Food Chemistry 128 (2011) 1051–1057

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Food Chemistry

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Formation of Strecker aldehydes between protein carbonyls – a-Aminoadipic andc-glutamic semialdehydes – and leucine and isoleucine

Mario Estévez a,b,⇑, Sonia Ventanas b, Marina Heinonen a

a Department of Food and Environmental Sciences, Food Chemistry, University of Helsinki, 00014, Helsinki, Finlandb Department of Animal Production and Food Science, Food Technology, University of Extremadura, 10003 Cáceres, Spain

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 November 2010Received in revised form 9 March 2011Accepted 6 April 2011Available online 12 April 2011

Keywords:AASGGSCarbonyl-amine reactions2-Methylbutanal3-MethylbutanalStrecker-type reactions

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.04.012

⇑ Corresponding author. Address: Department of AScience, Food Technology, University of Extremadura+34 927257122; fax: +34 927257110.

E-mail address: mariovet@unex.es (M. Estévez).

The potential implication of protein carbonyls, a-aminoadipic (AAS) and c-glutamic semialdehydes (GGS)on the formation of Strecker aldehydes from leucine (LEU) and isoleucine (ILE) was investigated in thepresent work. Solution mixtures containing the semialdehydes and each of the amino acids, were incu-bated at 80 �C and analysed for the volatiles in the headspace using SPME–GC–MS at sampling times (1,12 and 24 h). The addition of the semialdehydes in solutions containing LEU and ILE led to a significantincrease of the corresponding Strecker aldehydes, 3-methylbutanal and 2-methylbutanal. According tothe present results, AAS is more reactive than GGS and the reaction of both semialdehydes with LEU ismore effective than that with ILE. The ability of the semialdehydes to promote the formation of the Strec-ker aldehydes is inhibited by blocking their carbonyl moiety and significantly affected by the pH of themedia. Beyond their role as protein oxidation markers, AAS and GGS may be active compounds in foodsystems and their interactions with other food components could lead to relevant consequences on foodquality.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The oxidation of lipids and the Maillard reaction have beenhighlighted as the most important reactions in Food Science owingto the positive and negative impact of their products on numerousquality features such as food flavour, colour, and nutritional value(Belitz, Grosch, & Schieberle, 2009; Mottram, 1998; Zamora & Hi-dalgo, 2005). The Strecker degradation of amino acids is one ofthe main reactions leading to the final aroma compounds in theMaillard reaction (Belitz, Grosch, & Schieberle, 2009). It involvesthe oxidative deamination and decarboxylation of the amino acidin the presence of a-dicarbonyl compounds formed in the Maillardreaction and the formation of the corresponding Strecker aldehyde(Mottram, 1998). Each amino acid yields a specific Strecker alde-hyde which consists of a molecule with one carbon atom lessthan the amino acid from which it is formed. Strecker aldehydessuch as 3-methylbutanal (malty flavour) and phenylacetaldehyde(honey-like flavour), derived from leucine and phenylalanine,respectively, are commonly reported as volatile components andaroma contributors in foods such as meat and dairy products(Delgado, Gonzalez-Crespo, Cava, Garcia-Parra, & Ramirez, 2010;

ll rights reserved.

nimal Production and Food, 10003 Cáceres, Spain. Tel.:

Estévez, Morcuende, Ventanas, & Cava, 2003; Jurado, Garcia,Timon, & Carrapiso, 2007). Both, lipid oxidation and the Maillardreaction interact in complex food systems and share commonchemical mechanisms and intermediate compounds (Zamora &Hidalgo, 2005). In fact, certain carbonyls derived from lipidperoxidation such as alkadienals and ketodienes have beendemonstrated to promote the oxidative degradation of amino acidsand yield the corresponding Strecker aldehydes via Strecker-typereactions (Zamora, Gallardo, & Hidalgo, 2007, 2008). This routemight be of high significance in particular foods in which theamount of reducing sugars to undergo Maillard reactions and yielda-dicarbonyl compounds is relatively low and the occurrence oflipid oxidative reactions is considerably high. For instance, theformation of high amounts of Strecker aldehydes in dry-cured meatshas mainly been ascribed to the interaction between oxidisedlipids and free amino acids derived from the proteolytic degradationof myofibrillar proteins (Jurado et al., 2007; Ventanas, Ventanas,Tovar, García, & Estévez, 2007; Ventanas et al., 1982).

