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Research Note

Does High Hydrostatic Pressure Affect Fruit Esters?Evripidis Lambadarios and Ioannis Zabetakis*

University of Leeds, Procter Department of Food Science, Leeds LS2 9JT (U.K.)(Received April 5, 2001; accepted July 19, 2001)

The effect of high hydrostatic pressure on ester formation and hydrolysis was studied. Six esters and the corresponding carboxylicacids and alcohols were subjected to high-pressure treatments of 400 and 800 MPa under three different pH conditions (namely,buffer solutions of pH 4, 6 and 8). The selected compounds were dissolved into buffer solutions, subjected to the pressure treatmentand then extracted using dichloromethane. The analysis and quantication were carried out by gas chromatography with ameionization as detector. High pressure appeared to have no effect on ester formation or hydrolysis under the investigated conditions. Inall cases, a small decrease at the levels of carboxylic acids and esters was observed without any evidence of further reaction. Thisdecrease, referred to as decomposition, depended on pressure and pH conditions. Ester decomposition was minimised when a high- pressure treatment of 400 MPa in basic conditions (pH 8) was applied. Carboxylic acid decomposition was minimal in basicconditions and it was independent of the pressure applied.

r 2002 Elsevier Science Ltd. All rights reserved.

Keywords: strawberry; fruits; esters; high hydrostatic pressure; avour

Introduction

High hydrostatic pressure (HHP) technology is a novel

nonthermal food processing technology where foods aresubjected to HHP, generally in the range of 100600 MPa,at or around room temperature (Mertens, 1995) .In general, nowadays, food consumers have a highexpectation of food quality. There is a strong demandfor more natural minimally processed, additive freefoods and the reported benecial and unique effects of high pressure appear to offer particular potential inthese respects (Johnston, 1994) .Esters are among the most widespread of all naturallyoccurring compounds. They are moderately polar andthus insoluble in water with dipole moments in the1.52.0 D range. Dipoledipole interactions contributeto the intermolecular attractive forces that cause estersto have boiling points slightly higher than hydrocarbonsof similar molecular weight. Because they lack hydroxylgroups, they cannot form hydrogen bonds witheach other; consequently, esters have lower boilingpoints than alcohols of comparable molecular weight(Yamamoto et al ., 1991).Many esters, especially the volatile ones of lowmolecular weight, have characteristically pleasant

odours and are responsible for the avour and fragranceof many fruits, owers and articial avourings.Generally, the delicacy of natural avours and fra-

grances is due to complex mixtures, for instance, morethan 100 substances contribute to the avour of ripestrawberries (Zabetakis & Holden, 1997) . On the otherhand, cheap articial avourings are often singlecompounds or at the most simple mixtures of estersand ketones.Among the most common esters contributing to avourand aroma are: pentyl acetate which resembles theodour of bananas, octyl acetate that of oranges, ethylbutanoate that of pineapples, pentyl butanoate that of apricot and isopentyl valerate that of apples (McMurry,1994) . Our particular interest lies towards: ethyl andmethyl acetate, ethyl and methyl butanoate and ethyland methyl hexanoate which contribute to the esteryand green notes of odour in strawberries. We are alsointerested in their formation and hydrolysis to car-boxylic acids and alcohols (Zabetakis & Holden, 1997;Sumitani et al ., 1994; Zabetakis et al ., 2000; Larsenet al ., 1992).On a study on the effect of HHP on strawberry a-vour, volatile esters were not identied (Zabetakiset al ., 2000a). It was suggested that esters werehydrolysed to the respective acids and alcohols duringHHP treatment. The absence of esters was explainedon the basis of a possible hydrolytic effect that HHP

causes (Tauscher, 1995) . In this work, we report our

*To whom correspondence should be addressed. Stratigou Makriyanni6, Nea Filadela, Athens, GR-143 42 Greece. Tel./fax: +30 102510589; E-mail: dryz@subdimension.com

0023-6438/02/$35.00 doi:10.1006/fstl.2001.0863r 2002 Elsevier Science Ltd. All rights reserved. All articles available online at http://www.idealibrary.com on

