
disulfide bonds and a well-defined C-terminal arm with no
regular secondary structure [110] which is shown in Fig.2,
where a 3D and surface protein of barley LTP native
protein (here called 1LIP, red) is shown [111] In
com-parison with other plant lipid transfer proteins, the barley
protein has a small hydrophobic cavity but is capable of
binding different lipids such as fatty acids and acyl-CoA
[25,112, 113] According to molecular mass, this
multi-gene family is subdivided into two subfamilies, ns-LTP1
(9 kDa) and ns-LTP2 (7 kDa); both located in the aleurone
layer of the cereal grain endosperm [56, 114] LTP1 and
LTP2 are expressed in barley grain but only LTP1 has been
able to be detected in beer LTP1 is claimed to be an
inhibitor of malt cysteine endoproteases [14,115] The role
of LTP1 in beer is described more detailed in the sections
foam and haze formation
Protein Z and LTP1
Evans [116,117] investigated the influence of the malting
process on the different protein Z types and LTP1 He
discovered that the amount of LTP1 did not change duringgermination but a significant proportion of the bound/latentprotein Z was converted into the free fraction He claimsthat during germination, proteolytic cleavage in the reac-tive site loop converts protein Z to a heat and proteasestable forms, and hence, they can survive the brewingprocess He ascertained also that kilning reduced theamount of protein Z and LTP1 [66,118]
Evans [116] analyzed feed and malting barley varietiesand could not find any differences in the level of protein Zand LTP1 He also ascertained malt-derived factors thatinfluence beer foam stability, such as protein Z4, b-glucan,viscosity, and Kohlbach index Beer components (pro-tein Z4, free amino nitrogen, b-glucan, arabinoxylan, andviscosity) were correlated with foam stability [117] Pro-tein Z4, protein Z7, and LTP1 have been shown to act asprotease inhibitors [116,119,120]
Proteins in wort and beerProteins influence the whole brewing process not only inthe form of enzymes but also in combination with othersubstances such as polyphenols As enzymes, they degradestarch, b-glucans, and proteins In protein–protein linkages,they stabilize foams and are responsible for mouthfeel andflavor stability, and in combination with polyphenols, theyare thought to form haze As amino acids, peptides, and salammoniac, they are important nitrogen sources for yeast[121] Only about 20% of the total grain proteins are watersoluble Barley water-soluble proteins are believed to beresistant to proteolysis and heat coagulation and hence passthrough the processing steps, intact or somewhat modified,
to beer [116, 122, 123] Several aspects of the brewingprocess are affected by soluble proteins, peptides, and/orFig 2 3D and surface protein of barley LTP native protein (1LIP, red) is shown [ 111 ]
Trang 7amino acids that are released No more than one-third of
the total protein content passes into the finished beer which
is obtained throughout mainly two processes; mashing and
the wort boiling Mashing is the first biochemical process
step of brewing and completes the enzymatic degradation
started during malting Enzymes synthesized during
malt-ing are absolutely essential for the degradation of large
molecules during mashing These enzymes are displayed in
Table1 [1, 7] The three biochemical basic processes
taking place during malting are cytolysis, proteolysis, and
amylolysis, which are indicated by b-glucan, FAN, and
extract concentration, respectively In order to get good
brews, part of the insoluble native protein must be
con-verted into ‘‘soluble protein’’ during malting and mashing
This fraction comprises a mixture of amino acids, peptides,
and dissolved proteins, and a major portion of it arises by
proteolysis of barley proteins [23] During the brewing
process, there are three possibilities to discard the
(unwanted) proteinic particles The first opportunity is
given during wort boiling, where proteins coagulate and
can be removed in the ‘‘whirlpool’’ The second, during
fermentation, where the pH decreases and proteinic
parti-cles can be separated by sedimentation The third step is
during maturation of beer During maturation, proteins
adhere on the yeast and can be discarded [124]
It has also been demonstrated that yeast proteins are
present in beer, but only as minor constituents [73] Beer
contains *500 mg/L of proteinaceous material including a
variety of polypeptides with molecular masses ranging
originate from barley proteins, are the product of theenzymatic (proteolytic) and chemical modifications(hydrogen bonds, Maillard reaction) that occur duringbrewing, especially during mashing, where proteolyticenzymes are liable for those modifications [125] A beerprotein may be defined as a more or less heterogeneousmixture of molecules containing the same core of peptidestructure, originating from only one distinct protein present
in the brewing materials [126] Jones [13–17] surveyedproteinases and their behavior during malting and mashing.Proteinases are not active in beer anymore; hence, they areinactivated when the temperature rises above 72°C, whichhappens already during mashing [1,7,13,25–30].Proteins influence two main quality aspects in the finalbeer: 1st haze formation and 2nd foam stability In thefollowing lines, these quality attributes are described in amore detailed way
Haze formationProteins play a major role in beer stability; hence, they are,beside polyphenols, part of colloidal haze There exist twoforms of haze; cold break (chill haze) and age-related haze[127] Cold break haze forms at 0°C and dissolves athigher temperatures If cold break haze does not dissolve,age-related haze develops, which is non-reversible Chillhaze is formed when polypeptides and polyphenols arebound non-covalently Permanent haze forms in the same
Table 1 Enzymes in barley and barley malt [ 1 , 7 , 166 , 167 ]
Cytolysis b-glucan-solubilase Matrix linked b-glucan Soluble, high molecular weight b-glucan
Endo-b-(1-3)
glucanase
Soluble, high molecular weight b-glucan Low molecular weight b-glucan, cellobiose,
laminaribiose Endo-b-(1-4)
glucanase
Soluble, high molecular weight b-glucan Low molecular weight b-glucan, cellobiose,
laminaribiose Exo-b-glucanase Cellobiose, laminaribiose Glucose
Proteolysis Endopeptidase Proteins Peptides, free amino acids
Carboxypeptidase Proteins, peptides Free amino acids
Aminopeptidase Proteins, peptides Free amino acids
Amylolysis a-amylase High and low molecular weight a-glucans Melagosaccharides, oligosaccharides
Limit dextrinase Limit dextrins Dextrins
Pullulanase a-1,6- D -glucans in amylopectin, glykogen,
pullulan
Linear amylose fractions
Other Lipase Lipids, lipidhydroperoxide Glycerine, free fatty acids, fatty acid hydroperoxide
Lipoxygenase Free fatty acids Fatty acid hydroperoxide
Trang 8insoluble complexes are created which will not dissolve
when heated [128] Proanthocyanidins (condensed tannins)
from the testa tissue (seed coat) of the barley grain are
carried from the malt into the wort and are also found after
fermentation of the wort in the beer There they cause
precipitation of proteins and haze formation especially
after refrigeration of the beer, even if it previously had
been filtered to be brilliantly clear [129] Proteins, as the
main cause of haze formation in beer, can be divided into
two main groups: 1st proteins and 2nd their breakdown
products Protein breakdown products are characterized by
always being soluble in water and do not precipitate during
boiling Finished beer contains almost only protein
break-down products [126] The content of only 2 mg/L protein is
enough to form haze [118] Beer contains a number of
barley proteins that are modified chemically (hydrogen
bond formation, Maillard reaction) and enzymatically
(proteolysis) during the malting and brewing processes,
which can influence final beer haze stability Leiper et al
[130, 131] found out that the mashing stage of brewing
affects the amount of haze-active protein in beer If a beer
has been brewed with a protein rest (48–52°C), it may
contain less total protein but more haze-active proteins
because the extra proteolysis caused release of more haze
causing polypeptides Asano et al [62] investigated
dif-ferent protein fractions and split them in 3 categories: 1st
high, 2nd middle and 3rd low molecular weight fractions
being high molecular weight fractions: [40 kDa, middle
molecular weight fraction: 15–40 kDa and low molecular
weight fraction: \15 kDa Nummi et al [132] even
sug-gested that acidic proteins derived from albumins and
globulins of barley are responsible for chill haze formation
(Table2)
Researchers proofed that proline-rich proteins are
involved in haze formation [63,65,124,127,128,130,131,
133–137] Outtrup et al [138] say that haze-active proteins
are known to be dependent on the distribution of proline
within the protein Nadzeyka et al [127] suggested that
proteins in the size range between 15–35 kDa comprised the
highest amount of proline It was also investigated that
proline and glutamic acid-rich hordeins, in the size range
between 10–30 kDa, are the main initiators causing haze
development [63,74] b-Amylase, protein Z, and two
chy-motrypsin inhibitors have relatively high-lysine contents
[100] Barley storage proteins that are available for lysis are all proline-rich proteins [15] Dadic and Belleau[139,140] on contrary say that there is no specific aminoacid composition for haze-active proteins Leiper [130,131]even says that not only the mainly consistence of proline andglutamic acid of the glycoproteins is responsible for causinghaze but also that the carbohydrate component consistslargely of hexose It was found out that the most importantglycoproteins for haze formation are 16.5 and 30.7 kDa insize Glycation is a common form of non-enzymatic modi-fication that influences the properties of proteins [109] Non-enzymatic glycation of lysine or arginine residues is due tothe chemical reactions in proteins, which happen during theMaillard reaction [109] It is one of the most widely spreadside-chain-specific modifications formed by the reaction ofa-oxoaldehydes, reducing carbohydrates or their derivativeswith free amine groups in peptides and proteins, such ase-amino groups in lysine and guanidine groups in arginine[141, 142] The proteins in barley malt are known to beglycated by D-glucose, which is a product of starch degra-dation during malting [108] D-glucose reacts with a freeamine group yielding a Schiff base, which undergoes a rapidrearrangement forming more stable Amadori compounds.Haze-sensitive proteins
hydro-Polypeptides that are involved in haze formation are alsoknown as sensitive proteins They will precipitate withtannic acid, which provides a mean to determine theirlevels in beer Proline sites of these polypeptides bind tosilica gel hydroxyl groups so that haze-forming proteins areselectively adsorbed, since foam proteins contain littleproline and are thus not affected by silica treatment [143].Removal of haze forming tannoids can be effected usingPVPP [143] To assure colloidal stability, it is not neces-sary to remove all of the sensitive proteins or tannoids.Identification of a tolerable level of these proteins can beused to define a beer composition at bottling that deliverssatisfactory haze stability [94,99] To prolong stability ofbeer, stabilization aids are used Haze-forming particles areremoved with: (a) silica, which is used to remove proline-rich proteins that have the ability to interact with poly-phenols to form haze in bright beer, or (b) PVPP, which isused to remove haze-active polyphenols
Evans et al [144] investigated the composition of thefractions which were absorbed by silica This analysisrevealed that the mole percentage of proline rangedbetween 33.2 and 38.0%, and of glutamate/glutaminebetween 32.7 and 33.0%, consistent with the proline/glu-tamine–rich composition of the hordeins [144] Iimure
et al [65] stated in their studies that proteins adsorbed ontosilica gel (PAS) are protein Z4, protein Z7, and trypsin/amylase inhibitor pUP13 (TAI), rather than BDAI-1
Table 2 Distribution of hordeins in barley according to their size
Trang 9(a-amylase inhibitor), CMb, and CMe La´zaro et al [145]
investigated the CM proteins CMa, CMb, and CMe The