Whereas the implication of lipid oxidation on food flavourand the overall quality of foods is well-established, the impactof protein oxidation on these issues is still poorly understood.The formation of carbonyl compounds is a well-documentedconsequence of the metal-catalysed oxidation of food proteins(Requena, Chao, Levine, & Stadtman, 2001). In a recent study,Estévez, Ollilainen, and Heinonen (2009) detected two specificcarbonyl compounds, namely, a-amino adipic semialdehyde

1052 M. Estévez et al. / Food Chemistry 128 (2011) 1051–1057

and c-glutamic semialdehyde (AAS and GGS, respectively), inseveral oxidised food proteins, including dairy, soy, and myo-fibrillar proteins. AAS derives from the oxidative degradationof lysine whereas GGS is formed from arginine and proline(Requena, Levine, & Stadtman, 2003). The formation of thesemialdehydes involves the oxidative deamination of the aminoacid side-chain and the eventual formation of an aldehyde moi-ety in the presence of transition metals (Requena et al., 2003).Both semialdehydes have been highlighted as protein oxidationmarkers in biological (Daneshvar et al., 2001) and food systems(Armenteros, Heinonen, Ollilainen, Toldrá, & Estévez, 2009; Esté-vez, Ollilainen, and Heinonen, 2009). However, the impact ofthese semialdehydes on nutritional, sensory or toxicological as-pects of food is ignored. Although the implication of these pro-tein carbonyls in further reactions has recently been suggested(Akagawa et al., 2003; Armenteros et al., 2009; Estevez & Heinonen,2010), there is no certainty about the reactivity of these carbonylderivatives and their fate in food systems is unknown.

The present study aims to shed light on the role played by AASand GGS on the formation of specific Strecker aldehydes, 3- and2-methylbutanal, from leucine and isoleucine, respectively.

2. Material and methods

2.1. Material

All chemicals were supplied by J.T Baker (Deventer, Holland),Riedel de-häen (Seelze, Germany), and Sigma Aldrich (Steinheim,Germany). All chemical were of analytical grade except HPLC-grade methanol. Water used was purified by passage through aMilli-Q system (Millipore Corp., Bedford, MA). Egg shell membranewas isolated from fresh white leghorn hen eggs, thoroughlywashed with distilled water, cut into small pieces (5 � 5 mm)and finally dried with filter paper before using.

2.2. Synthesis of AAS and GGS

AAS and GGS were synthesised from Na-acetyl-L-lysine and Na-acetyl-L-ornithine using lysyl oxidase activity from egg shell mem-brane following the procedure described by Akagawa et al. (2006).Briefly, 5 mM Na-acetyl-L-lysine and 5 mM Na-acetyl-L-ornithinewere independently incubated with constant stirring with 2 gegg shell membrane in 25 ml of 20 mM sodium phosphate buffer,pH 9.0 at 37 �C for 24 h. The egg shell membrane was then re-moved by centrifugation. The resulting AAS and GGS solutionswere filtered through hydrophilic polypropylene GH Polypro(GHP) syringe filters (0.45 lm pore size, Pall Corporation, USA)and further purified by using silica gel column chromatographyand ethyl acetate/acetic acid/water (20:2:1, v/v/v) as elution sol-vent. The purified residue was reconstituted with Milli-Q waterand the pH adjusted to whether 5.0, 7.0 or 9.0 by using 1 M HCl.Freshly made semialdehydes solutions were employed withinone hour for the in vitro assays. In order to confirm the implicationof the carbonyl moiety of the semialdehydes in the studied reac-tions, in some samples, this functional group was blocked througha reductive amination with 3 mmol p-amino-benzoic acid (ABA) inthe presence of 4.5 mmol sodium cyanoborohydride (NaCNBH3) asdescribed by Akagawa et al. (2006). The purity of the solutions andthe identification of the synthesised compounds were confirmedby using Fast Atom Bombardment coupled to mass spectrometry(FAB-MS). The spectra and fragmentation patters of the com-pounds were published elsewhere (Estévez, Ollilainen, and Hei-nonen, 2009). The structures of the authentic compounds assynthesised following the aforementioned procedures have been

analysed using 1H NMR techniques and reported by Akagawa, Suy-ama, and Uchida (2009).

