Amino acid profile and Maillard compounds of sun-dried pears ...

Eur Food Res Technol (2011) 233:637–646 639at the village of Ervedal da Beira (Oliveira do Hospital,Portugal) at the commercial maturity stage (Fig. 1a). Onaverage, the fresh fruits had a mass of 64 g, with a diameterof 4.3 cm and height of 6.5 cm. The moisture content was78%.For fresh pear analysis, some fruits were frozen justafter harvesting. The remaining fruits were processedaccording to the different methodologies under study.According to the traditional sun-drying process, thefresh fruits were peeled and allowed to dry in an openspace with sunshine incidence, as described by Ferreiraet al. [5] (Fig. 1b). After being peeled, fresh pears werealso allowed to dry in two greenhouses that differed in theirsize, structure and location. GH1 was provided with airconvection, with a flux of 900 m 3 h -1 , it was located atground level and, in comparison to GH2, had the largestcargo capacity. Its area was 6.3 m 2 (3.19 m long by 1.93 mwide), and the height in the sides was 1.24 m, whereas inthe middle was 1.97 m. The structure was aluminium withhorticulture glass and there were two roof windows for airextraction. The drying time for GH1 was approximately7 days (Fig. 1c). The GH2 greenhouse consisted of insulatedglass panels and had the form of a box (1.20 m heightby 1.20 m long and 1.00 m wide). Inside, it was composedby a U-shaped structure, parallel to the floor, where pearswere let to dry. GH2 was located on a roof of a buildingand contained a step structure coated with reflective film,where pears were allowed to dry. This interior structureallowed a greater incidence of the light and consequentlypromoted the drying process by natural air convection, withthe air entering in the lower level (first step) and leaving inthe higher level, by means of a girandole. The drying timefor GH2 was approximately 5 days (Fig. 1d), 2 days lessthat the drying time of GH1.For hot air tunnel drying (HAT), after peeled, pears werelet to dry at a constant temperature of 40 °C and an air flowof 1.2 m s -1 for about 7 days (Fig. 1e). The drying tunnelwas 2 m long, and the air convection was promoted by aventilator placed at the entrance of the tunnel. In thisstructure, pears were not exposed to light, although the airwas heated by a solar collector, and therefore the sun wasalso the main source of energy.Independently of the type of processing used, the processedfruits reached a mass of 10–14 g with a moisturecontent of 20% and had a maximum width of 2.5–3.4 cm,height of 3.9–4.7 cm and thickness of 1.2–1.5 cm. Thesensory descriptive profile of the traditional product, whenevaluated by a sensory panel test, is the following: brown–red uniform colour (Fig. 1b), sweet and slight acid, with anot too hard–not too soft texture and presenting elasticity.Concerning GH1, it was considered very similar to thetraditional product, with the same rate of global appreciation,but with lower colour uniformity (Fig. 1c), sweetnessand elasticity. GH2 was similar to GH1, although obtaininga lower global appreciation, possibly due to an even loweracidity (Fig. 1d). HAT presented the sensory profile closeto the traditional product concerning colour uniformity,sweetness and toughness but it was not appreciated due tothe colour tonality (yellow-orange) (Fig. 1e) and slight lessacidity. This product, although having the accurate organolepticcharacteristics except the colour, was rejected bythe panellists due to the inaccurate colour when comparedwith the traditional product.Before analysis, the pulp of the fresh and dried pearswas collected, ground and freeze-dried.Amino acids analysisThe methodology for acid hydrolysis of protein materialwas adapted from Zumwalt et al. [22]. To a test tube with ascrew cap with PTFE coating was rigorously weighed10 mg of freeze-dried material. To each sample, 2 mLhydrochloric acid (HCl) 6 M was added; the content of thetubes was frozen in liquid nitrogen, evacuated during 1 minwith a vacuum pump and refilled with nitrogen. Afterthawing, the suspension was sonicated for 5 min in anultrasound bath at room temperature. The procedure offreezing–vacuum–sonication was repeated twice. Thehydrolysis took place during 24 h at 110 °C using a heatingblock. After cooling to room temperature, 500 lL of theFig. 1 The S. Bartolomeu pear.a Fresh pears and pears dried bydifferent technologies:b Traditional, c GH1, d GH2and e HAT123