CM proteins are a group of major salt-soluble endosperm
proteins encoded by a disperse multigene family and act as
serine proteinase inhibitors Genes CMa, CMb, and CMe
are located in chromosomes 1, 4, and 3, respectively
Protein CMe has been found to be identical with a
previ-ously described trypsin inhibitor Furthermore, Iimure et al
[64] analyzed proline compositions in beer proteins, PAS,
and haze proteins It was proofed that the proline
compo-sitions of PAS were higher (ca 20 mol%) than those in the
beer proteins (ca 10 mol%), although those of the
haze-active proteins such as BDAI-1, CMb, and CMe were
6.6–8.7 mol% These results suggest that BDAI-1, CMb,
and CMe are not predominant haze-active proteins, but
growth factors of beer colloidal haze Serine proteinase
inhibitors have also been called trypsin/a-amylase
inhibi-tors, and it has been proposed that some of them might
inhibit the activities of barley serine proteinases However,
none have been shown to affect barley enzymes [16]
Robinson et al [146] identified a polymorphism for beer
haze-active proteins and surveyed by immunoblot analysis
throughout the brewing process In this polymorphism,
some barley varieties contained a molecular weight band at
12 kDa, while in other varieties, this band was absent Pilot
brewing trials have shown that the absence of this 12 kDa
protein conferred improved beer haze stability on the
resulting beer This band was detected by a polyclonal
antibody raised against a haze-active, proline/glutamine–
rich protein fraction; it was initially assumed that the band
was a member of the hordein protein family [144,147]
Foam formation
Beer foam is an important quality parameter for customers
Good foam formation and stability gives an impression of a
freshly brewed and well-tasting beer Therefore, it is
nec-essary to investigate mechanisms that are behind foam
formation Beer foam is characterized by its stability,
adherence to glass, and texture [148] Foam occurs on
dispensing the beer as a result of the formation of CO2
bubbles released by the reduction in pressure The CO2
bubbles collect surface-active materials as they rise These
surface-active substances have a low surface tension, this
means that within limits they can increase their surface
area and also, after the bubbles have risen, they form an
elastic skin around the gas bubble The greater the amount
of dissolved CO2 the more foam is formed But foam
formation is not the same as foam stability Foam is only
stable in the presence of these surface-active substances
[1] Beer foam is stabilized by the interaction between
hop a-acids, but destabilized by lipids [30, 148] Theintention is to find a good compromise of balancing foam-positive and foam-negative components Foam-positivecomponents such as hop acids, proteins, metal ions, gascomposition (ratio of nitrogen to carbon dioxide), and gaslevel, generally improve foam, when increased Whereasfoam negatives, such as lipids, basic amino acids, ethanol,yeast protease activity, and excessive malt modification,decrease foam formation and stability Free fatty acids,which are extracted during mashing, have a negative effect
on foam stability [64,65,80,85,88,128–131,166].Foam-positive proteins can be divided into highmolecular weight proteins (35–50 kDa) and low molecularweight proteins (5–15 kDa) which primary originate frommalt but in small amount can also originate from yeast [62,
73, 148] It is thought that during foam formation, phiphile proteins surround foam cells and stabilize them byforming a layer They arrange themselves into bilayers, bypositioning their polar groups toward the surroundingaqueous medium and their lipophilic chains toward theinside of the bilayer, defining a non-polar region betweentwo polar ones [149] There are two main opinions con-cerning the nature of foaming polypeptides in beer Thefirst position claims the existence of specific proteins whichbasically influence foam stability Those proteins areknown as protein Z and LTP1 [150, 151] The secondargument claims the existence of a diversity of polypep-tides which stabilize foam; the more hydrophobic theirnature, the more foam active they are [122, 152], likehordeins that are rich in proline and glutamine content andexhibit a hydrophobic b-turn-rich structure [74] KAPP
am-[153] investigated the influence of albumin and hordeinfractions from barley on foam stability, because both areable to increase the foam stability The ability to form morestabile foams seems to be higher by albumins than byhordeins Denaturation of these proteins causes an increase
in their hydrophobic character and also in their foam bility This confirms the already known opinion that themore hydrophobic the protein, the better is the foam sta-bility [122,152] The foams from albumins are more stablethan those from hordeins This may also be the reason forthe increased ability of albumin fractions to withstand thepresence of ethanol The foam stability of both albuminsand hordeins is increased by bitter acids derived from hops.Whereas the barley LTP1 does not display any foamingproperties, the corresponding beer protein is surface active.Such an improvement is related to glycation by Maillardreactions on malting, acylation on mashing, and structuralunfolding on brewing which was ascertained by Perrocheau
sta-et al [25] During the malting and brewing processes,LTP1 becomes a surface-active protein that concentrates inbeer foam [55] LTP1 is modified during boiling and this
Trang 10forms have been recovered in beer with marked chemical
modifications including disulfide bond reduction and
rear-rangement and especially glycation by Maillard reaction
The glycation is heterogeneous with variable amounts of
hexose units bound to LTPs [112] The four lysine residues
of LTP1 are the potential sites of glycation [112]
Alto-gether, glycation, lipid adduction, and unfolding should
increase the amphiphilic character of LTP1 polypeptides
and contribute to a better adsorption at air–water interfaces
and thus promote foam stability
Van Nierop et al [30] established that LTP1
denatur-ation reduces its ability to act as a binding protein for foam
damaging free fatty acids and therefore boiling and boiling
temperature are important factors in determining the level
and conformation of LTP1 and so enhance foam stability
Perrocheau et al [25] showed that unfolding of LTP1
occurred on wort boiling before fermentation and that the
reducing conditions are provided by malt extract Van
Nierop et al [30] showed that the wort boiling temperature
during the brewing process was critical in determining the
final beer LTP1 content and conformation It was
discov-ered that higher wort boiling temperatures (102°C)
resul-ted in lower LTP1 levels than lower wort boiling
temperatures (96°C) Combination of low levels of LTP1
and increased levels of free fatty acids resulted in low foam
stability, whereas beer produced with low levels of LTP1
and free fatty acids had satisfactory foam stability LTP1
has been demonstrated to be foam promoting only in its
heat denatured form [55,150,154]
Perrocheau et al [26] investigated heat-stable,
water-soluble proteins that influence foam stability Most of the
heat stable proteins were disulfide-rich proteins, implicated
in the defense of plants against their bio-aggressors, e.g.,
serpin-like chymotrypsin inhibitors (protein Z), amylase
and amylase-protease inhibitors, and lipid transfer proteins
(LTP1 and LTP2) Leisegang et al [95–97] identified
LTP1 as a substrate for proteinase A, which degrades
LTP1, but does not influence protein Z and may have a
negative influence on beer foam stability Iimure et al [32]
invented a prediction method of beer foam stability using
protein Z, barley dimeric a-amylase inhibitor-1 (BDAI-1)
and yeast thioredoxin and confirmed BDAI-1 and protein Z
as foam-positive factors and identified yeast thioredoxin as
a possible novel foam-negative factor Jin et al [155,156]
found out in their research that structural changes of
pro-teins during the wort boiling process are independent of the
malt variety It was discovered that barley trypsin inhibitor
CMe and protein Z were resistant to proteolysis and heat
denaturation during the brewing process and might be
important contributors to beer haze formation Vaag et al
[28] found a new protein of 17 kDa which seemed to
influence foam stability even more than protein Z and
barley like LTP1 She could support this theory by the
correlation of the content of this so called 17 kDa proteinand the foam half-life of lager beers LTP1 and the 17 kDaprotein exhibit some similarities; their tertiary structuresare characterized by disulfide bridges, both are rich incysteine and are modified during heating to a more foampromoting form Ishibashi et al [80] agrees that bothmalting and mashing conditions influence the foam-activeprotein levels in experimental mashes Proteinaceousmaterials in beer have as well been implicated in the sta-bilization of beer foam Molecular weight has beenreported to be important for foaming potential, while thehydrophobicity of polypeptides has been cited as a con-trolling factor [62] Kordialik-Bogacka et al [157, 158]investigated also foam-active polypeptides in beer Incontrary to Osman et al [123] in this investigation, it wasconfirmed that fermentation influences the protein com-position of beer and particularly in beer foam
Yeast polypeptides were also found in beer foam It wasnoted that, especially during the fermentation of highgravity wort, excessive foaming may occur, and this may
be one of the reasons why beer brewed at higher gravitieshas a poor head It was detected that polypeptides ofmolecular weight about 40 kDa present in fermented wortand foam originated not only from malt but also from yeastcells Okada et al [159] studied on the influence of proteinmodification on foam stability during malting They foundthat the foam stability of beer samples brewed from barleymalts of 2 cultivars decreased as the level of malt modifi-cation increased, but the foam stability of another cultivardid not change In this research, they defined BDAI-I as animportant contributor to beer foam stability
ConclusionProteins do not only influence haze formation; furthermore,they play an important role for mouthfeel and foam sta-bility These aspects are important for brewers, sinceconsumer judge beer also according to these quality attri-butes As it is known, most foam-positive proteins are alsohaze active, Evans et al [144] made an investigation toimmunologically differentiate between those two proteinforms (foam and haze-active proteins) and concluded that
no barley variety or growing condition have any significantinfluence on beer stability It was also demonstrated in aregression analysis that a prediction of foam stability is notpossible, which underlines the complexity of these prob-lems It is suggested that both foam-active and foam-neg-ative components should be measured and that the amount
of hordeins and protein Z4 are somehow related It wasalso ascertained that foam and haze-active proteins sharesome epitopes and that oxygen during the brewing processinfluence haze stability of beer [147]
Trang 11Leiper et al [130, 131] studied beer proteins that are
involved in haze and foam formation All proteins were
found to be glycosylated to varying degrees The size
range of the polypeptides which make up the glycoprotein
fraction of beer is relatively narrow and the range was
found to be from 10 to 46 kDa The glycoproteins were
found to consist of proteins, six carbon sugars (hexoses),
and five carbon sugars (pentoses) Beer glycoproteins
were found to exist in three forms; those responsible for
causing haze, those responsible for providing foam
sta-bility, and a third group that appeared to have no role in
physical or foam stability Approximately 25% of beer
glycoproteins are involved in foam and foam stability As
3–7% of beer glycoproteins have been identified as being
involved in haze formation, this leaves around 70% of
beer glycoproteins that appear to have no role in either
physical and/or foam stability This fraction contains the
most abundant beer polypeptide, protein Z, which is
glycosylated with both hexoses and pentoses It has been
estimated that about 16 % of the lysine content of