2.3. Reaction between semialdehydes and amino acids

Solutions of 5 mM leucine and 5 mM isoleucine were freshlyprepared in 25 ml of 20 mM sodium phosphate buffer at several fi-nal pH: 5.0, 7.0 and 9.0. An aliquot (1 ml) of the amino acid solution– whether leucine (LEU) or isoleucine (ILE) – was dispensed in a4 ml-Solid-Phase Microextraction (SPME) vial together with 1 mlof the semialdehyde solution - whether AAS or GGS- resulting in4 different reaction mixtures; LEU + AAS, LEU + GGS, ILE + AAS,ILE + GGS. Each of these mixtures were allowed to react at threedifferent pH – acidic (pH = 5.0), neutral (pH = 7.0) or basic(pH = 9). Each experimental mixture (12 in total) was prepared intriplicate and the whole experiment was carried out twice (n = 6for each experimental mixture). Each mixture was allowed to reactin a thermoblock at 80 �C and constant stirring during 1, 12 and24 h. At sampling times, vials were allowed to cool down at37 �C while immersed in a water bath as a previous step to the vol-atiles extraction using the SPME. Vials containing only the aminoacids (LEU; ILE), the semialdehydes (AAS; GGS) and the mixturesof amino acids with the derivatised forms of the semialdehydes(LEU + [AAS�ABA]; LEU + [GGS�ABA]; ILE + [GGS�ABA];ILE + [GGS�ABA]) were also prepared and processed followingthe aforementioned procedure.

2.4. GC analysis of Strecker aldehydes

The Headspace (HS) sampling was performed following a meth-od previously described (Estévez et al., 2003). The SPME fibre,coated with a divinylbenzene-carboxen-poly(dimethylxilosane)(DVB/CAR/PDMS) 50/30 lm, was preconditioned prior analysis at220 �C during 45 min. The SPME fibre was exposed during30 min to the headspace of the vial immersed in water at 37 �C.The SPME was then transferred to a HP5890GC series II gas chro-matograph (Hewlett–Packard, USA) coupled to a mass-selectivedetector (Agilent model 5973). Volatiles were separated using a5% phenyl-95% dimethyl polysiloxane column (Restek, USA)(30 m � 0.25 mm id., 1.0 mm film thickness). The carrier gas wasHelium at 18.5 psi, resulting in a flow of 1.6 ml min�1 at 40 �C.The SPME fibre was desorbed and maintained in the injection portat 220 �C during the whole chromatography run. The injector portwas in the splitless mode. The temperature programme was iso-thermal for 10 min at 40 �C and then raised at the rate of7 �C min�1 to 250 �C, and held for 5 min. The GC/MS transfer linetemperature was 270 �C. The mass spectrometer operated in theelectron impact mode with an electron energy of 70 eV, a multi-plier voltage of 1650 V and collecting data at a rate of 1 scan s�1

over a range of m/z 40–300. Strecker aldehydes detected in thechromatograms were positively identified by comparing theirspectra and linear retention indexes (LRI) with those from standardcompounds (Sigma–Aldrich, Steinheim, Germany). Chromato-graphic areas from MS are provided as area units (AU).

2.5. Data analysis

All types of mixtures were made in triplicate and all analyseswere made in duplicate (n = 6). Data obtained from MS were com-puted in a Two-Way Analysis of Variance using SPSS for Windowsver. 15 in order to study the effect of the addition of the semialde-hyde and the incubation time or the pH of the medium, andthe corresponding interaction effects, on the formation of 3- and2-methylbutanal from leucine and isoleucine, respectively.