640 Eur Food Res Technol (2011) 233:637–646internal standard solution (norleucine 5.0 mM in HCl0.1 M) was added and the tubes content was evaporated todryness under vacuum in a centrifugal evaporator. Theresulting material was dissolved in 1 mL HCl 0.1 M andfiltered with 0.45 lm filters. For the analysis of free aminoacids, 10 mg of each sample was suspended in 2 mL of asolution of HCl 0.1 M and spiked with 500 lL of theinternal standard solution. The suspension was left stirringfor several hours and then was filtered with 0.45 lm filters.The solutions containing the released amino acids weredried under vacuum using a centrifugal evaporator.The derivatization of amino acids for GC analysis wasperformed according to the methodology described byMacKenzie et al. [23]. The resultant solid residue wasdissolved in 200 lL of a solution of 3 M HCl in isobutanol.This solution was prepared by adding 270 lL of acetylchloride per mL of dry isobutanol; the isobutanol was driedwith calcium hydride, distilled and stored with molecularsieves 4 Å. The mixture was heated to 120 °C for 10 minand, after shaking in a vortex, was heated for further30 min. After cooling to ambient temperature, the excess ofreagent was evaporated under vacuum using a centrifugalevaporator. Then, 200 lL of a solution of 0.2 mg mL -1BHT prepared in ethyl acetate was added and the solventwas removed under vacuum in a centrifugal evaporator.Afterwards, 100 lL of heptafluorobutyric anhydride wasadded and the mixture was heated during 10 min at 150 °C.After cooling to room temperature, the excess of solventwas removed under vacuum and the material obtained wasdissolved in 50 lL of ethyl acetate and analysed immediatelyor frozen at -20 °C until analysis.Separation of amino acids was achieved by gas chromatography,carried out in a PerkinElmer Clarus 400instrument (PerkinElmer, Massachusetts, USA) equippedwith a flame ionisation detector (FID). The injector waskept at 250 °C and the detector at 260 °C. Hydrogen wasused as carrier gas. A DB-1 (30 m, 0.25 mm i.d. and0.15 lm thickness) fused-silica capillary column (J & WScientific) was used with the following temperature programme:1 min hold at 70 °C, increase to 170 °C at 2.0 °C/min and then to 250 °C (5 min hold) at 16 °C min -1 . Thecompounds were identified by their retention times andchromatographic comparison with authentic standards.Quantification was based on the internal standard methodusing L-norleucine, and the calibration curves were built for18 amino acids. For asparagine (Asn) and aspartic acid(Asp), as well as for glutamine (Gln) and glutamic acid(Glu), the methodology does not allow the distinctionbetween the amide and carboxylic acid functions. As such,those amino acids were quantified together as Asx and Glx,respectively. Also, the methodology used did not allow thedetection of His. The limit of quantification of analysedamino acids was determined to be ten times the value of theresidual signal peaks.Analysis of carboxymethyllysine (CML),carboxyethyllysine (CEL) and furosineThe acid hydrolysis for the CML and CEL release fromthe protein material and furosine conversion fromfructosyl-lysine residues present on the protein materialwas performed based on the methodology described byBuser et al. [24]. To a test tube with a screw cap withPTFE coating was rigorously weighed 75 mg of freezedriedmaterial. To each sample was added 3 mL of 7.8 MHCl, and the resulting suspension was sonicated for15 min in an ultrasound bath. The tubes were sealed undernitrogen atmosphere, and the hydrolysis took place during24 h at 110 °C using a heating block. After cooling toroom temperature, the tubes