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condition for methyl butanoate is a high-pressuretreatment of 400 MPa at pH 4. In the case of ethylhexanoate, the percent decomposition varied between0.1 and 12%. The highest degree of decomposition wasobserved in pH 4 and 800 MPa conditions, while thelowest appears to be at pH 8 when a pressure of 800 MPa was, respectively, applied. As a result, theoptimum conditions for ethyl hexanoate is a high-pressure treatment of 800 MPa at pH 8. For methylhexanoate, the percent decomposition varied between0.1% and 18.5%. The highest degree of decompositionwas observed in pH 8 and 800 MPa conditions, whilethe lowest appears to be at pH 8 when a pressureof 400 MPa was applied. As a result, the optimumcondition for methyl hexanoate is a high-pressuretreatment of 400 MPa at pH 8. These results aresummarised in Table 1 .

Effect of HHP on acids and alcohols

As far as carboxylic acids and alcohols are concerned,there was no sign of ester formation at all. It was foundthat there was no ester present after the samples weresubjected to high-pressure treatment, while there wasonly a small decrease in the carboxylic acid concentra-tion further described as decomposition.In the case of acetic acidethanol, the percent decom-position of the acetic acid varied between 0.1 and 8%.The highest degree of decomposition was observed inpH 4 and 800 MPa conditions, while the lowest appearsto be at pH 8 when a pressure of 800MPa was applied.As a result, the optimum conditions, for acetic acid inacetic acidethanol solutions is a high-pressure treat-ment of 800MPa at pH 8. For the mixture of aceticacidmethanol, the percentage decomposition of theacetic acid varied between 0.5 and 6%. The highestdegree of decomposition was observed in pH 4 and800 MPa, while the lowest in pH 6 and 400 MPaconditions. As a result, the optimum conditions foracetic acid in acetic acidmethanol solutions is a high-pressure treatment of 400 MPa in pH 6. In the case of butanoic acidethanol, the percent decomposition of thebutanoic acid varied between 0.5 and 5.5%. The highestdegree of acid decomposition was observed at pH 4when the sample was subjected to 800 MPa pressure,

while the lowest was at pH 8 under 400 MPa pressuretreatment. As a result, the optimum conditions for

butanoic acid in butanoic acidethanol solutions is ahigh-pressure treatment of 400 MPa in pH 8. For themixture of butanoic acidmethanol, the percent decom-position of the butanoic acid varied between 0.5 and10%. The highest degree of acid decomposition wasobserved at pH 4 and when the sample was subjected to800MPa pressure, while the lowest was at pH 8 under400 MPa pressure treatment. As a result, the optimumconditions for butanoic acid in butanoic acidmethanolsolutions is a high-pressure treatment of 400 MPa in pH8. In the case of hexanoic acidethanol, the percentdecomposition of the hexanoic acid varied between 2and 14%. The highest degree of acid decomposition wasobserved at pH 4 when the sample was subjected to a400MPa pressure, while the lowest was found at pH 4under 800 MPa pressure treatment. As a result, theoptimum condition for hexanoic acid in hexanoic acid methanol solutions is a high-pressure treatment of 400MPa in pH 8. For the mixture of hexanoic acid methanol, the percent decomposition of the acid varied

between 0.1 and 19%. The highest degree of aciddecomposition was observed at pH 4 and when thesample was subjected to a 400 MPa pressure, while thelowest was at pH 6 under 400 MPa pressure treatment.As a result, the optimum condition for hexanoic acid inhexanoic acidethanol solutions is a high-pressuretreatment of 400 MPa in pH 6. In general, thedecomposition of hexanoic acid was very low when theacid was subjected to various pressures under differentpH conditions. These results are shown in Table 2 .A pattern describing the decomposition of the esters andacids cannot be established. In fact, there seems to be anantagonistic effect between two factors when pressure isapplied. In the case of high-pressure treatment, a volumedecrease is expected to occur when pressure increases.But as far as high-pressure treatment on the overallesterication reaction is concerned, the effect cannot beeasily predicted.In the case of ester solutions, only a small decrease in theester concentration was observed without any evidence of further reaction. There was no carboxylic acid formationthat would indicate ester hydrolysis. Esters did nothydrolyse when pressure was applied because whenhydrolysis occurs there is a volume increase. Accordingto the principle of Le Chatelier, when pressure increases a

decrease in volume is expected (McMurry, 1994) . Esterhydrolysis leads to the formation of a carboxylic acid and