M. Estévez et al. / Food Chemistry 128 (2011) 1051–1057 1053

3. Results and discussion

3.1. Detection of Strecker aldehydes in reaction mixtures

The implication of protein carbonyls, namely AAS and GGS, onthe formation of Strecker aldehydes from leucine and isoleucine,is investigated through a set of in vitro reactions. The analysis ofthe HS of the solutions containing the synthesised semialdehydesand leucine revealed a main peak at min. 4.82 which was positivelyidentified as the Strecker aldehyde 3-methylbutanal (Fig. 1A). Inboth reaction mixtures, LEU–AAS and LEU–GGS, the area of thisvolatile aldehyde increased significantly over time to reach ahighest value by the end of the assay (24 h) (Table 1). Besides3-methylbutanal, the HS of the solutions had other volatilecomponents which corresponded to laboratory pollutants such asethanol, chloroform and hexane (Fig. 1). In contrast to the Streckeraldehydes, the areas of the interphering solvents did not increaseover time confirming their origin as external artifacts. The HS ofthe reaction mixtures containing the semialdehydes and isoleucinehad a main peak at min. 5.12 which was positively identified as theStrecker aldehyde 2-methylbutanal. The area of this volatile com-pound also increased significantly during the incubation of thereaction mixtures to reach a highest point after 24 h.

Strecker aldehydes, such as 2- and 3-methylbutanal, are potentodorants and common volatile components of a large variety offoods in which they contribute specific aroma notes. The formationof these compounds in food systems as a result of the degradationof amino acids in the presence of dicarbonyls derived from Mail-lard reaction has been profusely documented (Belitz, Grosch, &Schieberle, 2009). Other carbonyl compounds such as certain alka-dienals formed from lipid oxidation have been proven to react withamino acids through Strecker-type reactions and yield the corre-sponding Strecker aldehydes, which highlights the timely interac-tion between lipid oxidation and the Maillard reaction (Zamora,Gallardo, & Hidalgo, 2007). The present data suggests that the

3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

0.5x105

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2x105

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3.5x105

4x105

4.5x105

5x105

5.5x105

Retention Time

Abundance

6x105

LEU

3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

0.5x105

1x105

1.5x105

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3x105

3.5x105

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Retention Time

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6x105

3-methylbutanal

P1P2

P4

(A)

(C)

(LEU-AAS

3-methylbutanal

P1

P2

P4P3P4

Fig. 1. Total ion chromatograms corresponding to the HS of the following solutions: LEU2 = hexane; 3 = ethanol; 4 = siloxanes.

amino acids leucine and isoleucine could also react with the pro-tein oxidation carbonyls, AAS and GGS, to yield the Strecker alde-hydes 2- and 3-methylbutanal, respectively.

In order to confirm that the 3-methylbutanal was formed as aresult of the reaction mixtures, the HS of the amino acid and thesemialdehydes solutions were also analysed using GC–MS (Table3). The HS of the semialdehyde solutions only had minor amountsof laboratory pollutants and no traces of Strecker aldehydes(Fig. 1B). On the other hand, the incubation of the amino acid solu-tions for 24 h at 80 �C eventually led to the formation of slightlyrelative amounts of the corresponding Strecker aldehydes(Fig. 1C). The formation of these small quantities of Strecker alde-hydes may have been caused by the thermal degradation of theamino acids or as a result of the reaction of the amino acids withtrace amounts of compounds able to trigger the Strecker reaction.The addition of the solutions containing the synthesised proteincarbonyls significantly increased the formation of the Streckeraldehydes. In particular, the addition of AAS increased the forma-tion of 2- and 3-methylbutanal more than 40 and 20-fold timescompared to the respective amino acid solutions, whereas theaddition of GGS enhanced the formation of the same Strecker alde-hydes 24 and 14-fold times, respectively.