content was evaporated todryness under vacuum. The resulting material was dissolvedin 4 mL of 2 M HCl and filtered with 0.45 lmpore filters.The acid hydrolysates were purified by solid-phaseextraction (SPE) to improve the baseline in the procedurefor quantification by GC–MS [25]. The solid-phaseextraction cartridges with 500 mg of C-18 stationary phasewas packed with 5 mL of ethanol, followed by 5 mL ofdistilled water and finally with 5 mL of a 2 M HCl solution.The samples were dissolved in 4 mL of 2 M HCl,filtered and applied in the C-18 cartridges, recovering theeluent in a test tube. The column was washed with 3 mL of2 M HCl and the eluent recovered in the same test tube.The solvent was removed under vacuum using a centrifugalevaporator.The furosine, CML and CEL residues were derivatisedby esterification with isobutanol and acylation with heptafluorobutyricanhydride as described for amino acidsderivatization, with the exception that the BHT solutionwas not added after the esterification with isobutanol [24].The furosine, CML and CEL residues were analysed bygas chromatography–quadrupole mass spectrometry (GC–MS, Agilent). The column and the temperature programmeused were the same as for amino acids analysis. Selectedion monitoring (SIM) was used for the detection of thecompounds. The ions at m/z 110, 621 and 578 were used,respectively, for furosine, CML and CEL detection. Forqualification, additional ions at m/z 280 and 240, m/z 565and 519 and m/z 533 and 511, respectively, were used.Quantification was performed using the lysine residues asinternal standard. The MS was operated in the electronimpact mode with an electron impact energy of 70 eV anddata collected at a rate of 3 scan s -1 . The ion source andthe transfer line were kept at 230 °C [20].123

642 Eur Food Res Technol (2011) 233:637–646Table 2 Protein amino acidscontent (mg 100 g -1 ) of 2009fresh and dried pear fruitsValues are mean ± standarderror (3 replicates)Protein amino acids content (mg 100 g -1 )Amino acid Fresh 09 Traditional 09 GH1 09 GH2 09 HAT 09Ala 15.21 ± 1.22 15.01 ± 0.62 18.94 ± 5.89 17.77 ± 2.55 11.10 ± 2.45Gly 9.11 ± 0.19 8.52 ± 0.06 10.24 ± 2.07 8.02 ± 2.71 8.47 ± 0.79Val 22.32 ± 2.24 21.58 ± 0.57 26.09 ± 0.34 23.30 ± 0.06 19.81 ± 3.05Thr 12.92 ± 1.17 10.50 ± 1.86 11.95 ± 0.61 9.78 ± 3.32 13.32 ± 0.61Leu 64.16 ± 11.64 58.52 ± 5.51 60.25 ± 12.11 66.22 ± 11.93 55.25 ± 4.44Ile 33.28 ± 10.71 29.82 ± 3.64 28.87 ± 0.69 38.28 ± 3.82 34.98 ± 4.74Pro 19.77 ± 6.82 9.22 ± 2.05 14.89 ± 3.12 4.76 ± 2.04 12.45 ± 0.65Asx 44.72 ± 12.23 41.48 ± 2.80 51.99 ± 8.96 49.99 ± 4.64 44.65 ± 2.32Phe 11.48 ± 2.68 9.01 ± 4.37 11.87 ± 0.11 12.45 ± 0.97 11.99 ± 1.02Glx 79.58 ± 13.87 93.48 ± 2.12 90.92 ± 12.39 83.84 ± 6.69 89.92 ± 8.16Lys 24.88 ± 1.13 19.34 ± 2.52 18.99 ± 6.59 23.53 ± 0.78 24.02 ± 3.25Total 337.43 ± 63.89 316.46 ± 13.47 345.98 ± 10.66 336.92 ± 25.21 325.97 ± 30.27amino acid in higher amount, followed by Glx. Consideringtheir relative molar percentages, Pro accounts for39–25% of the free amino acids, whereas Glx accounts for24–15%. Although no data are available for free aminoacids of dried pears, the results are comparable with thoseobtained for the dehydration of grapes where it wasobserved the decrease in the total free amino acid contentas well as a decrease in Asx [11]. These authors attributedthe decrease in the free amino acid content to the low wateractivity of samples, favouring the Maillard reaction atroom temperature. Also, the higher relative amount of freePro has been observed in pear juice [14]. In fact, theincrease in the synthesis of Pro has been reported as aresponse of the fruits to stress conditions [19, 26, 27],namely due to the modulation of expression of proline-richglycoproteins (extensins).Eleven amino acids were found in proteins from freshand traditional sun-dried pears (Table 2; Fig. 2b). In total,they account for 4.23 mg per g of dry weight of the pulp inthe samples from fresh fruits in 2008 season and3.37 mg g -1 in 2009. A value of 2.31 mg g -1 (dry weight)for fresh pear (Pyrus communis