pro-tein Z are glycated during the brewing process through
Maillard reaction [61,126]
There are three major groups of proteins in beer The
first consists of a group of proline-rich fragments
origi-nating from hordein ranging in size from 15–32 kDa which
are involved with haze formation The second is LTP1
(9.7 kDa in pure form) that is involved in foam stability
and the third is protein Z (40 kDa) that appears to have no
direct function, but may play a role in stabilizing foam
once it has been formed [130, 131] Several authors [25,
30,49,66,70,73,125,126,160,161] investigated
haze-active proteins in beer Two major proteins in beer are
claimed to cause haze formation and influence foam
sta-bility; protein Z and LTP 1 Protein Z and LTP1 are heat
stable and resistant to proteolytic modification during beer
production and appear to be the only proteins of barley
origin present in significant amounts in beer It is presumed
that protein Z causes haze and is all the same positive for
foam stability [70,73] LTP1 is claimed not to influence
foam stability but the quantity of foam generated [55,117]
Protein Z is homologous to serine protease inhibitors and
these inhibitory properties might be the reason that protein
Z is not degraded by proteolytic enzymes during malting
and mashing [104, 126, 162, 163] Curioni et al [125]
showed that glycation of protein Z improved foam stability
and might prevent precipitation of protein during the wort
boiling step Both glycation and denaturation increase the
amphiphilicity of LTP1 polypeptides and contribute to a
better adsorption at air–water interfaces of beer foam [55,
164] Jin et al [155,156] found out in their research that
structural changes of proteins during the wort boiling
process are independent of the malt variety It was
dis-were resistant to proteolysis and heat denaturation duringthe brewing process and might be important contributors tobeer haze formation It is known that foam-active hydro-phobic protein fractions in beer can be hydrolyzed byproteinases leading to a decrease in foam stability.Besides proteins, other beer constituents such as iso-alpha acids, peptides, amino acids, proteinase, fatty acids,and melanoidins were suggested to influence haze forma-tion and foam properties [154,165] The contents of theseconstituents in beer were influenced by brewing materialvariables such as barley varieties, malt types, hop usage,yeast strains, and malting and brewing processes
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Trang 16O R I G I N A L P A P E R
Determination of celiac disease-specific peptidase activity
of germinated cereals
Benedict Geßendorfer•Georg Hartmann •
Herbert Wieser• Peter Koehler
Received: 8 July 2010 / Revised: 17 September 2010 / Accepted: 4 October 2010 / Published online: 19 October 2010
Ó Springer-Verlag 2010
Abstract A method to determine the celiac
disease-spe-cific peptidase activity of different germinated cereals was
developed Kernels of common wheat, spelt, emmer,
ein-korn, rye, and barley were germinated, lyophilized, and
milled into flour and bran The latter was extracted at pH
4.0 to obtain a solution enriched with peptidases The
synthetic a-gliadin peptide with the amino acid sequence
PQPQLPYPQPQLPY (peptide IV), which has been shown
to be toxic for celiac disease patients, was selected as
substrate for bran peptidases It was quantified by
reversed-phase high-performance liquid chromatography on C18
silica gel For kinetic studies, rye bran extract was
incu-bated with peptide IV at 50°C and pH 6.5 The peptide
was degraded continuously, and only 30.2% of the original
peptide was detected after 90 min Accordingly, the bran
extracts of all cereals were investigated The incubation
time was set to 60 min at 50°C, and the degradation of
peptide IV was performed at pH 4.0 and 6.5, respectively
Except for rye, peptide degradation was faster at pH 4.0
than at pH 6.5 At pH 4.0, emmer extract was most active,
followed by spelt, common wheat, and einkorn extracts
The activity of rye and barley extracts was significantly
lower In conclusion, the method is easy to perform, quick,and provides reproducible results It can be applied to otherpeptidase sources such as bacterial or fungal cultures tooptimize peptidase preparations suitable for detoxifyinggluten-containing food or for drugs to treat celiac disease.Keywords Cereals Germination Peptidases
Celiac disease
IntroductionCeliac disease (CD) is a frequent inflammatory disease ofthe small intestine triggered by the storage proteins(gluten) from wheat, rye, barley, and possibly oats [1],although oats are currently considered as safe for CDpatients, if they are pure Gluten proteins are not com-pletely degraded by human gastrointestinal enzymesresulting in peptides that stimulate T cells in the laminapropria of CD patients [2] Most of these toxic peptidesare derived from glutamine- and proline-rich sections ofgluten proteins and have a minimum length of nine aminoacid residues required for T-cell recognition [3] Thecurrent essential therapy is a lifelong abandonment ofwheat, rye, barley, and oat products by means of a strictgluten-free diet; this means a severe restriction of lifequality Therefore, there is an urgent need to develop safeand effective alternatives ‘Peptidase therapy’ has beenproposed as a future therapeutic option for CD, in whichspecific ‘prolyl endopeptidases’ are used to degradeproline-rich gluten peptides into small fragments that donot stimulate intestinal T cells any more [4] In addition,these peptidases have been recommended for decreasingthe level of CD-toxic proteins and peptides in rawmaterial and food For these purposes, peptidases from
H Wieser P Koehler ( &)
Deutsche Forschungsanstalt fu¨r Lebensmittelchemie,
Lise-Meitner-Straße 34, 85354 Freising, Germany
Trang 17bacteria (e.g Flavobacterium meningosepticum,
sour-dough lactobacilli) and fungi (Aspergillus niger) have
been suggested [2,5,6] Our previous investigations have
shown that also peptidases from germinated cereals
(common wheat, rye, barley) have the ability to degrade
CD-toxic proteins and peptides very quickly [7] These
peptidases have distinct advantages when compared to
bacterial or fungal peptidases and are promising
candi-dates for the detoxification of gluten-containing foods [8]
and possibly for oral therapy of CD patients They are
composed of endopeptidases and exopeptidases and have
unique specificities optimized by nature for the
frag-mentation of gluten proteins and peptides [7] and are
derived from a naturally safe and cheap raw material
Their production is part of well-established technological
processes (malting, brewing, milling), and the extraction
of highly active peptidases from bran is simple In
con-trast to bacterial and fungal peptidases, no genetic
engi-neering is necessary for production However, up to date,
the activity of these peptidases is mostly determined by
using protein substrates such as hemoglobin and casein or
short synthetic peptide substrates of very limited length
These substrates provide no or only limited information
whether the peptidases under study are able to degrade
CD-toxic peptides Therefore, the aim of the present work
was to develop a method to determine CD-specific
pep-tidase activity by using a CD-toxic peptide as substrate
and to show that it can be applied to screen the
CD-specific peptidase activity of different germinated wheat
species, rye, and barley
Materials and methods
Germination
Kernels (150 g) of common wheat (cultivar (cv.)
Tom-mi), spelt (cv Oberkulmer Rotkorn), emmer (cv Osiris),
einkorn (cv Tifi), rye (cv Nikita), and barley (cv Barke)
were germinated for seven days at 15°C in two phases
[7] During the first one, kernels were immersed with
distilled water (450 mL) for 5 h at 20°C and 70% air
humidity using a wide glass vessel (Ø 18 cm) After the
water was decanted, the kernels were transferred into a
stainless steel sieve (mesh size = 1.2 mm), washed with
distilled water, and allowed to equilibrate for 19 h at
13°C and 100% air humidity After washing with
dis-tilled water, the kernels were again soaked for 4 h and
equilibrated for 20 h as described earlier The wet kernels
were finally soaked for 10 min, transferred into a plastic
container (14 9 14 9 6 cm) with a perforated bottom,
and subjected to a temperature of 15°C for 120 h (second
distilled water twice a day Germination was stopped bypouring liquid nitrogen onto the kernels, which were thencrushed by means of a blender (300 W, Krups, Solingen,Germany) and lyophilized The dried material was milledinto flour (particle size \ 0.2 mm) and bran ([ 0.2 mm)using a Quadrumat Junior mill (Brabender, Duisburg,Germany) and stored at -18°C
Extraction of peptidasesBran (100 mg) was extracted with 1.0 mL of buffer A(sodium acetate 0.2 mol/L, pH 4.0 with HCl) at roomtemperature (RT & 22°C) by means of subsequent vor-texing (1 min) and magnetic stirring (30 min) The sus-pension was centrifuged (RT, 20 min, 3,850 g), and thesupernatant was decanted and filtered through a 0.45-mmmembrane (= peptidase solution)
Peptide substrateSynthetic peptide IV [7] (amino acid sequencePQPQLPYPQPQLPY, molecular mass 1,665.9 g/mol) waspurchased from GenScript Corporation, Piscataway, NJ,USA; the grade of purity was 95% The peptide (0.7 mg)was dissolved in 1.0 mL of buffer A or buffer B (sodiumhydrogen sulfate 0.2 mol/L, pH 8.0), respectively (=sub-strate solutions A and B)
IncubationAbout 150 lL of peptidase solution was mixed with
150 lL of substrate solution A (final pH = 4.0) or strate solution B (final pH = 6.5) and magnetically stirredfor 60 min at 50°C The reaction was stopped by heatingfor 10 min at 90 °C The samples were stored at 4 °Cbefore reversed-phase high-performance liquid chroma-tography (RP-HPLC) analysis In a preliminary experi-ment, the peptidase solution prepared from rye bran wasincubated with substrate solution B and aliquots (100 lL)
sub-of the assay were taken after 0, 10, 20, 30, 45, 60, 75, and
90 min incubation time and inactivated by heating tidase solution, substrate solution, and incubation assaywere filtered through a 0.45-mm membrane prior toRP-HPLC analysis, which was performed under the fol-lowing conditions: instrument, solvent module 126 with aSystem Gold Software (Beckman, Munich, Germany);column, Nucleosil 100–5 C18, 3 9 250 mm (Macherey–Nagel, Dueren Germany); temperature, 50°C; injection,
Pep-30 lL; elution system, (A) triethylammonium formiate(TEAF) (0.01 mol/L, pH 3.0), (B) acetonitrile ? TEAF(0.01 mol/L) linear gradient, 0 min 0% B, 30 min 40% B;flow rate, 0.8 mL/min; detection, UV absorbance at
Trang 18Choice of celiac-active peptide
The synthetic peptide IV consisting of fourteen amino acid
residues with the sequence PQPQLPYPQPQLPY was
selected as substrate for the determination of CD-specific
peptidase activity [7] This peptide occurs in the
N-termi-nal domain of a-gliadins (positions a62–75) and has been
shown to be toxic for CD patients in vivo [9, 10]
More-over, it is part of the CD-toxic so-called 33mer-peptide
(a56–88), which also cannot be degraded by human
gas-trointestinal enzymes [2] The reason for choosing peptide
IV instead of the 33-mer peptide was its shorter length and
thus the fact that in further work, it can be more easily
synthesized and purified than the 33-mer Its amino acid
composition is characterized by a high proline content
(43 mol-%), which is responsible for the resistance to
digestion In the present work, peptide IV was quantified
by RP-HPLC on C18 silica gel via the absorbance area
measured at 210 nm wavelength (Fig.1) Due to a purity
grade of 95%, the elution profile of peptide IV showed two
minor peaks beside the major peak and the total area of the
three peaks was taken for integration
Germination and peptidase preparation
Kernels from pure cultivars of different wheat species
(common wheat, spelt, emmer, einkorn), rye, and barley
were used as starting materials for the production of
pep-tidase preparations To induce peppep-tidase activity, the
ker-nels were germinated for seven days at 15°C, freeze-dried,
and milled into flour and bran According to previous