3.2. Plausible reaction mechanisms

As a plausible proposal, Fig. 2 depicts the set of reactions thatmay take place during the Strecker degradation of leucine and iso-leucine in the presence of AAS and GGS. According to this proposal,the aldehyde moiety of the semialdehydes would react with theamine group of the amino acid to form a Schiff base structure. Thisintermediate compound would be eventually hydrolysed to yield adiamino- and an oxo-carboxilic acid. The decarboxylation of thelatter would lead to the formation of the Strecker aldehyde. The fi-nal products of these reactions namely, the Strecker aldehyde andthe corresponding diamino-carboxylic acid, were detected by using

20

3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

0.5x105

1x105

1.5x105

2x105

2.5x105

3x105

3.5x105

4x105

4.5x105

5x105

5.5x105

Retention Time

Abundance

6x105

AAS

P1

P2

P4P3

3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

0.5x105

1x105

1.5x105

2x105

2.5x105

3x105

3.5x105

4x105

4.5x105

5x105

5.5x105

Retention Time

Abundance

6x105

LEU+[AAS-ABA]

3-methylbutanal

P1P2

P3

B)

(D)

+ AAS (A), AAS (B), LEU (C) and LEU+[AAS�ABA] (D). P: Pollutant; 1 = chloroform;

Table 1Strecker aldehydes (AU) detected in the HS of solutions containing protein carbonyls, amino acids and the mixture of amino acids and derivatised protein carbonyls incubated at80 �C during 24 h.

AAS GGS LEU ILE LEU + [AAS–ABA] ILE + [GGS–ABA]

2-Methylbutanal n.d. n.d n.d 0.42 n.d 0.473-Methylbutanal n.d. n.d 0.92 n.d 0.83 n.d.

n.d., not detected.

Table 23-Methylbutanal detected in the HS of solutions containing mixtures of leucine (LEU)and protein carbonyls (AAS, GGS) incubated at 80 �C for 1, 12 and 24 h.

LEU + AAS LEU + GGS p-value1

1 h 12 h 24 h 1 h 12 h 24 h C T CxT

1.21d 6.51b 10.92a 1.00d 4.36c 6.54b <0.001 <0.001 <0.001

a,b,c,dMeans with different superscripts are significantly different (p < 0.05).1 Effects in two-way ANOVA; C, carbonyl compound (AAS vs. GGS); T, incubation

time; CxT, interaction between carbonyl and incubation time.

1054 M. Estévez et al. / Food Chemistry 128 (2011) 1051–1057

GC–MS and LC–MS (Estévez, Ollilainen, and Heinonen, 2009),respectively.

As far as we know, the reaction between AAS and GGS and par-ticular amino acids to yield Strecker aldehydes has never been de-scribed before. However, the reactivity of the AAS aldehyde groupis known to be considerably high and the aldosamine condensationproposed in Fig. 2 has been profusely described for protein-boundAAS and lysine residues (Dolz & Heidemann, 1989). According toDolz and Heidemann (1989), the carbonyl moiety of the AAS mol-ecule reacts promptly with protein-bound lysine as well as withother AAS residues to form Schiff bases and aldol condensationstructures, respectively. These reactions are known to take placein vivo to form, under physiological conditions, cross-links betweenpeptides chains within collagen and elastine proteins (Eyre, 1984).The carbonyl–amine condensation between oxidised amino acidsand amino acid residues also occur as a result of diverse age-re-lated disorders which involve increased protein oxidation, intenseAAS formation and uncontrolled protein cross-linking (Eyre, 1984;Sell, Strauch, Shen, & Monnier, 2007). AAS and GGS have neverbeen reported to be involved in such reactions in food systemsand this may be due to the fact that these compounds were, untilrecently, not thought to be present in food systems. Soon afterthese semialdehydes were found to be formed as a result of foodprotein oxidation (Estévez, Ollilainen, and Heinonen, 2009), severalstudies highlighted the likely implication of these semialdehydes infurther reactions in food systems (Armenteros et al., 2009; Estévez,Ollilainen, and Heinonen, 2009; Fuentes Ventanas, Morcuende,Estévez, & Ventans, 2010; Estevez & Heinonen, 2010; Ganhaoand Estévez, 2010). According to one of these studies (Estévez,Ventanas, and Heinonen, 2009), the formation of high quantities ofAAS and GGS during the in vitro oxidation of BSA, a-lactalbumin,myofibrillar and soy proteins occurred along with a concomitantincrease of Strecker aldehydes in the HS of the protein insolates.The absence of reducing sugars and oxidising lipids in the modelsystems emphasised the role that some other carbonyl compoundssuch as those formed from oxidised proteins may play in the for-mation of Strecker aldehydes. The results from the present studysuggest that the carbonyl moieties of AAS and GGS could react withthe amine group of free amino acids, namely leucine and isoleu-cine, to form Schiff base structures similar to those reported to takeplace in vivo, and eventually trigger the formation of the corre-sponding Strecker aldehydes. In order to prove the timely implica-tion of the carbonyl moiety of the semialdehydes in the formationof the Strecker aldehydes, the amino acid solutions were allowedto react with derivatised forms of AAS and GGS (Table 1). The HS