L.) is reported in literature[7], although from Beurré variety, which is lower than thevalues for protein found for S. Bartolomeu fruits from bothharvests. In traditional sun-dried fruits, the amount ofprotein amino acids was higher (18%) than in fresh in 2008(5.00 mg g -1 ) and lower (6%) in 2009 (3.17 mg g -1 ).For the fresh fruits, the most abundant protein aminoacids are Glx and Leu (20–19 mol%, each) and Asx(17–13 mol%) (Fig. 2b). Also in relevant amount occur Ile(10–7 mol%), Ala (8–7 mol%) and Val (7 mol%). Asx wasreported by Pilipenko et al. [7] as the predominant aminoacid in Pyrus communis L. fruits of Beurré variety(35 mol%), showing that S. Bartolomeu pears present lowerrelative amounts, although similar amounts are observedwhen expressed in mg g -1 . All amino acids found as constituentsof S. Bartolomeu pears have been also found inBeurré variety. The exception is Hyp, not found in amountsto be detected in S. Bartolomeu pears. Hyp is an amino acidcharacteristic of extensins and arabinogalactan-rich glycoproteins.Pyrus communis L. arabinogalactan-rich glycoproteinsfrom suspension cultures are composed mainly byGlx (16 mol%) [28]. In Chinese species, reporting eightpear varieties, Chen et al. [8] showed large variations in theamino acid profile for the different pear varieties. Anyway,Asn accounted for near 20% of total amino acids. In thesepears, Ser is the main constituent, although not always,which is not the case of Pyrus communis L., where no Serwas found.No significant changes were observed in the amino acidsprofile of proteins from traditional sun-dried pears whencompared to the fresh samples (Fig. 2b). However, inrelation to the same mass of protein, a decrease in thecontent of Pro (-16% in 2008 and -50% in 2009), Phe(-15 and -16%), Lys (-9 and -17%) and Thr (-10 and-13%) was observed in both harvests, contrasting with theincrease in Glx (?17 and ?25%). The higher differencewas Pro, in accordance with the higher increase in Pro foundas free amino acid. Also, the increase in the content in Glxin proteins is in accordance with the lower amounts found asfree amino acid. As no data are available concerning thecomposition of the amino acids of proteins of dried fruits,the origin of this phenomenon remains unknown, althoughit can be suggested that modulation in Pro-rich glycoproteinsynthesis and/or degradation seems to be involved. Concerningthe decrease in Phe, Lys and Thr, it is possible that itis related with the occurrence of Maillard reactions. Thishypothesis will be verified taking into account the analysisof other independent samples, namely the pears driedaccording to different methodologies, which present123

644 Eur Food Res Technol (2011) 233:637–646increase in the relative content of Val and Ala was alsoobserved in traditional, GH1 and GH2 processed fruits.Detection of carboxymethyllysine (CML),carboxyethyllysine (CEL) and furosine in pearsIn order to observe whether the browning of sun-driedpears also results from the contribution of Maillard reactionproducts, the analysis of carboxymethyllysine (CML),carboxyethyllysine (CEL) and furosine in dried pears wassearched and fresh pears were also analysed to be used ascontrol. CML quantification was performed together withCEL due to the fact that the applied methodology does notallow the separation between these two compounds.Although literature does not report the occurrence offurosine, CML and CEL in unprocessed materials usingthis methodology, it was noticed their occurrence in freshpears. In fact, furosine content detected for fresh pears was871 mg per 100 g of protein present in the sample and theamount of CEL ? CML detected was 275 mg 100 g -1protein. Some authors [29, 30] reported that this kind ofcompounds might partially be formed from degradationproducts derived from lipids or sugars during samplehydrolysis. As these fruits have not been processed, theoccurrence of these compounds may be resultant of anartefact of the methodology used, namely from the acidhydrolysis to which the samples were submitted. In