experiments [7], in which germination was conducted at 15and 30°C, the lower temperature was selected for germi-nation, because under these conditions, higher peptidaseactivities were obtained for most cereal grains and the riskfor mold spoilage was considerably lower at 15 whencompared to 30°C After germination, peptidase solutionswere prepared by extracting the bran with a sodium acetate/HCl buffer at pH 4.0
Peptidase activity test
In a preliminary test, the bran extract of rye was incubatedwith peptide IV at 50°C and pH 6.5 [7] and aliquots weretaken after 0, 10, 20, 30, 45, 60, 75, and 90 min to followpeptide degradation by germination-induced CD-specificpeptidases The reaction was stopped by heating, and thequantity of residual peptide IV was determined by RP-HPLC The elution patterns of the assays demonstrated thatthe constituents of the bran extract did not disturb peptidequantitation (Fig 1) This was confirmed by comparativeinjections of peptide standard and sample solutions (pH4.0, 0.35 mg/mL each), the latter containing the branextract from germinated wheat The results for peptidases
in rye bran extract shown in Fig.2 indicated that specific peptidases were present because peptide IV wascontinuously degraded during incubation with 30.2% of thestarting peptide being detectable after 90 min For rye branextract, these data were fitted to a first-order exponentialdecay typical for enzymatic reactions with a coefficient ofcorrelation of r = 0.997 Usually, enzyme activity is cal-culated from the change of the measured parameter duringthe first few minutes of the reaction However, in this case,this was not possible because the standard deviations of thepeptide absorbance areas after an incubation time of 10 and
celiac-20 min were subject to variation Standard deviations werebetter, if peptide degradation rates of more than 50% wereconsidered Therefore, it was decided to use an incubationtime of 60 min for the following experiments even thoughthis leads to a slight underestimation of the enzymaticactivity In general, shorter incubation times wouldhave been possible, but with higher standard deviations.Thus, the difference of the peptide concentrations at 0 and
60 min was used for the calculation of the activity In thecase of highly active peptidase preparations, shorter incu-bation times or dilution of the peptidase solution isrecommended
pH Dependence of activityPrevious studies demonstrated that pH optima of wheat andrye peptidases were 4.0 and 6.5 [7]; thus, these pH valueswere applied in parallel assays The degradation rates andpeptidase activities (units per mg bran, nkat per mLFig 1 HPLC chromatogram of rye bran extract mixed with peptide
IV at the beginning of incubation at pH 6.5
Trang 19extract) are summarized in Table1 The results from
twelve analyses performed as duplicates indicated a good
reproducibility for the method (average coefficient of
var-iation = ±3.7%) With the exception of rye, the peptidase
extracts caused a faster degradation and a higher activity at
pH 4.0 when compared to pH 6.5 At pH 4.0, the emmer
extract had the highest degradation rate (75.6%) and
activity (88.3 nkat) followed by spelt (75.0%/87.6 nkat),
common wheat (61.4%/71.7 nkat), and einkorn (55.9%/
65.3 nkat) The effects of rye extract (29.4%/34.3 nkat)
and barley extract (29.7%/34.7 nkat) were significantly
weaker The values for pH 6.5 were particularly lowered in
the case of common wheat, spelt, and emmer, but
drasti-cally increased in the case of rye extract (55.0%/64.2 nkat)
It has to be emphasized that these data have been obtained
under standardized conditions of germination not
opti-mized for the different grains Because different cereals
may substantially differ in their germination preferences,substantially higher peptidase activities can be expected ifgermination is carried out under optimized conditions
DiscussionThe experiments have demonstrated that it is possible todetermine the activity of peptidases with substrates that arepresent in the small intestine after digestion of gluten bygastric, pancreatic, and brush border enzymes In this case,
a peptide derived from a-gliadins with in vivo CD toxicityhas been selected Using a selection of two or three CD-toxic peptides as substrate would have increased thespecificity of the method; however, to keep the assay assimple as possible, it was decided to use only one peptide.Thus, using a CD-toxic peptide as substrate appears to bemost suited for determining CD-specific peptidase activitywhen compared to synthetic substrates containing chro-mophores for spectrophotometric detection, which mightinterfere with a proper interaction of enzyme and substrateand thus might provide incorrect information on the trueCD-specific enzyme activity
When compared to fungal or bacterial sources, cerealgrains are a good alternative for producing CD-specificpeptidases Peptidase activity can be induced or increased
by germinating the grains under standardized conditions Inthis work, peptidases were enriched in the bran showingactivities ranging from 21 to 63 U/kg bran They wereaffected by both the pH value and the bran source Themeasured peptidase activities would be well sufficient to beused for the degradation of residual gluten (‘detoxifica-tion’) in foods or raw materials such as wheat starch, beer,
or wheat bran, since the incubation time is not as limited as
in therapeutic applications In food applications, the branitself could be used as the enzyme sample; on the other
Fig 2 Degradation of peptide IV during incubation with a rye bran
extract at pH 6.5 Fitting of the data to an experimental decay The
coefficient of correlation r was 0.997
Table 1 Residual peptide IV after 60 min incubation with bran extracts from different germinated cereals (0 min = 100%) and peptidase activity of the bran as well as of the extract at pH 4.0 and pH 6.5a
(%) (U/kg)b (U/kg)c (nkat/L)d (%) (U/kg)c (U/kg)b (nkat/L)dCommon wheat 61.4 ± 1.8 43.0 ± 1.3 172.5 ± 5.2 71.7 ± 2.1 44.6 ± 3.3 125.2 ± 9.2 31.2 ± 2.3 52.1 ± 3.9 Spelt 75.0 ± 3.4 52.5 ± 2.4 186.4 ± 8.5 87.6 ± 4.0 53.7 ± 2.8 133.5 ± 7.1 37.6 ± 2.0 62.7 ± 3.3 Emmer 75.6 ± 1.1 62.9 ± 0.8 262.2 ± 3.3 88.3 ± 1.3 61.7 ± 2.2 180.1 ± 6.3 43.2 ± 1.5 72.0 ± 2.6 Einkorn 55.9 ± 0.5 39.1 ± 0.4 258.4 ± 2.6 65.3 ± 0.6 52.3 ± 1.3 241.9 ± 5.9 36.6 ± 0.9 61.1 ± 1.5 Rye 29.4 ± 2.2 20.6 ± 1.5 103.7 ± 7.6 34.3 ± 2.6 55.0 ± 0.8 193.9 ± 3.0 38.5 ± 0.6 64.2 ± 0.9 Barley 29.7 ± 0.8 20.8 ± 0.6 90.6 ± 2.6 34.7 ± 0.9 23.9 ± 1.1 72.7 ± 3.5 16.7 ± 0.8 27.9 ± 1.3
a Mean value of two determinations ± standard deviation
b Units per kg bran
c Units per kg bran extract
Trang 20hand, a buffered extract of the bran would possibly be more
suitable For therapeutic use (gluten detoxification in the
stomach), a CD-specific peptidase activity of 5–50 U/mg in
the drug has been suggested [11], which is 106times higher
than the peptidase activity of the bran From this, it is clear
that the peptidases present in bran of germinated cereals
need to be enriched to be suitable for the use as a drug to
detoxify gluten in the gastrointestinal tract
Conclusion
The developed method for the determination of
CD-specific peptidase activity of germinated cereals can be
performed relatively simple and quick and produces
repeatable results In principle, this method is also
appli-cable for other sources of peptidases, e.g cultures of
bac-teria and fungi or sourdough, and supports the production
of peptidase preparations optimal for the detoxification of
gluten-containing raw materials and foods, and even for the
oral therapy of CD Future studies will investigate
differ-ences between cultivars within a cereal species
Acknowledgments The authors thank Leibniz-Gemeinschaft
(WGL) for financial support, Mrs A Axthelm for excellent technical
assistance, and Dr C Kling, Hohenheim, and Dr K.-J Mueller for
supplying us with spelt, emmer, and einkorn kernels.
References
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LM, Khosla C (2002) Science 297:2275–2279
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C, Silano M, de Vincenzi M, Losito I, Gobetti M (2006) Biochim Biophys Acta 1762:80–93
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9 Arentz-Hansen H, Ko¨rner R, Molberg Ø, Quarsten H, Vader W, Kooy YMC, Lundin KEA, Koning F, Roepstorff P, Sollid LM,
Mc Adam SN (2000) J Exp Med 191:603–612
10 Ellis HJ, Pollock EL, Engel W, Fraser JS, Rosen-Bronson S, Wieser H, Ciclitira PJ (2003) Gut 52:212–217
11 Shan L, Bethune M, Khosla C, Gass J, Pyle GG, Gray GM, Isaacs
I, Strohmeier G (2008) US Patent Application US 2008095710 A1 20080424
Trang 21Abstract Five commercial grape seed extracts (GSEs)
were put under pasteurisation (HTST and LTLT), cooking,
baking and sterilisation conditions After each treatment,
the tannin content, antioxidant activity, browning and
characteristics of eight phenolic compounds were
deter-mined For nearly all quantified parameters, significant
differences (p \ 0.05) were found between at least two
treatments The gallic acid, gallocatechin and browning
parameters showed a greater tendency to increase in the
treatments, and the antioxidant activity showed a greater
tendency to decrease A positive correlation between the
tannin content and browning and a negative correlation
between the gallic acid and antioxidant activity were
found The GSEs were clearly grouped based on their
composition; nevertheless, a grouping based on the
treat-ments did not exist It can be concluded that the thermal
treatments affected the stability of all GSEs in a different
manner depending on the phenolic profile of each extract
Keywords Polyphenols Grape seed Thermal
treatments Antioxidant activity Flavan-3-ols
TC Tannin content
AA Antioxidant activityA420 Browning absorbance at 420 nm
IntroductionInterest in the research of polyphenols from different nat-ural sources has grown because polyphenols can be utilised
as antioxidants in the food industry, and they benefit humanhealth in various ways The beneficial effects of naturalantioxidants on human health come mainly from thecapability of polyphenols to scavenge free radicals andtherefore protect cells from the damage caused by freeradicals [1] Polyphenols can also act as anti-inflammatoryagents [2, 3], they can inhibit the progression of athero-sclerosis [4,5], and they can prevent the development andprogression of cancer [6]
Waste by-products from the wine industry have proven
to be one source that is rich in phenolic compounds.Among the different wine industry by-products, grapeseeds contain the highest amount of total phenolic com-pounds Catechins and their isomers and polymers are themain phenolic components in the seeds [7 9]
These grape seed extracts (GSE) can be employed asfunctional ingredients in different nutraceutical products.Functional ingredients must fulfil certain parameters For
G Davidov-Pardo ( &) I Arozarena M R Marı´n-Arroyo
Department of Food Technology, Public University of Navarre,
Campus Arrosadia s/n Edificio de los Olivos, Navarre, Spain
DOI 10.1007/s00217-010-1377-5
Trang 22to elaborate the product to which it was added, the
func-tional ingredient must retain its characteristics and
properties
Many of the elaboration processes in the food industry
involve temperature elevation, making the thermal stability
of the components within the product essential It is known
that the polyphenolic content from GSEs in food can be
affected by many factors, such as grape variety [10, 11],
environment [12], food storage conditions [13, 14] and
food processing, which includes heating Heating studies
have been conducted on different food products to evaluate
these changes For example, the epimerisation and
degra-dation of flavan-3-ols have been evaluated and modelled
for a green tea extract at high temperatures ranging from
100 to 165°C with various durations of up to 120 min
These tests showed that the epimerisation and degradation
of the tea’s catechins followed first-order reactions, and the
rate constants of the reaction kinetics followed the
Arrhe-nius equation [15] The polymerisation of phenolic
com-pounds is also known to occur with food processing and
storage, which leads to the formation of brown-coloured
macromolecules [16] Other studies [17–19] have
evalu-ated the phenolic content and antioxidant power of
differ-ent products like red grape skins, oak nuts and apple and