Table 1 The effect of HHP on ethyl acetate, methyl acetate, ethyl butanoate, methyl butanoate, ethyl hexanoateand methyl hexanoate expressed as percent decomposition of the ester

% Decomposition a

pH 4 pH 6 pH 8

400 MPa 800 MPa 400 MPa 800 MPa 400 MPa 800 MPa

Ethyl acetate 5.2 7 0.31 7.8 7 0.42 10.1 7 0.55 8.2 7 0.44 2.1 7 0.11 13.7 7 0.56Methyl acetate 7.8 7 0.39 10.1 7 0.45 8.2 7 0.48 10.2 7 0.51 6.4 7 0.48 5.8 7 0.38Ethyl butanoate 0.1 7 0.01 3.1 7 0.11 0.3 7 0.02 7.5 7 0.33 3.4 7 0.23 4.3 7 0.28Methyl butanoate 0.1 7 0.01 14.1 7 0.43 18.0 7 0.45 4.1 7 0.11 4.4 7 0.23 6.2 7 0.38Ethyl hexanoate 1.6 7 0.12 11.8 7 0.34 4.4 7 0.29 5.9 7 0.44 4.3 7 0.22 0.1 7 0.01Methyl hexanoate 0.9 7 0.03 2.8 7 0.11 10.1 7 0.31 6.2 7 0.29 0.1 7 0.01 18.5 7 0.59a Values are means and standard deviations for N =3.

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an alcohol, which causes a volume increase. Thus, esterhydrolysis is not favoured by pressure.On the other hand, solvolytic reactions are enhanced bypressure. Solvolytic reactions are nucleophilic substitu-tions by the solvent, which, in this case, is water

(McMurry, 1994) . As a result, the solvent nucleophili-cally attacks the ester and enhances its hydrolysis.However, the results that we obtained did not show anysolvolysis in the system we studied.The same effect also appears in the case of carboxylicacids and alcohol solutions. Carboxylic acids andalcohols should react to produce esters when pressureis applied due to the oncoming decrease in volume.Moreover, interactions between the acid and the solventlead to a further increase in volume. The acid interactswith water and hydrates. This hydration leads to afurther increase in volume.On the other hand, hydrogen bonds are formed amongthe acid, the alcohol and water. The formation of hydrogen bonds can lead to a small volume contraction.Consequently, when pressure is applied, a volumedecrease can occur due to hydrogen bond formationand not necessarily due to ester formation.In any case, the role of pH is very important in esterformation and hydrolysis and thus the selection of theexperimental pH conditions is of great importance. Inthe case of base-induced hydrolysis, a mineral acid, forinstance HCl, is needed in the last step to protonate thecarboxylate ion and release the carboxylic acid. In thecase of acid-catalysed hydrolysis, a protonation is

required to activate the carbonyl group during the rststep. In both cases, low pH values favour hydrolysis asthe degree of protonation increases. As a result, low pHvalues favour hydrolysis.In general, it seems that pH has the same effect on bothformation and hydrolysis reactions. In low pH valuesboth reactions are favoured, while as pH increases therate of both reactions is decreased, expecting higherrates of reaction in more acidic conditions.The obtained results indicate a dependency of decom-position on pH. To be more specic, the degree of decomposition is decreased as pH increases. This is dueto the fact that in high pH values the rate of bothreactions, hydrolysis and formation, is reduced. As aresult both ester and acid solutions were more stable inbasic conditions. In the case of esters, the optimum

condition under which the degree of decompositionminimised and thus appeared to be more stable was aHHP treatment of 400 MPa at pH 8. In the case of carboxylic acids, high pressure appeared to have nosignicant effect on the degree of decomposition.

Minimal loss was observed at pH 8, independent of the pressure applied. Acids appeared to be stable at alower pH (e.g. pH 6) where the decomposition was notaffected by pressure. The optimum conditions for estersare 800 MPa at pH 8 and for acids are either 400 or800MPa at pH 8.Hydrogen bonding and compound volatility are alsoaffected by pH conditions. Hydrogen bonding is affectedonly in the case of carboxylic acids because esters do notform this type of bonds. In this case, when pH decreases,the degree of acid dissociation also decreases. Thus,[ROOH] becomes the predominant form. [ROOH] hasthe ability to form more hydrogen bonds than [ROO

]

species. As a result, acidic conditions enhance hydrogenbond formation.Ester and carboxylic acid volatility are both affected bypH. In the case of esters, the effect is rather complicated.As pH conditions become more acidic, hydrolysis isenhanced, ROOR 0 concentration is decreased whileROOH concentration is increased. But ROOH speciesare relatively more volatile than ROOR 0 species. So,even though ester volatility is not decreased, esters arehydrolysed into more volatile acids. A low-pressuretreatment in basic conditions is thus required to ensureminimum damage.