of these reaction mixtures contained similar amounts of Streckeraldehydes than those found in the HS of the amino acids(Fig. 1D). According to these results, blocking the carbonyl groupof AAS and GGS through the reaction with ABA, disable the semial-dehydes to react with the amino acids and promote the formationof the Strecker aldehydes. Like this, the present study originallyhighlights that protein degradation products – free amino acidsand free oxidised amino acids – would be sources of Strecker alde-hydes in the absence of reducing sugars and oxidising lipids. Someparallel experiments confirmed that these protein carbonyls couldpromote the formation of Strecker aldehydes from other aminoacids such as phenylalanine and methionine (data not shown).Whereas this paper shows novel and previously unknown dataon the reactivity of protein carbonyls, the present results werenot unexpected at all as the reactions between carbonyls and freeamino acids to yield Strecker aldehydes have been described ashighly unspecific (Zamora, Gallardo, & Hidalgo, 2008). In fact, alarge variety of lipid oxidation products containing carbonylmoieties such as hydroperoxides, hydroxydienes, ketodienes andshort-chain aldehydes have been proved to be implicated in theStrecker-type degradation of several amino acids (Hidalgo, Gallardo,& Zamora, 2005; Hidalgo & Zamora, 2004; Zamora et al., 2008). Thepresent results also emphasise the connection between the oxida-tion of proteins and the Maillard reaction. It is noteworthy that themechanism involved in the formation of AAS and GGS from thecorresponding amino acids is similar to the oxidative deaminationwhich takes place during the Strecker degradation of amino acids.In fact, Akagawa, Sasaki, Kurota, and Suyama (2005) proved thatAAS and GGS can be formed in the presence of glucose and Mail-lard-derived dicarbonyls such as methylglyoxal through Maillard-type reactions. Unlike the formation of Strecker aldehydes fromfree amino acids, the formation of AAS and GGS can take place fromprotein-bound amino acids as the concerned amino group is lo-cated in the side chain of susceptible amino acids. Hence, AASand GGS may promote the formation of Strecker aldehydes fromamino acids, with these semialdehydes being, themselves, oxida-tion products from Maillard-type reactions.

The HS of the solutions containing either of the amino acids andAAS had significantly higher amounts of the Strecker aldehydes at12 and 24 h than the HS of the GGS counterparts (Tables 2 and 3).The highest reactivity of AAS would explain the involvement of thissemialdehyde in the equivalent in vivo reactions that, as aforemen-tioned, take place under physiological and pathological conditions.Whereas GGS is known to be formed as a result of the oxidation ofplasma and liver proteins in vivo and in vitro (Akagawa et al., 2006;Daneshvar, Frandsen, Artrup, & Dragsted, 1997) the involvement ofthe aldehyde moiety of this compound in further reactions hasnever been described. According to other supportive works, AASand GGS accumulated during the in vitro oxidation of food proteinsuntil a certain point at which the amounts of both semialdehydesdecreased (Estevez & Heinonen, 2010; Estévez, Ollilainen, andHeinonen, 2009. The loss of semialdehydes coincided with aconcomitant formation of Strecker aldehydes (Estévez, Ollilainen,and Heinonen, 2009) and was interpreted as an involvement ofthese compounds in further reactions. In those studies, the lossof AAS was considerably more intense than that of GGS. Despite

Table 32-Methylbutanal detected in the HS of solutions containing mixtures of isoleucine(ILE) and protein carbonyls (AAS, GGS) incubated at 80 �C for 1, 12 and 24 h.