strawberry juice After submitting the products to
temper-atures above 80°C for different periods of time, reductions
in their phenolic content and antioxidant power were
observed Other studies have shown that after
steam-cooking vegetables such as broccoli and potatoes, their
phenolic content increased perhaps due to an enhanced
availability for extraction [20–22]
The stability of a functional ingredient is fundamental to
elaborate a nutraceutical product because changes in the
ingredient may affect its nutritional value (e.g antioxidant
capacity [17–19], composition [15,23] and bioavailability
[20–22]) or its organoleptic quality (e.g colour [16])
Considering the possibility of using GSE as a functional
ingredient in products that could be subjected to heat, the
aim of this study is to evaluate the stability of polyphenolic
extracts from grape seeds when the extracts are subjected
to common thermal processing conditions used in the food
industry The stability of the GSE was evaluated based on
changes in their main individual phenolic compounds and
tannin con concentration, as well as changes in their
anti-oxidant activity and browning
Materials and methods
Samples
Five different commercial GSEs were used, and they
were chosen based on their total phenolic content in all
cases higher than 85% All the GSE had a similar oration procedure that involved a hydroalcoholic extrac-tion and a spray drying The GSEs provided by Nutraland(China), HongJiu Biotech (China), Ethical Natural(USA), Exxentia (Spain) and Bioserae (France) wereused The extracts were dissolved in a hydroalcoholicsolution (20% v/v) at a concentration of 5 g/L and wereput in hermetic closed glass bottles to be submitted todifferent heat conditions After the thermal treatments,the samples were stored at 4°C without oxygen for1–2 days prior to analysis Before the essays, the sampleswere centrifuged at 10.7 9 103g for 5 min in a Sigma3K30 (GMBH, Germany) centrifuge For the phenolicand tannin content analyses, the samples were diluted 10times, and for the antioxidant activity, the samples werediluted 50 times
elab-ChemicalsMethanol HPLC grade, Folin-Ciocalteu reagent, 1-butanol,hydrochloric acid 37%, perchloric acid 60%, sodium car-bonate, ferric sulphate heptahydrate and gallic acid (GA)were purchased from Panreac (Spain) 2,2-Diphenyl-1-picrylhydrazyl (DPPH), (?)-catechin (CAT) and (-)-gallocatechin (GC) were purchased from Sigma Chemical
Co (Germany) (-)-Epicatechin (EPI) was purchased fromFluka (Germany) (-)-Epicatechingallate (ECG), (-)-epi-gallocatechin gallate (EGCG), (-)-epigallocatechin (EGC)and procyanidins B1 (ProB1) and B2 (ProB2) were pur-chased from Extrasynthese (France) 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was pur-chased from Aldrich Chemical (Germany)
Food processing conditionsFive thermal treatments that involved a temperature risewere chosen to simulate conditions used in the foodindustry either to create or transform a product such ascooking or baking and/or to preserve that product such aspasteurisation or sterilisation The conditions were asfollows
LTLT (low-temperature long-time) pasteurisation Thesamples were kept in a water bath until they reached
65 ± 2°C for 30 min, and then they were placed in an icebath
HTST (high-temperature short-time) pasteurisation Thesamples were kept in a water bath until they reached
75 ± 1°C for 20 s, and then they were placed in an icebath
Cooking The samples were kept in a water bath until theyreached 93 ± 2°C for 30 min
Trang 23Baking The samples were baked in a Kowell D8AFY
(Kowell, Korea) oven at 180°C for 90 min The samples
reached a temperature of 98 ± 1°C
Sterilisation The samples were sterilised in an HJ
Mar-rodan L1581 (Navarra, Spain) water bath pressure
auto-clave at 120°C for 20 min
To compensate for the solvent loss after submitting the
extracts to the thermal conditions, the volume was restored
to 100 mL
To approximately quantify and compare the intensity of
the thermal treatments, the D-z model for the degradation
of microorganisms, enzymes or quality attributes in food
products was used following the Eq.1 proposed by
Patashnik [24], where T is the temperature at every minute
of the thermal treatment; T* is the reference temperature;
z is the kinetic parameter that defines the thermoresistance
of a microorganism, enzyme or quality attribute in a food
product; and FTis the following degradation relation:
FTX
The reference values used to calculate the FT were those
published by Mishkin and Saguy [25] These values were
obtained after evaluating the thermal degradation of grape
anthocyanins, and they are as follows: z = 54.7°C;
T* = 121°C
Total phenolic content (TPC)
The Folin-Ciocalteu method was employed [26] to obtain
the TPC In a 100-mL volumetric flask, 1 mL of the diluted
extract, 50 mL of deionised water, 5 mL of the
Folin-Ci-ocalteu reagent and 20 mL of a sodium carbonate solution
20% (w/v) were added, in that order The volumetric flask
was filled to its volume with deionised water After 30 min,
the absorbance of the samples was measured at 750 nm in a
Cintra 20 (GMBH, Germany) double beam
spectropho-tometer The phenolic content was expressed in gallic acid
equivalents after the preparation of a standard curve of
gallic acid from 0 to 600 mg/L
Tannin content (TC)
The acidic butanol technique was used to quantify the
procyanidin content of the extracts [27] A stock solution of
FeSO47H2O 0.07% (w/v) dissolved in 1-butanol:HCl 95-5
(v/v) was prepared In a test tube, 7 mL of the stock
solution and 0.5 mL of the diluted sample were mixed and
heated for 50 min at 95°C The mixtures were cooled in an
ice bath, and the absorbance was measured at 550 nm using
a Cintra 20 (GMBH, Germany) double beam
spectropho-tometer A blank reagent was prepared following the same
The TC was expressed in cyanidin equivalents after thepreparation of a standard curve of cyanidin from 0 to
1 mL per minute throughout the analysis The injectionswere made with a 20-lL fixed loop The elution pro-gramme used was as follows: from 100% A to 78% A in
55 min, from 78% A to 0% A in 10 min and then isocraticfor another 10 min Quantification was performed byestablishing calibration curves for each compound deter-mined using the standards
Antioxidant activity (AA)The antiradical activity of the extract was evaluated based
on the technique by Rivero-Pe´rez et al [28] A 60 lMmethanolic solution of DPPH (2,940 lL) was mixed with
60 lL of the extract in a polystyrene cuvette The bance at 515 nm was measured at that exact moment, andafter 60 min, using a Cintra 20 (GMBH, Germany) doublebeam spectrophotometer The antioxidant activity wasreported as mMoles of Trolox equivalents per gram of dryextract after the elaboration of a standard curve of Trolox.Browning absorbance at 420 nm (A420)
absor-The A420 of the samples was performed directly on theextracts by measuring the absorbance at 420 nm using aCintra 20 (GMBH, Germany) double beam spectropho-tometer The measurement was taken at 420 nm becausethe aim was to evaluate the browning of the extracts, which
is related to the yellowish colours [29]
Statistical analysisStatistical analyses were conducted using SPPS 16.0 (SPPSInc., Chicago, Il) Differences between the treatments were
Trang 24confidence level of 95% To look for relationships between
the samples and the treatments, a principal component
analysis (PCA) and a cluster analysis were performed
Results and discussion
Characterisation of extracts
Table1 shows the content of all individual phenolic
compounds identified by HPLC of all GSEs before the
thermal treatments Among the identified compounds, the
ECG was above the detection limit only for extracts D and
E The EGCG was below the detection limit for extract C,
the EGC was below the detection limit for extract A, and
the GC was below the detection limit for extract B
The total amount of individual phenolic compounds for
extracts B, C, D and E was more than 100 mg/gdw of their
whole dry weight (dw), while extract A had the lowest
amount at nearly 80 mg/gdw GA was the compound that
presented the highest content heterogeneity between
extracts mainly because extract B had four times more GA
than the other extracts GC was the compound with the
most homogeneous concentration among the extracts
Based on the mean results of each quantified compound
in all five extracts, CAT had the highest amount, followed
by EPI These results are in agreement with those
pub-lished by other authors who have analysed GSEs by HPLC
[11, 30, 31] The compounds that showed the lowestamounts had a gallate group in their structures (EGCG, AGand ECG)
Table2shows the parameters calculated to characterisethe GSEs The five tested extracts had a similar pH Extract
B had the highest pH value, but it was only half of a pHunit above the other four extracts It is known that pHaffects the stability of polyphenolic compounds and that a
pH between 4 and 5 confers more stability to catechins andtheir isomers and polymers than more alkaline or acidicvalues [14,32,33]
The TPC for all GSEs was more than 800 mg GAE pergram of dry weight Extract E had the highest amount, andextract C had the lowest Extracts A and C presented thehighest TC and the lowest amount of individual phenoliccompounds (Table 1) Therefore, the polyphenols in theseextracts may have been more polymerised than the poly-phenols in the other extracts The ratio between the TPCand TC was approximately double compared with theresults obtained by Makris et al [7], who used the samemethods to quantify both parameters In contrast, the AAvalues in this work were lower than the ones they obtained.The extracts with the highest amount of TC also showedthe highest values on the A420 parameter, which meansthat these samples had a darker brown colour It is knownthat the progressive polymerisation of phenolic compoundsresults in the formation of brown-coloured macromolecules[16]
Table 1 Individual phenolic content identified by HPLC of all GSEs before thermal treatments
Extracts Gallic acid (GA)
(mg/gdw*)
Gallocatequin (GC) (mg/gdw)
Procyanidin B1 (ProB1) (mg/gdw)
Catechin (CAT) (mg/gdw)
Epigallocatechin (EGC) (mg/gdw)
Epicatechin (EPI) (mg/gdw)
Epicatechin gallate (ECG)(mg/gdw)
Total amount of identified compounds (mg/gdw)
* Milligrams per gram dry weight
a–e Different letters within a column are significantly different (p \ 0.05)
Trang 25The CVs of the parameters were generally lower than
the CVs of the individual phenolic compounds, resembling
the homogeneity that the TPC and the AA showed The
heterogeneity of these results demonstrates the influence ofthe source and the extraction procedures had on the finalcharacteristics of the extracts
Table 2 Characterisation indexes of all GSEs before thermal treatments
Extract pH Total phenolic content
(TPC) (mgGAE/gdw)*
Tannin content (TC) (mgCyE/gdw)**
Antioxidant activity (AA) (mmol trolox/gdw)
* Gallic acid equivalents (GAE) per gram dry weight
** Cyanidin equivalents (CyE) per gram dry weight
a–e Different letters within a column are significantly different (p \ 0.05)
a,b c
b
c
d
a c
c
a
c,d
a,b a
a
a,b
a,b
b,c b
a
a,b
a
b,c a
b
b
a,b
a,b a
b
a,b
a,b
a a
a
c
b
b,c b
Fig 1 Individual phenolic content identified by HPLC of all GSEs
after all thermal treatments a Catechin; b Epicatechin; c Procyanidin
Baking, Sterilisation.a–eDifferent letters are significantly different for each extract separately (p \ 0.05)
Trang 26Effects of thermal treatments on the GSE profiles
The FTvalues obtained after using Eq.1can be group into three
magnitude orders The least intensive treatments included
both pasteurisations (HTST 0.02 min and LTLT 0.04 min); the
group second in intensity was formed by the cooking and bakingprocedures (1.20 and 3.80 min, respectively); and the sterili-sation group (12.00 min) was the most intensive treatment.The concentration of CAT was more stable than theconcentration of the rest of compounds (Fig.1a) It is
a
0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50
E D
a a
b b
a,b b
b a,b
b b
c c
c c
d d
d d
e e
e e
Fig 2 Individual phenolic content identified by HPLC of all GSEs
after all thermal treatments a Gallic acid; b Epigallocatechin;
c Epigallocatechin gallate; d Epicatechin gallate and e Gallocatechin.
Control, HTST, LTLT, Cooking, Baking, Sterilisation.