On the other hand, pH conditions affect carboxylic acidsin the same way as in hydrogen bond formation. Inacidic conditions ROOH, which is the predominantspecies, is more volatile than ROO

species. So, as pH

decreases, acid volatility is increased.Here, the reported decomposition (i.e. loss) could bepossibly attributed to the high volatility of thecompounds used and the inherent experimental error.In all cases, both HHP treatments of 400 and 800 MPahad no effect on the overall reaction. The degree of decomposition varied with pressure. Ester decomposi-tion minimised during 400 MPa HHP treatment, whileacid decomposition appeared to be independent of thepressure applied.The results reported here could be explained on the basisthat high pressure was applied for a short period of time

Table 2 The effect of HHP on mixtures of acetic acid/ethanol, acetic acid/methanol, butanoic acid/ethanol,butanoic acid/methanol, hexanoic acid/ethanol and hexanoic acid/methanol expressed as percent decomposition of the acid

% Decomposition a

pH 4 pH 6 pH 8

400 MPa 800 MPa 400 MPa 800 MPa 400 MPa 800 MPa

Acetic acid/ethanol 2.2 7 0.11 8.2 7 0.33 4.3 7 0.28 2.9 7 0.14 0.41 7 0.05 0.1 7 0.01Acetic acid/methanol 1.2 7 0.04 5.8 7 0.29 0.5 7 0.04 0.7 7 0.05 5.8 7 0.23 0.6 7 0.05Butanoic acid/ethanol 2.1 7 0.14 5.5 7 0.23 1.2 7 0.14 2.3 7 0.19 0.5 7 0.05 0.9 7 0.07Butanoicacid/methanol 4.1 7 0.24 10.1 7 0.43 2.1 7 0.14 8.1 7 0.44 0.5 7 0.02 7.8 7 0.38Hexanoic acid/ethanol 14.1 7 0.44 2.8 7 0.34 6.8 7 0.44 5.5 7 0.33 2.0 7 0.11 7.0 7 0.22Hexanoic acid/methanol 19.1 7 0.55 0.5 7 0.08 0.1 7 0.02 0.9 7 0.06 0.3 7 0.04 1.5 7 0.23a Values are means and standard deviations for N =3.

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(15 min) and thus it can be considered as a mildtreatment. The selection of the processing time wasbased on the previous work on high-pressure treatment.High pressure was applied for a period of 1030min tostrawberry jam (Kimura et al ., 1994) , 15 min to guava juice (Yen & Lin, 1999) , 15 min to strawberry (Zabetakiset al ., 2000a ,b), 10min to sh and sh products(Ohshima et al ., 1993) , 10 min to kimchi (Sohn & Lee,1998) and 10 min to peach (Sumitani et al ., 1994). Theprocessing time in our work was selected as an averageof the above.Comparing the obtained results with that of theprevious research in this area, 400600 MPa pressuretreatments seem to have no or minor effects. A 400 MPatreatment on strawberry jam preserved the fresh avourand natural colour (Kimura et al ., 1994) , 600 MPasterilised guava juice (Yen & Lin, 1999) , 500 MPapreserved the original avour and taste of sh (Ohshimaet al ., 1993) , 400MPa did not affect the structure of kimchi (Sohn & Lee, 1998) and did not change the

composition of aromatic compounds in peach (Sumitaniet al ., 1994) . As a result, 400 MPa HHP treatment isconsidered to be a mild process. Here, the reportedresults are in good agreement with that of the previouswork on the effect of HHP on strawberry avourcompounds (Zabetakis et al ., 2000 a). In that case,highest avour stability was observed when sampleswere treated with low pressures of 200400 MPa.In fact, the best avour retention was observed at400 MPa.

Acknowledgements

The excellent technical support of Ian Boyes isappreciated.

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