order toavoid this fact, a preliminary protein extraction would berequired to remove all interferences in the analysis methodology.Nevertheless, the amount of furosine andCML ? CEL was higher in dried samples. Using freshpears as control of the methodology applied, it was possibleto determine the amount of these compounds producedafter the different drying technologies used (Fig. 4).For dried pears, furosine amount ranged between247 mg 100 g -1 protein for traditional and 80 mg 100 g -1protein for HAT. For GH1 and GH2, the furosine amountwas 182 and 136 mg 100 g -1 proteins, respectively. Theamount of furosine detected for dried pears was similar tothat described in literature for products to which moderateContent (mg / 100 g Protein)300250200150100500Fur CML+CEL Fur CML+CEL Fur CML+CEL Fur CML+CELTraditional GH1 GH2 HATFig. 4 Furosine (Fur) and carboxymethyllysine (CML) ? carboxyethyllysine(CEL) content (mg 100 g -1 protein) in dried pearsfrom 2009 harvestheat treatment is applied, such as UHT milk products [31,32] (35–269 mg 100 g -1 protein), jams and fruit-basedinfant foods [18] (44–448 mg 100 g -1 protein) and cookedbeef and salmon [29] (100–200 mg 100 g -1 protein). ForCML ? CEL quantification in dried pears, the amount ofthese compounds ranged between 96 mg 100 g -1 proteinfor traditional and 37 mg 100 g -1 protein for HAT. ForGH1 and GH2, the furosine amount was 94 and 71 mg100 g -1 proteins, respectively. The values determined forthe content of CML ? CEL in dried pears were, asexpected, higher compared to those described in literaturefor CML even in severely heat-treated foods, such assterilised evaporated milk [17] (43–52 mg 100 g -1 protein)and hypoallergenic powdered infant formulas [30](13–32 mg 100 g -1 protein). This fact may be due to CELinterference in CML quantification.In both analysis (furosine and CML ? CEL), there wasa tendency for the occurrence of higher levels of thesecompounds in traditional pears and lower contents in HATdried pears, while GH1 and GH2 dried pears presentedintermediate levels (Fig. 4), showing that the traditionalwas the most severe processing of all studied and the HATwas the less severe.Taking into account the amount of the decrease in thecontent of each amino acid with the different treatments(Table 2) and the amounts of Maillard reaction productsquantified (Fig. 4), it is possible to observe a positivecorrelation between the contents of these compounds andthe decrease in the relative amount of Lys and Phe. On thecontrary Val, Ala, Ile and Glx showed a negative correlation.The decrease in the relative content of Pro does notshow any relation with the occurrence of these compounds.The Lys degradation observed in Traditional and GH1pears is comparable to that occurring in condensed milk butis lower than that of chocolate, processed breakfast cerealsand bread [33] and fruits and vegetables in baby foods [34],which allows to include the pears in moderate thermallyprocessed foods if processed according to the traditionalprocess or with GH1. Using this parameter as marker of thethermal damage [11, 13, 35], it can be stated that the pearsproduced by HAT do not lose the nutritional value concerningthe stability of Lys. However, Maillard reactionproducts, when in a moderate amount as observed in traditionaland GH1 pears, may contribute to beneficial antioxidantproperties [36, 37].The differences observed between the colour of traditionalpears (reddish brown colour) and HAT dried pears(yellow-orange colour) are also in accordance with thehigher amount of Maillard reaction products in traditionalprocessed fruits. For GH1 and GH2 dried pears, the colourationwas similar to the traditional dried pears, althoughGH2 fruits presented a not so intense colour as traditionaland GH1 ones (Fig. 1). These results allow to infer that the123