ILE + AAS ILE + GGS p-value1

1 h 12 h 24 h 1 h 12 h 24 h C T CxT

0.93d 4.29bc 8.74a 0.60d 3.77c 5.11b 0.007 <0.001 0.003

a,b,c,d,eMeans with different superscripts are significantly different (p < 0.05).1 Effects in two-way ANOVA; C, carbonyl compound (AAS vs. GGS); T, incubation

time; CxT, interaction between carbonyl and incubation time.

GGS

AAS

O

NH2

O

OH

+

Isoleucine

-H2O

NH2

NH2

O

OH

OO

OH CH3H2O

-C02

Diamino-carboxilic acid

ONH2

O OH

+ -H2O

OO

OH CH3H2O

NH2

O

OH

NH2

Schif f Base (stable Imine)

-C02

Diamino-carboxilic acid

Schif f Base (stable Imine)

CH3

NH2

O

OH

Isoleucine

CH3

NH2

O

OH

CH3

NH2O

OH

N

OOH

CH3

NH2

O

OH

N

O OH

CH3

O

2-methylbutanal

GGS

AAS

O

NH2

O

OH

+NH2

O

OH

Leucine NH2O

OH

N

OOH

-H2O

NH2

NH2

O

OH

OO

OHH2O

-C02

Diamino-carboxilic acid

ONH2

O OH

+NH2

O

OH

Leucine

-H2O

OO

OHH2O

O

3-methylbutanal

NH2

O

OH

N

O OH

NH2

O

OH

NH2

Schif f Base (stable Imine)

-C02

Diamino-carboxilic acid

Schif f Base (stable Imine)

(A)

(B)

Fig. 2. Proposal of the reaction between leucine (A) and isoleucine (B) and protein carbonyls (AAS and GGS) to form 2- and 3-methylbutanal, respectively.

Table 43-Methylbutanal detected in the HS of solutions at different pHs containing mixturesof leucine (LEU) and protein carbonyls (AAS, GGS) and incubated at 80 �C for 24 h.

LEU + AAS LEU + GGS p-value1

pH 5 pH 7 pH 9 pH 5 pH 7 pH 9 C pH CxpH

2.77d 10.92a 8.84b 1.13e 6.54c 6.26c <0.001 <0.001 <0.001

a,b,c,d,eMeans with different superscripts are significantly different (p < 0.05).1 Effects in two-way ANOVA; C, carbonyl compound (AAS vs. GGS); pH, incuba-

tion pH; CxT, interaction between carbonyl and incubation pH.

M. Estévez et al. / Food Chemistry 128 (2011) 1051–1057 1055

the reactivity of the carbonyl moieties of the semialdehydes,both were more effective at promoting the formation of 3-methyl-butanal from leucine than at promoting the formation of 2-meth-ylbutanal from isoleucine (Tables 2 and 3).

3.3. Influence of pH on the reaction mechanism

According to the present study, the likely reaction between thesemialdehydes and leucine and isoleucine for the formation of thecorresponding Strecker aldehydes is highly influenced by the pH of

the reaction media (Tables 4 and 5). The relative amounts of 3-methylbutanal in the HS of the LEU + AAS solutions were signifi-cantly affected by the pH and followed the order pH 7 > pH9 > pH 5. Similarly, the formation of 3-methylbutanal from themixture of GGS and LEU was significantly less intense at pH 5 thanat higher pHs. Comparable effects were observed for the amountsof 2-methylbutanal from the solutions containing ILE and thesemialdehydes. Whereas the reaction between the semialdehydesand the amino acids is slightly delayed at basic pHs, this reactionis noticeably hindered when the pH of the media shifts from

Table 52-Methylbutanal detected in the HS of solutions at different pHs containing mixturesof isoleucine (ILE) and protein carbonyls (AAS, GGS) incubated at 80 �C for 24 h.