a–e Different letters are significantly different for each extract rately (p \ 0.05)
Trang 27sepa-known that non-epi structures of catechins are more stable
than epi structures because the epimerisation frequency is
104times greater than inverse epimerisation [15, 34–36]
This observation is reinforced by the EPI content in our
study showing a clear tendency to decrease with the
ther-mal treatments for all of the extracts (Fig.1b) and the
tendency to increase shown by GC after the epimerisation
of the EGC (Fig.2e) The ProB1 and ProB2 contents for
extracts A and C tended to increase The ProB2 content for
extract B did not present significant differences between
the thermal treatments (Fig.1c, d) For extracts B, D and E,
the tendency was to decrease Extracts A and C had in
common the highest TC Perhaps the increase of
procy-anidins in these extracts was due to a hydrolysis of tannins
as a consequence of the thermal treatments [19,37]
GA had a clear behaviour of increasing in all extracts
(Fig.2a) The more intense the treatment, the higher the
GA concentration The tendency of GA to increase may
have been due to a hydrolysis of the gallotannins withraising the temperature, thus realising the methyl gallateunits [38–40] Another possible explanation is the excision
of the gallate group attached to the central ring of theflavonoids The EGC showed small increases with the mostintensive treatments, except for GSE D, where it did notpresent significant differences between any of the treatments,and EGCG and the ECG showed decreases (Fig.2b–d) Thisbehaviour reinforces the idea that the gallate excision fromposition 3 in the C ring and the epimerisation process are bothinduced by heat The fact that EGC showed small increasesrather than more obvious ones may be because the losses thatmay have resulted from epimerisation were compensated for
by the gallate excision in EGCG
The TC generally showed decreases after the thermaltreatments (Fig.3a), but they were not always significant(p \ 0.05) These results are in concordance with thoseobtained by Van Der Sluis [19] In contrast, Piva et al [41]
b
a
a
a a,b
b,c
a
b
a a
a,b
a a,b
Extracts
Fig 3 Characterisation indexes of all GSEs after all thermal
treatments a Tannin content; b Antioxidant activity; c Browning
Sterilisation. a–eDifferent letters are significantly different for each extract separately (p \ 0.05)
Trang 28and Manzocco et al [16] found that heat induced the
acceleration of the polymerisation process of individual
phenolic compounds The slight decrease in the TC index
may have been due to the acidified butanol method not
responding to the terminal units of the oligomeric or
polymeric tannins These units of the molecule are not able
to generate the C-4 carbo-cation necessary for the conversion
to anthocyanidin The result is a low response from tannins
with a lower polymerisation degree, while tannins with a high
polymerisation degree give high responses [42]
The AA in all cases showed a decrease compared with
the control sample (Fig.3b) These results are contrary to
those found by Kim et al [43], who worked with whole and
powdered grape seeds The difference may be due to the
phenolic compounds in the grape seeds being bound to
other components of the seeds and the heat liberating them,
while in the GSE, the phenolic compounds are free and
therefore more susceptible to the thermal treatments The
LTLT treatment had the lowest values for the A, B and C
GSEs For GSEs D and E, there was a significant difference
between each thermal treatment and the control, but not
among the thermal treatments The decrease in ECG and
EGCG may have affected the AA of the extracts because
these phenolic compounds are known to have more
scav-enging power than the flavan-3-ols that clearly increased:
(GA, GC and CAT), due to their stearic conformation and
the presence of the gallate group joined to the C ring
[36,44,45] The decrease in TC may also have influenced the
decrease in AA Tannins have been proved to have more
scavenging power than simple phenolic compounds [46]
Finally, the A420 had a clear tendency to increase, whichindicates a darkening of the samples’ brown colour (Fig.3c).The increased absorbance at 420 nm was common for allextracts and was directly related to the intensity of the thermaltreatments The browning of the samples was mainly due tothe progressive polymerisation and oxidation of the phenoliccompounds, which resulted in macromolecules that conferred
a brown colour [16] The radical scavenging power of theseformed macromolecules is contradictory; in the research made
by Manzocco et al [16] after pasteurising a tea extract, itschain-breaking activity measured by the DPPH techniqueincreased, but on the contrary in the research made byJayabalan et al [47], the radical scavenging activity of aKombucha tea decreased after being pasteurised; in bothcases, the tea became darker after the heat treatments.Principal component analysis (PCA)
To reduce the data numbers and to find correlationsbetween the measured parameters and the extracts, a PCAwas performed Principal component 1 (PC1) explained51.9% of the total variance, while PC2 explained 22.1%.The biplot of PC1 versus PC2 is shown in Fig.4 It can
be seen that, except for GC, the individual phenolic pounds grouped together on the opposite side, but along thesame component (PC1) as TC and A420 CAT, EPI andboth procyanidins were linked, probably because CAT andEPI are epimers and the procyanidins are their dimmers
com-TC and A420 were also linked, reinforcing the idea thatthe polymerisation of phenolic compounds results in the
ECG; 2,7; -3,9 EGC; 1,0; -3,5
EGCG; 2,9; 3,8 GA; 1,1; 5,6
PC1 51.9%
A C E
Fig 4 PCA biplot of the matrix of mean values of all evaluated parameters The first number next to the parameter abbreviation means the loading for the first component and the second number for the second component
Trang 29formation of macromolecules with a brown colour The
placement of GC on the opposite side of the individual
phenolic compounds may be due to the content of GC being
higher in the extracts with the greatest amount of tannins and
darker colour, resulting in an association of these three
parameters GA was orthogonal to both the catechin and
tannin groups, and it was negatively correlated with AA
In accordance with Tabart et al [45], and contrary to the
results obtained by Guendez et al [11], the correlation
(r2= 0.309) between the TPC and the AA was not significant
(p [ 0.05) There are compounds with a strong reducing
power but a weak scavenging power; these compounds may
have interfered with the correlation between TPC and AA
One example of these compounds are CAT and EPI which
compared to EGC have a stronger reducing power but a lower
antioxidant capacity measured by the DPPH technique [45]
This biplot clearly shows that the samples were
sepa-rated in terms of extracts and not in terms of thermal
treatments Extracts A and C are darker, and they have a
higher amount of TC Extracts D and E have a higher
amount of individual phenolic content Finally, extract B
clearly has the highest amount of GA and the lowest AA
The different geographic origins of the extracts are not
reflected in the groups identified after the PCA analysis
Conclusions
It can be concluded that, in most cases, submitting GSEs to the
thermal treatments commonly used in the food industry in
most cases changes their phenolic profile as well as their
physical and chemical properties, in this case, colour and
antioxidant activity The differences in pH of the samples,
especially the higher pH of the B extract, did not affect the rate
of change in their phenolic composition after the thermal
treatments compared with the other four extracts The stability
of the GSEs was affected differently depending on the
phe-nolic profile of each extract, and the compounds with a gallate
group attached to their structure were more sensitive to heat It
is not possible to point to one treatment as the most aggressive
for all of the measured parameters on all the extracts; the
changes in the characteristics of the GSEs are not always
directly related to the intensity of the thermal treatment
Acknowledgments Nutraland (China), HongJiu Biotech (China),
Ethical Natural (USA), Exxentia (Spain) and Bioserae (France) for
kindly providing the samples This work was partially financed by
the Mexican Science and Technology Council (CONACYT) and by
the Public University of Navarra (Spain).
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Trang 31O R I G I N A L P A P E R
The effect of Fenton’s reactants and aldehydes on the changes
of myoglobin from Eastern little tuna (Euthynnus affinis) dark
Abstract The influences of Fenton’s reactants (H2O2and
FeCl2) and aldehydes (hexanal and hexenal) on changes of
oxymyoglobin and metmyoglobin from Eastern little tuna
(Euthynnus affinis) dark muscle were studied In the
pres-ence of H2O2, both oxymyoglobin and metmyoglobin were
rapidly oxidized into ferrylmyoglobin based on spectra
patterns In the presence of Fe2?and/or H2O2, the changes
in fluorescent intensity of myoglobin were noticeable, but
there were no changes in aggregation ratio Release of
non-heme iron from myoglobin was mainly governed by H2O2
When aldehydes were incorporated, the oxidation of
oxy-myoglobin and conformational changes of globin were
more pronounced No release of non-heme iron was
noticeable, suggesting the stability of heme moiety toward
aldehydes Hexenal had a great impact on cross-linking of
oxymyoglobin and metmyoglobin via covalent
modifica-tion Alteration of myoglobin redox state might be
enhanced by conformational changes of globin induced by
both Fenton’s reactants and aldehydes
Keywords Myoglobin Ferrylmyoglobin H2O2
Cross-linking Hexanal Hexenal
IntroductionMyoglobin, a predominant pigment protein found in darkfleshed-fish species, has been suggested for its relationshipwith lipid oxidation [1,2] Lipid oxidation is a major cause
of deterioration of food and food products, especially thosecontaining high content of unsaturated fatty acids Oxida-tion of myoglobin is associated with the accelerated lipidoxidation [1] The role of hemin dissociation in the ability
of different heme proteins to promote lipid oxidation cesses was reported by Richards et al [2]
pro-Deoxymyoglobin and oxymyoglobin are in the ferrousstate (Fe2?), whereas metmyoglobin is in the ferric state(Fe3?) Autoxidation of oxymyoglobin results in the for-mation of metmyoglobin and superoxide (•OOH/O2•-),which rapidly dismutate to H2O2and O2[3] The interaction
of H2O2with metmyoglobin led very rapidly to generation
of active species, perferrylmyoglobin (•MbFe(IV) = O)and ferrylmyoglobin species (MbFe(IV) = O), which couldinitiate lipid peroxidation [3, 4] The ferrous myoglobinspecies, deoxymyoglobin and oxymyoglobin, can likewisereact with H2O2, resulting in the formation of ferrylmyo-globin by direct two-electron oxidation [5] Additionally,
O2•-can also reduce dissociated hemin to heme that furtherreacts with H2O2to form either hydroxyl radical or a non-protein bound ferryl-heme [6] These products are able toabstract a hydrogen atom from a polyunsaturated fatty acidand initiates lipid oxidation Chan et al [3] revealed that
H2O2, presumably generated during oxymyoglobin tion, played an important role in mediating oxymyoglobinand lipid oxidation in liposome system The oxidation ofphosphatidylcholine by ferrylmyoglobin was found to beapproximately sevenfold greater than that observed fornative myoglobin [7] The potential of ferrylmyoglobin to
oxida-Y Thiansilakul S Benjakul (&)
Department of Food Technology, Faculty of Agro-Industry
Prince of Songkla University, Hat Yai, Songkhla 90112,
Thailand
e-mail: soottawat.b@psu.ac.th
M P Richards
Department of Animal Sciences, Meat Science and Muscle
Biology Laboratory, University of Wisconsin-Madison,
DOI 10.1007/s00217-010-1370-z
Trang 32production, the concentration of reducing agents, and their
compartmentalization in the muscle cells [8]
Lipid oxidation could generate a variety of secondary
products, predominantly n-alkanals, trans-2-alkenals,
4-hydroxy-trans-2-alkenals, and malondialdehyde [9] The
aldehyde products are more water-soluble than their parent
compounds and could potentially interact with myoglobin
[9, 10] Hexanal, hexenal, and 4-hydroxynonenal were
reported to enhance the oxidation of tuna oxymyoglobin
[11] Porcine metmyoglobin formation was greater in the
presence of 4-hydroxynonenal [12] Lynch and Faustman
[10] also determined the effect of aldehyde lipid oxidation
products on oxymyoglobin oxidation, metmyoglobin
reduction, and the catalytic activity of metmyoglobin as a
lipid prooxidant in vitro Aldehydic lipid oxidation
prod-ucts covalently attached to myoglobin and subsequently
cause structural alterations, which would make the protein
more susceptible to oxidation [9]
The process of ferrous myoglobin converting into ferric
metmyoglobin is responsible for discoloration of meat and
acceleration of lipid oxidation [8,11] However, less
infor-mation regarding the influence of the oxidation products of
myoglobin and lipid on stability of fish myoglobin has been
reported A better understanding of this subject would
pro-vide promising approaches for inhibition of lipid oxidation
as well as improving overall quality of fish or meat Eastern