Eur Food Res Technol (2011) 233:637–646 645drying process promotes the occurrence of Maillard reactionsin the S. Bartolomeu pears and that in the traditional,GH1, and GH2 drying processes, they contribute to thereddish brown colour of the dried pears. For HAT drying, itis possible that the heating of the fruits has not been highenough to promote the occurrence of Maillard reactions inthe most advanced steps, when the brown colouration isproduced.Influence of pH, temperature and moisture on driedpear colour formationAs the Maillard reaction is known to be favoured duringthe dehydration process systems [13] under conditions ofpH ranging 5–7, with intermediate moisture content andtemperatures over 50 °C using long processing times, a fewsets of simple experiments were performed on pear tissuesto relate the occurrence of Maillard compounds with thereddish brown colouration.In order to test the influence of pH on the developmentof the colour of the pears, pieces of freeze-dried pulp of afresh pear were immersed in buffer solutions in the rangeof the pH scale. It was observed that the tissues immersedin the solutions with higher pH became darker than theother ones. The major colour variations were observed inthe interval of pH 4–6, whereas at pH 5.0, the colour of thetissue approached the reddish brown colour characteristicof the traditional product (Fig. 5a).abclowmoisturepH 4 pH 5 pH 6sunlightdarkness30 °C 50 °ChighmoisturehighmoistureFig. 5 Effect of a pH variation, b sunlight exposure and c moistureand temperature in the development of the colour of pear tissuesThe freeze-dried pear tissues that have been immersed inthe buffer solution at pH 5.0 were placed (in a wet form) intwo plastic containers, one exposed to sunlight and theother one was placed in the dark. This experiment allowedto observe that the sample exposed to sunlight presented anincrease in the intensity of the colour when compared withthe sample placed in the dark (Fig. 5b). Because watercondensation was observed over the walls of the containerswhere the tissues were exposed to the light and not in thecontrol ones, it was hypothesised that the increase intemperature of the wet tissues could be an important factorfor pear tissues colouration. To test this hypothesis, piecesof freeze-dried pear tissues were immersed in the buffersolution at pH 5.0 and the wet material was cut in twohalves. One part was placed in a Petri dish that was closedto preserve the moisture and the other one was left in directcontact with the air. Both samples were placed in an ovenat 30 and 50 °C. It was observed that the tissues that weremaintained closed, keeping the moisture, presented moreintense colourations and the colour intensity was higher for50 °C than for 30 °C (Fig. 5c).These results allow concluding that the pH, the dryingtemperature and the tissues moisture are importantparameters for the development of the characteristic colourof the traditional product.ConclusionThe results obtained allowed concluding that drying ofS. Bartolomeu pears affects the free amino acid profile,increasing Pro content and decreasing Asx and Glx.Although the proteic amino acid profile was not significantlymodified with the drying process, a loss of therelative content of Lys is observed. This loss is related withthe increase in the amount of furosine, CML and CELformed during the drying process.Although all drying methods tested promoted theincrease in the amount of CML, CEL and furosine, thisincrease was higher in the case of the traditional sun-dryingand greenhouse processing, where the fruits presentedreddish brown colour and lower for the hot air tunnelprocessing, where the fruits became yellow-orange. Theseresults allow to conclude that the drying processing ofS. Bartolomeu pears promotes Maillard reactions, possiblycontributing to the characteristic reddish brown colour ofthis product. These reactions are formed under conditionsof pH, temperature and moisture that promote the developmentof this characteristic colour.Acknowledgments We thank FCT for financial support throughproject PTDC/AGR-ALI/74587/2006, Research Unit 62/94-QOPNA(PEst-C/QUI/UI0062/2011) and Cláudia Nunes Post-Doc grantSFRH/BPD/46584/2008.123

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