ILE + AAS ILE + GGS p-value1

pH 5 pH 7 pH pH 5 pH 7 pH 9 C T CxT

1.37e 8.74a 6.86b 0.86e 5.11c 3.21d <0.001 <0.001 <0.001

a,b,c,d,eMeans with different superscripts are significantly different (p < 0.05).1 Effects in two-way ANOVA; C, carbonyl compound (AAS vs. GGS); pH, incuba-

tion pH; CxT, interaction between carbonyl and incubation pH.

1056 M. Estévez et al. / Food Chemistry 128 (2011) 1051–1057

neutral to acid pHs. In agreement with the present results, otherauthors have previously reported the impact of the pH media onthe occurrence of carbonyl–amine condensation reactions (Hidalgo,Alaiz, & Zamora, 1999). Hidalgo and Zamora (2004) reported thatthe Strecker-type degradation of phenylalanine in the presence of4,5-epoxy-2-alkenals is maximum at neutral pHs and decreaseswhen the pH of the reaction media is whether decreased orincreased. It is plausible that protonated a-amino groups fromthe amino acids are unable to form Schiff bases with aldehydemoieties and therefore, the initial step for the Strecker degradationof amino acids would be highly improbable. The present resultssuggest that lowering the pH from neutral values would decreasethe potential role of the semialdehydes as promoters of Streckeraldehydes formation. The previously reported differences betweenAAS and GGS for their potential reactivity with amino acidsremained the same at acid and basic pHs.

3.4. Role of AAS and GGS in the formation of Strecker aldehydes

Whereas the actual implication of AAS and GGS in the Strecker-type degradation of free amino acids in food systems remainsunknown, it is plausible that protein semialdehydes would play arelevant role as sources of these odorants in particular ripenedmeats such as dry-cured products. The simultaneous occurrenceof protein proteolytic and oxidative reactions and the formationof Strecker aldehydes during ripening of meat products suggeststhat protein semialdehydes may be implicated in the formation ofStrecker aldehydes by reacting with neighbouring free amino acids(Coutron-Gambotti & Gandemer, 1999; Martín, Córdoba, Antequera,Timón, & Ventanas, 1998; Toldrá, 2004). In fact, Armenteros et al.(2009) reported considerably high levels of AAS and GGS indry-cured hams and loins. In accordance with the results fromthe present study, the pH of the food system may play a relevantrole on the reactivity of the protein carbonyls. In this sense, thepH of most foods, including dry-cured meats (�pH = 6) is withinthe range in which the carbonyl–amine condensation may takeplace. This extent, however, may be studied in depth and eventuallyproved by developing specific studies on real food products.

The present work is, in conclusion, a first approach to shed lighton the role that particular protein oxidation products such as AASand GGS, may play in reactions with relevant consequences onfood quality. Beyond their role as protein oxidation markers in foodsystems, these molecules may be active compounds which interactwith other food components. Further studies could be developedon the basis of the present findings to investigate the actual impactof protein oxidation products on the occurrence of these relevantreactions in specific food systems. The kinetics of the presentreactions and quantitative data will also be aims to accomplishin future works.

Acknowledgement

M. Estévez thanks the European Community for the economicalsupport from two consecutive Marie Curie Fellowships; Intra-

European (MEIF-CT-2006-039555-Pox-MUSCLE) and ReintegrationGrant (FP7-PEOPLE-2009-RG-248959-Pox-MEAT). M. Estévez iscurrently employed through the Spanish RYC-MINN program(RYC-2009-03901). Authors also acknowledge the technical sup-port from Miikka Olin, Kirsti Risunen and Maija Ylinen.

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