little tuna (Euthynnus affinis) is a species available in the
Gulf of Thailand and the Indian Ocean It provides the high
global economic value for canning and sashimi [13] This
species contains red muscle with a high amount of
myo-globins, which may undergo oxidation with ease, especially
in the presence of prooxidative compounds, such as Fenton’s
reactants and aldehydic lipid oxidation products Therefore,
the objective of this investigation was to study the stability of
myoglobin from Eastern little tuna dark muscle as influenced
by Fenton’s reactants and selected aldehydes
Materials and methods
Chemicals
b-Mercaptoethanol (bME), Triton X-100, pyridine, hexanal,
trans-2-hexen-1-al, low-range protein markers, and
bath-ophenanthroline disulfonic acid were purchased from Sigma
(St Louis, MO, USA) Sodium dithionite and ferrous chloride
were obtained from Riedel (Seeize, Germany) Hydrogen
peroxide, trichloroacetic acid, sodium nitrite, and iron standard
solution were procured from Merck (Damstadt, Germany)
Fish collection and preparation
Twenty Eastern little tuna (Euthynnus affinis) with the
average weight of 0.5–0.55 kg were obtained from the
dock in Songkhla province, Thailand The fish were loaded 24 h after capture, placed in ice with a fish/ice ratio
off-of 1:2 (w/w), and transported to the Department off-of FoodTechnology, Prince of Songkla University, Hat Yai,Songkhla within 1 h Upon arrival, fish were washed withcold water (5C), beheaded, and eviscerated Thereafter,the fish were filleted, and the dark muscle was manuallyexcised and collected within the same batch The com-posite sample was minced until the uniformity wasobtained
Purification of myoglobin from Eastern little tunaExtraction and purification of myoglobin were performedaccording to the method of Thiansilakul et al [14] Easternlittle tuna mince (100 g) was mixed with 300 mL of coldextracting medium (10 mM Tris–HCl, pH 8.0 containing
1 mM EDTA, and 25 g/l Triton X-100) The mixture washomogenized for 1 min at a speed of 13,000 rpm using anIKA Labortechnik homogenizer (Selangor, Malaysia).After centrifugation at 9,600 9g for 10 min at 4C using
an Avanti J-E centrifuge (Beckman Coulter, Palo Alto, CA,USA), the supernatant was filtered through a Whatman No
4 filter paper (Whatman International Ltd., Maidstone,UK) The filtrate referred to as ‘‘crude myoglobin extract’’was subjected to ammonium sulfate fractionation(65–100% saturation) The precipitate obtained after cen-trifugation at 20,000 9g for 60 min was dissolved in aminimal volume of cold 5 mM Tris–HCl buffer, pH 8.5,which was referred to as ‘‘starting buffer’’ The mixturewas then dialyzed against 10 volumes of the same bufferwith 20 changes at 4C The dialysate was immediatelyapplied onto a Sephadex G-75 column (2.6 9 70 cm;Amersham Bioscience, Uppsala, Sweden), which waspreviously equilibrated with the starting buffer The sepa-ration was conducted using the starting buffer at a flow rate
of 0.5 mL/min Fractions of 3 mL were collected andmeasured at 280 and 540 nm using a UV-1800 spectro-photometer (Shimadzu, Kyoto, Japan) The fractions withthe high absorbance at 540 nm were pooled and used as
‘‘tuna myoglobin’’
Preparation of oxymyoglobin and metmyoglobinOxymyoglobin and metmyoglobin were prepared accord-ing to the method of Tang et al [15] with some modifi-cations An aliquot (5 mL and 0.25 mM) of tunamyoglobin was converted to oxymyoglobin by the addition
of 0.1 g of sodium dithionite Metmyoglobin was prepared
by adding 0.1 g of potassium ferricyanide into 5 mL of thetuna myoglobin solution The sodium dithionite andpotassium ferricyanide were then removed by dialysis ofthe sample against 10 volumes of cold 50 mM phosphate
Trang 33buffer, pH 7.0 with 20 changes of dialysis buffer The
concentrations of oxymyoglobin and metmyoglobin were
adjusted to 0.2 mM
Determination of myoglobin concentration
The concentration of myoglobin (mM) was determined by
measuring the absorbance at 525 nm The molar extinction
coefficient of 7.6 9 10-3was used for calculation [15]
Effect of Fenton’s reactants on the changes of Eastern
little tuna myoglobin
The changes in oxymyoglobin and metmyoglobin as
affected by Fenton’s reactants were monitored according to
the method of O’Grady et al [1] with a slight modification
In a final volume of 4 mL, the solutions of oxymyoglobin
and metmyoglobin (0.2 mM) were incubated with FeCl2
(0.2 mM) and/or H2O2(0.1 mM) The different procedures
for addition of FeCl2and/or H2O2into myoglobin solutions
were used Those included myoglobin ? FeCl2 (MF),
myoglobin ? H2O2 (MH), myoglobin ? FeCl2? H2O2
(MFH), myoglobin ? H2O2? FeCl2 (MHF),
myoglo-bin ? the mixture of FeCl2, and H2O2 previously mixed
for 1, 5, and 10 min (MM1, MM5, and MM10) After the
thorough mixing, all samples were stored at 4C for 1 h
and analyzed for the changes of myoglobin
Effect of aldehydes on the changes of Eastern little tuna
myoglobin
To study the effect of hexanal and hexenal on the changes
of oxymyoglobin and metmyoglobin, the solutions of
oxymyoglobin and metmyoglobin (0.15 mM) were added
with hexanal or hexenal (1 mM) at a ratio of 1:1 to obtain a
final concentration of 0.5 mM aldehyde Control was
aldehyde-free and was prepared in the same manner, except
an equivalent volume of ethanol was used instead The
treated samples were collected at 0, 1, 2, 4, 6, and 8 days of
incubation at 4C for analysis To determine the
cross-linking effect of aldehydes, the mixture containing hexanal
and hexenal at a final concentration of 12.5 mM was also
prepared and incubated at 4C for 1 day prior to
SDS-PAGE analysis
Analyses of heme protein degradation and myoglobin
oxidation
Heme protein degradation was determined by the changes in
absorption spectra and Soret band [16] The proportions of
three myoglobin forms, deoxymyoglobin, oxymyoglobin,
and metmyoglobin, were calculated following the modified
myoglobin by the effect of Fenton’s reactants, the ratio ofabsorbance at 580 and 525 nm (A580/A525) was calculated[11] A high A580/A525value indicates a high proportion ofoxymyoglobin
Tryptophan fluorescent intensityTryptophan fluorescent intensity of treated myoglobinsolutions was measured using an RF-1501 fluorometer(Shimadzu, Kyoto, Japan) at an excitation wavelength of
280 nm and an emission wavelength of 325 nm according
to the method of Chanthai et al [17]
Myoglobin aggregation ratioMyoglobin solutions were filtered through a Millipore filter(pore size, 0.45 lm) (Millipore, Bedford, MA, USA) Thefiltrate was determined for the protein content [15] Thepercentage of the decrease in protein content relative tothat of the initial sample (time = 0) was calculated asmyoglobin aggregation ratio [18]
Non-heme iron contentNon-heme iron content of myoglobin solutions was deter-mined according to the method of Schricker et al [19] with
a slight modification One milliliter of oxymyoglobin andmetmyoglobin solutions was pipetted into a screw cap testtube, and 50 lL of 0.39% (w/v) sodium nitrite was added.Four milliliters of a mixture of 40% trichloroacetic acidand 6 M HCl (ratio of 1: 1 (v/v), prepared freshly) wereadded The tightly capped tubes were incubated at 65Cfor 22 h and then cooled at room temperature for 2 h Thesupernatant (400 lL) was mixed with 2 mL of the non-heme iron color reagent, a mixture of bathophenanthrolinedisulfonic acid, double-deionized water, and saturatedsodium acetate solution at a ratio of 1:20:20 (w/v/v), pre-pared freshly After vortexing and standing for 10 min, theabsorbance was measured at 540 nm The non-heme ironcontent was calculated from iron standard curve The ironstandard solutions (Fe(NO3) in HNO3), with the concen-trations ranging from 0 to 2 ppm, were used
SDS-polyacrylamide gel electrophoresis
To determine the protein pattern of myoglobins, the treatedmyoglobin solutions were subjected to SDS-PAGEaccording to the method of Laemmli [20] Samples (20 lg)with reducing and non-reducing conditions were loadedonto polyacrylamide gels comprising 4% stacking geland 17.5% running gel and subjected to electrophoresisusing a Mini Protean II unit (Bio-Rad Laboratories, Inc.,
Trang 34standards including bovine serum albumin (66 kDa),
oval-bumin (45 kDa), glyceraldehyde-3-phosphate
dehydroge-nase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen
(24 kDa), trypsin inhibitor (20 kDa), a-lactalbumin
(14.2 kDa), and aprotinin (6.5 kDa) were used for molecular
weight estimation
Statistical analysis
Experiments were run in triplicate using three different
batches of samples Data were subjected to analysis of
variance (ANOVA) Comparison of means was carried out
by Duncan’s multiple range test For pair comparison, t-test
was used [21] Statistical analysis was performed using the
Statistical Package for Social Science (SPSS 11.0 for
Windows, SPSS Inc., Chicago, IL, USA)
Results and discussion
Effect of Fenton’s reactants on the changes of Eastern
little tuna myoglobin
Changes in absorption spectra
Oxymyoglobin solution contained oxymyoglobin as the
predominant form (68.91%), followed by metmyoglobin
(23.64%) and deoxymyoglobin (7.45), respectively For
metmyoglobin solution, a complete metmyoglobin
forma-tion (100%) was found The strong absorpforma-tion of myoglobin
was located in a wavelength region of 350–450 nm or Soret
band, in which the intense peaks at 413 and 406 nm were
observed for the control oxymyoglobin and metmyoglobin,
respectively (Fig.1) The typical spectrum in a region of
450–750 nm representing the redox state of myoglobin had
the peaks at wavelengths of 542 and 577 nm for
oxymyo-globin and of 502 and 630 nm for metmyooxymyo-globin
When Fe2? and/or H2O2were added, myoglobin redox
state was altered as evidenced by the changes in absorption
characteristics of oxymyoglobin and metmyoglobin
solu-tions Absorbance spectra of MF for oxymyoglobin and
metmyoglobin solution were similar to that of the control
oxymyoglobin solution, suggesting the reducing capability
of Fe2? The highest oxymyoglobin content was obtained
in the control and MF of oxymyoglobin solution (p \ 0.05)
(Table1) In the presence of H2O2with or without Fe2?
added, the great changes in absorption spectra were
observed in both oxymyoglobin and metmyoglobin
solu-tions MH, MFH, and MHF from oxymyoglobin and
met-myoglobin solutions presenting a Soret peak shifted to a
higher wavelength (*419 nm) Additionally, they had the
increased absorbance in the region of 500–600 nm along
with the unsharpened peak at wavelength of *545 nm
The similar spectra patterns were evidenced for myoglobin of sperm whale and horse heart [22,23] Prasad
ferryl-et al [24] reported the similar wavelength of absorptionpeaks between oxymyoglobin and ferrylmyoglobin fromhorse heart by which the latter had the drastic decrease inpeak height Therefore, both oxymyoglobin and metmyo-globin could be rapidly oxidized by H2O2, leading to theformation of ferrylmyoglobin via one or two-electronoxidation [1,5] For both oxymyoglobin and metmyoglo-bin solutions, MM1 more likely exhibited the typicalspectrum of ferrylmyoglobin Conversely, MM5 andMM10 tended to possess the different absorption spectra.Increasing time for Fenton’s reaction (Fe2??H2O2) mightdecrease the H2O2content and concurrently increased theamount of hydroxyl radicals that exhibited the prooxidativeeffect on myoglobin During the oxidation of oxymyoglo-bin solutions as accelerated by H2O2or hydroxyl radical,the decreases in oxymyoglobin contents were noticeable,and the lowest content was obtained in MM10 for bothoxymyoglobin and metmyoglobin solutions (p \ 0.05)(Table1) In the presence of H2O2 or Fenton’s reactants,
A580/A525, which indicated the proportion of bin form, was greater in MH, compared with the control formetmyoglobin solution (p \ 0.05) This possibly resultedfrom a presence of ferrylmyoglobin, an oxygen-containingmyoglobin (MbFe(IV) = O), which had a typical spectrumrelated to that of oxymyoglobin form [24] O’Grady et al.[1] revealed that the oxidation of oxymyoglobin in thepresence of both Fenton’s reactants may not involvehydroxyl radical production but proceeds via a directinteraction between H2O2and oxymyoglobin Thus, H2O2
oxymyoglo-was a necessary reactant in the conversion of globin or metmyoglobin to a hypervalent ferrylmyoglobin,which is known to be an effective prooxidant [1,8].Changes in tryptophan fluorescent intensity
oxymyo-Changes in tryptophan fluorescent intensity of bin and metmyoglobin solutions from Eastern little tuna asaffected by Fe2?and/or H2O2are depicted in Fig.2a Thehighest tryptophan fluorescent intensity was found in MH ofboth myoglobin solutions (p \ 0.05) H2O2was not onlycapable of oxidizing oxymyoglobin and metmyoglobin intoferrylmyoglobin, but also enhanced conformational changes
oxymyoglo-of apomyoglobin as evidenced by a marked increase influorescent intensity DeGray et al [25] reported that thereaction between H2O2 and recombinant sperm whalemyoglobin resulted in the formation of globin-centeredradicals, especially at tyrosine and tryptophan residues.Decreases in fluorescent intensity were observed in MF,MFH, MHF, MM1, MM5, and MM10 of both myoglobinsolutions, when compared with the corresponding controls(p \ 0.05) Heavy metal salt such as FeCl2 added into
Trang 35myoglobin solutions might act as the protein denaturant bydisruption of electrostatic interaction, resulting in an irre-versible conformational change or denaturation of protein[26] Hydroxyl radicals produced from Fenton’s reactioncould oxidize the proteins, contributing to either proteincross-linking or fragmentation [27, 28] Moreover, themodified protein might bury tryptophan or aromatic domaininside, as evidenced by the decrease in fluorescent intensity.However, slight differences in fluorescent intensity amongMFH, MHF, MM1, MM5, and MM10 of both myoglobinsolutions could be observed (p \ 0.05) This might berelated with the different prooxidant effect of hydroxylradicals toward apomyoglobin When comparing the impact
of Fenton’s reaction time on fluorescent intensity, it wasfound that reaction time had no influence on fluorescentintensity of metmyoglobin Nevertheless, the longer reac-tion caused the gradual decrease in fluorescent intensity ofoxymyoglobin solution Furthermore, the order of addition
MF Control
MH
MM10 MM5 MM1 MHF MFH
MF Control
MH
MM10 MM5 MM1 MHF MFH
MF Control
MH
MM10 MM5 MM1 MHF MFH
MF Control
MH
Fig 1 Effect of Fe2?and/or
H2O2on the absorption spectra
in a region of 350–450 and
450–750 nm of oxymyoglobin
(a and b) and metmyoglobin
(c and d) solutions from Eastern
little tuna dark muscle
Table 1 A580/A525value of oxymyoglobin and metmyoglobin
solu-tions from Eastern little tuna dark muscle as affected by Fe2?and/or
* Different superscripts in the same column indicate significant
dif-ferences (p \ 0.05) Different capital superscripts in the same row
Trang 36fluorescent intensity of oxymyoglobin (p \ 0.05) Thus, the
globin configuration was affected by Fenton’s reactants, but
the degree of changes depended on the form of myoglobin
Changes in aggregation ratio
Aggregation ratio of oxymyoglobin and metmyoglobin
from Eastern little tuna in the presence of Fe2? and/or
H2O2is shown in Fig 2b Following 1 h of incubation at
4 C, aggregation ratio of the control oxymyoglobin andmetmyoglobin was 19.84 and 14.00%, respectively Forboth myoglobin solutions, no differences in aggregationratio were observed for MH and its control (p [ 0.05).The lower ratios were noticeable in the presence of Fe2?,regardless of H2O2 added (p \ 0.05) Decrease in stabil-ization of the native structure of protein is presumed to bethe key step for protein aggregation [29] H2O2 showedthe insignificant effect on myoglobin aggregation, while
Fe2? and Fenton’s reactants could suppress the tion of myoglobin Oxymyoglobin and metmyoglobinsolutions, which had the low tryptophan fluorescentintensity (Fig 2a), might have hydrophilic amino residuesexisting at exterior portion along with charge shieldingfrom iron salt (FeCl2) Therefore, aggregation of globinenhanced via hydrophobic interaction could be reduced[29] The control and MH of oxymyoglobin solutionexhibited the higher aggregation ratios than those ofmetmyoglobin solution (p \ 0.05) This suggested a moreconformational change of oxymyoglobin in comparisonwith metmyoglobin, regardless of H2O2 incorporation.However, no differences in aggregation ratio wereobserved between oxymyoglobin and metmyoglobinsolutions when Fe2? was incorporated (p [ 0.05) Nodifference in protein patterns among all samples wasnoticeable (data not shown), indicating negligible change
aggrega-in the molecular weight of myoglobaggrega-in as aggrega-influenced byFenton’s reactants
Changes in non-heme iron contentThe released of non-heme iron content from oxymyoglobinand metmyoglobin solutions as affected by Fe2? and/or
H2O2 is shown in Fig.2c Non-heme iron contentincreased, when H2O2was added into oxymyoglobin andmetmyoglobin solution (p \ 0.05) Normally, heme group
is surrounded in a hydrophobic pocket-like structure ofglobin, in which iron has occupied four sites with nitrogen
of porphyrin ring and the other one site with histidineresidue of globin [30] H2O2with oxidizing power mightinduce the change of myoglobin redox state as well asstructure of globin, thereby enhancing iron released fromporphyrin ring In the presence of Fe2? with or without
H2O2 addition, no changes in iron released from bothmyoglobin solutions were observed, compared with thecontrol myoglobin (p [ 0.05) It suggested that porphyrinring of heme in hydrophobic pocket of myoglobin wasprobably stable in the presence of Fe2?or Fenton’s reac-tants For the same treatment, no difference in non-hemeiron content was noticeable between oxymyoglobin andmetmyoglobin except for MH that H2O2 might cause therelease of free iron from metmyoglobin at a higher extent
Ae Ad Ac Ae Bf Aa
Af
Bb
Bcd Bcd Ade
Ade Ae
Ab Ab Ab Ab Ab Ba
Ab
Ab Ab Ab Ab Aa
Ab Ab
Ab Ab Ab Ab
Ab Ab
Aa Aa
Ac
Ab Abc Abc
Ac Abc
Ba Ba
a
b
c
Fig 2 Effect of Fe2? and/or H2O2 on tryptophan fluorescent
intensity (a), aggregation ratio (b), and non-heme iron content
(c) of oxymyoglobin and metmyoglobin solutions from Eastern little
tuna dark muscle Bars represent the standard deviation (n = 3).
Different letters within the same myoglobin form indicate significant
differences (p \ 0.05) Different capital letters within the same
treatment indicate significant differences (p \ 0.05)
Trang 37Effect of aldehyde on the changes of Eastern little tuna
myoglobin
Changes in absorption spectra
The effect of hexanal and hexenal on changes in absorption
spectra of oxymyoglobin and metmyoglobin solutions from
Eastern little tuna is presented in Fig.3 For oxymyoglobin
solution, as the incubation time increased up to 8 days, the
peak of Soret bands was slightly increased in intensity and
shifted to a lower wavelength Concurrently, the typical
spectra of oxymyoglobin in a region of 450–750 nm
gradually disappeared These implied the alteration of
oxymyoglobin into an oxidized form induced by aldehyde,
especially with the sufficient incubation time After
incu-bation for 1 day, the proportion of metmyoglobin form in
the control and oxymyoglobin solution incorporated with
hexanal and hexenal increased to 31.03, 34.48, and
34.90%, respectively (p \ 0.05) With increasing
incuba-tion time up to 8 days, the highest formaincuba-tion of
metmyo-globin was found in oxymyometmyo-globin solution added with
hexenal (76.47%), followed by hexanal (71.07%) and thecontrol (68.73%), respectively (p \ 0.05) The result sug-gested that the oxidation of oxymyoglobin was induced byaldehydes, in which hexenal had a greater impact than itssaturated counterpart, hexanal Faustman et al [9] andLynch and Faustman [10] reported that metmyoglobinformation was greater in the presence of a9-b-unsaturatedaldehydes than their saturated counterparts having equiv-alent carbon chain length A negligible change in theabsorption spectra was observed for metmyoglobin solu-tions in both 350–450 and 450–750 nm regions, regardless
of incubation time, indicating that aldehydes had no effect
on the stability of metmyoglobin
Changes in tryptophan fluorescent intensityChanges in tryptophan fluorescent intensity of oxymyo-globin and metmyoglobin solutions in the presence ofhexanal and hexenal during the incubation period of up
to 8 days are presented in Fig.4a Within the first day
of incubation, an increase in fluorescent intensity was
Day8 Day6 Day4 Day2 Day1 Day0
Day8 Day6 Day4 Day2 Day1 Day0
Day8 Day6 Day4 Day2 Day1 Day0
Fig 3 Effect of hexanal and
hexenal on the absorption
spectra in a region of 350–450
and 450–750 nm of
oxymyoglobin (a and b) and
metmyoglobin (c and
d) solutions from Eastern little
tuna dark muscle as the function
of incubation time
Trang 38observed for both myoglobin solutions added with hexenal
(p \ 0.05), whereas negligible changes were found in the
controls and solutions containing hexanal (p [ 0.05)
Thereafter, fluorescent intensities of all samples gradually
increased as the incubation time increased (p \ 0.05)
Hexanal and hexenal had a great impact on conformational
changes of oxymyoglobin and metmyoglobin as indicated
by the higher fluorescent intensity after incubation for
2 days (p \ 0.05) In the presence of aldehyde, unfolding
of globin might be enhanced, causing the considerable
exposure of tryptophan residues A more susceptibility to
structural changes was found in oxymyoglobin, compared
with metmyoglobin Maheswarappa et al [31] reported thatalteration of myoglobin native form in adduction withaldehyde reduced the redox stability of oxymyoglobin.Therefore, the conformational changes of globin induced
by hexanal or hexenal were more pronounced in ferrousmyoglobin that was sensitive to oxidation
Changes in aggregation ratioAggregation ratio of oxymyoglobin and metmyoglobinsolutions added with hexanal and hexenal as a function oftime is shown in Fig.4b The increase in aggregation ratio
of all samples was observed with increasing incubationtime up to 8 days (p \ 0.05), except for the control met-myoglobin that had a constant value after 1 day of incu-bation time (p [ 0.05) For both myoglobin solutions, nodifference in aggregation ratio was found between myo-globin without and with the addition of aldehydes during
6 days of incubation (p [ 0.05) At day 8, both myoglobinsolutions incorporated with hexanal or hexenal had ahigher aggregation ratio, compared with the control(p \ 0.05) The result implied the profound impact ofhexanal and hexenal on conformational changes of myo-globin with extended time In addition, aggregation ratio ofoxymyoglobin was generally greater than metmyoglobincounterpart (p \ 0.05) Libondi et al [32] reported that theoccurrence of intermolecular cross-linking in protein wasinduced by malondialdehyde, accompanied by the change
in the secondary structure Therefore, aggregation ofmyoglobin was governed by the presence of aldehyde aswell as the reaction time
Changes in non-heme iron contentThe effect of hexanal and hexenal on changes in non-hemeiron content of oxymyoglobin and metmyoglobin solutions
at varying times is presented in Fig 4c As the incubationtime increased, no remarkable changes in non-heme ironcontent were obtained in all myoglobins, irrespective ofaldehydes (p [ 0.05) The result demonstrated that alde-hydes, both hexanal and hexenal, had no effect on ironreleased from heme complex Heme iron is normally cleftwithin porphyrin ring of heme pocket and is localized farfrom the surface of myoglobin molecule [30] Although theconformational changes of globin occurred to some extent,
it had less impact on iron release from porphyrin ring ofmyoglobin
Changes in protein patternOxymyoglobin and metmyoglobin from Eastern little tunadark muscle containing hexanal or hexenal at a concen-tration of 12.5 mM were determined for protein patterns
a
b
c
Fig 4 Effect of hexanal and hexenal on tryptophan fluorescent
intensity (a), aggregation ratio (b), and non-heme iron content (c) of
oxymyoglobin and metmyoglobin solutions from Eastern little tuna
dark muscle as the function of incubation time Bars represent the
standard deviation (n = 3)
Trang 39using SDS-PAGE as shown in Fig.5 The obvious band
appeared at molecular weight around 15 kDa was
pre-sumed to be a myoglobin Additionally, a band with
molecular weight of 30 kDa was observed under the
non-reducing condition in the control oxymyoglobin and
met-myoglobin (without addition of aldehydes) This protein
was most likely stabilized by the disulfide bond and might
be a dimer of myoglobin Hexanal had no effect on the
changes in protein pattern of both myoglobins This might
be owing to the negligible cross-linking effect of hexanal at
level used in this study Proteins with the molecular weight
of 30 kDa and the higher molecular weight components
were observed in oxymyoglobin and metmyoglobin
solu-tions added with hexenal (25 mM) under the reducing and
non-reducing conditions This result suggested that hexenal
could induce the cross-linking of myoglobin via
non-disulfide covalent bonds On the other hand, no changes in
protein pattern were observed when aldehydes were
incorporated at a low concentration (0.5 mM) (data not
shown) Lee et al [11] reported a mono-adduct of
4-hy-droxynonenal, a reactive aldehyde, to tuna myoglobin by
covalent modification that was expected to accelerate the
oxidation of oxymyoglobin
Conclusions
The oxidation of oxymyoglobin or metmyoglobin from the
dark muscle of Eastern little tuna mediated by H2O2
resulted in the generation of ferrylmyoglobin Fe2?and/or
H2O2could induce the conformational changes of globin
without aggregation of myoglobin H2O2might weaken the
porphyrin ring, leading to release of non-heme iron from
of globin but had no effect on the release of non-heme iron.Hexenal had a greater impact on the formation of met-myoglobin, compared with hexanal, and induced the for-mation of covalent cross-links of myoglobin Fenton’sreactants and aldehyde could therefore accelerate thechanges of fish myoglobin; however, it depended on itsforms
Acknowledgments This research was supported by the Thailand Research Fund under the Royal Golden Jubilee PhD Program to Yaowapa Thiansilakul (PHD/0101/2550) and TRF senior research scholar.
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kDa
Fig 5 SDS-PAGE pattern of
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containing 12.5 mM hexanal or
hexenal at 4 C for 1 day
under-reducing and non-under-reducing
conditions M
low-molecular-weight markers; 1 control
myoglobin; 2 myoglobin added
with hexanal; 3 myoglobin
added with hexenal
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