Chemical Papers 66 (10) 940–948 (2012)DOI: 10.2478/s11696-012-0200-0

ORIGINAL PAPER

Changes in lipid composition of apple surface layerduring long-term storage in controlled atmosphere‡

aKateřina Duroňová, a,bIvana Márová*, cMilan Čertík, bStanislav Obruča

aDepartment of Food Chemistry and Biotechnology, bCentre for Materials Research, Faculty of Chemistry, Brno University of

Technology, Purkyňova 118, 61200 Brno, Czech Republic

cFaculty of Biochemical Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia

Received 28 October 2011; Revised 5 April 2012; Accepted 6 April 2012

Apples are the most frequently consumed fruit and about 90 % of apple production is stored. Fattyacids and lipids are important constituents of plant cells. Disturbances in the lipid composition offruit may lead to various stress processes, resulting in some storage disorders. This work is focusedon an analysis of surface lipids of different varieties of apples stored in a normal atmosphere and amodified atmosphere with ultra-low oxygen content, for 4 months and 6 months. The major fattyacids in apple surface layers are palmitic acid, stearic acid, linoleic acid, and oleic acid. During the6-months storage period, a variety-specific decrease in the total fatty acids content and an increasein saturation degree was observed in all the varieties tested, when compared with the 4-monthsstorage. The greatest differences in saturation degree were observed in the Golden Delicious variety,in which the highest content of unsaturated fatty acids was also found. Microbial contaminationof apple surfaces increased gradually over the storage process. Higher fungi levels were found inapples stored in the regular atmosphere than in the modified atmosphere, which can be attributedto changes observed in the total lipid content and saturation degree of the surface fatty acids andalso to the sensitivity of microorganisms to the oxygen content in the storage room.c© 2012 Institute of Chemistry, Slovak Academy of Sciences

Keywords: apple, surface lipids, controlled atmosphere storage, oxygen content, microbial con-tamination

Introduction

Apples are among the most important fruits ex-tensively used for direct consumption and as a rawmaterial for the production of many food productsand preparations. Fresh apples should be rich in bio-logically active compounds, such as ascorbic acid andphenolic compounds, particularly flavanols, includingcatechins and proanthocyanidins; they also containfree amino acids and fatty acids, which have impor-tant roles to play in human health and in preservingfruit (Wu et al., 2007).

At present, more than 90 % of apples productionis stored and losses due to storage (despite the ap-

plication of modern storage technologies) range from5 % to 25 %. A decrease in the quality of produc-tion due to microbial contamination may be a fac-tor limiting storage duration. Economic losses causedby the development of various diseases during stor-age may be even greater than losses occurring inthe pre-harvest period. As far as the storage of ap-ples is concerned, the greatest losses are caused byeither physiological phenomena or by the develop-ment of fungal diseases (Gleosporium album, Peni-cillium expansum, Botrytis cinerea, Monilinia fructi-gena), even under low temperatures. Most of the in-fections remain latent up to harvesting. Visible symp-toms mostly appear after several months of storage

*Corresponding author, e-mail: marova@fch.vutbr.cz‡Presented at the 5th Meeting on Chemistry & Life 2011, Brno, 14–16 September 2011.

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when the natural resistance of apples decreases (Calvoet al., 2007).

The major part of market fruit spoilage occursafter the harvest (Lópes et al., 2007). Fungal infec-tions start developing, predominantly on the surfaceof bruised and damaged products. Although the fungiare sensitive to the storage atmosphere composition,the possibility of controlling decay is limited by thefruit’s tolerance to changes in O2 and CO2. Low tem-peratures and/or reduced levels of O2 in the storageenvironment during the first phase of fungal develop-ment prolong the initial period of infection and sup-press fruit decay (Lockhardt, 1967). During cold stor-age, the initiation of rotting is inhibited, but the fruittissue could lose resistance (Scholler et al., 2002).

Skin status is a most important factor in determi-nation of fruit quality. During long-term storage, massloss of about 5 g per 100 g can be observed, whichreduces fruit freshness and acceptability to the con-sumer. Sensory evaluation of the fruit skin status byvisual characterisation is relatively complicated andthe results are liable to be subjective. Thus, instru-mental measurements are preferable to sensory evalu-ations. To determine the 3D structure of selected fruitsurfaces, several advanced microscopy techniques wereused (Akyıldız & Ocal, 2006).

Fatty acids (FAs) and lipids are important struc-tural and metabolic constituents of plant and fruitcells. They are essential components of bio-membranesand are important for the function of most of the phys-ical and chemical reactions proceeding in a fruit cell.Disturbances in the composition of membrane lipidsoften exhibit serious effects on the cell’s adaptabilityto stress conditions, which may lead to various fruitstorage disorders (Song & Bangerth, 2003; Saquet etal., 2000). Along with structural roles, FAs and lipidsoften serve as precursors of important aroma volatileand regulatory compounds (Fellman et al., 2000).

It has currently proved difficult to find the eco-nomic and ecological balance between the level ofchemical treatment prior to harvest, the fruit storageconditions, and the degree of damage due to spoilagediseases. Within the context of this problem, it wouldbe useful to study the metabolic processes involved inthe production of apple defensive substances, to de-termine the composition of skin defensive barrier com-ponents, as well as to evaluate the effects of storageconditions and contaminating microorganisms.

This work is focused on the lipid and FA charac-teristics of different varieties of apples stored understandard atmospheric condition (regular atmosphere,RA) and modified atmosphere with ultra-low oxygenconcentration (ULO) for 6 months. The study is sup-plemented with microscopy analysis of surface layersand surface microflora to assess the impact of thestorage process on quality of the fruits. To the bestof our knowledge, the results of a similar long-termstudy have not previously been published. Stored ap-

ples were acquired from the collaborating Departmentof Post-Harvest Technology of Horticultural Products,Mendel University, Lednice, Czech Republic.

Experimental

Chemicals, reagents, and raw materials

All reagents were of analytical grade purity. Stan-dard lipids (mono-, di-, and triacylglycerols) anda calibration mixture of FA standards were pur-chased from Sigma–Aldrich (Prague, Czech Republic).Evirocheck� kits for surface microflora analysis wereobtained from Merck (Darmstad, Germany). All otherchemicals were purchased from Vitrum and Lach-Ner(Czech Republic).

Apple fruits (Malus domestica Borkh. cv. of thevarieties Golden Delicious, Granny Smith, and Cham-pion) were harvested in Holovousy (Czech Republic)and stored under RA (oxygen concentration of 21 %;6◦C; in darkness) and modified atmosphere with lowoxygen content; fluctuated anaerobiosis (FAN; oxygenconcentration of 1 %; carbon dioxide of 0.5 %) at 1◦Cfor 6 months. Samples for analysis were taken after4 months and 6 months of storage. Each time, threepieces of each apple variety were used for prepara-tion of a mixed sample. Controlled storage was realisedin collaboration with the Department of Post-HarvestTechnology.

Three popular local apple varieties were chosenfor analysis: Champion (short storage period), GoldenDelicious (middle to long storage period), and GrannySmith (long storage period). In each storage time,three fruits of each variety were taken, mixed sam-ples were prepared within two days and analyses wereperformed within the period of one week.

Isolation of lipids from apple tissues

Apples of the individual varieties were separatedto obtain the skin and the flesh. FA and lipid anal-yses were performed on about 40 cm2 of peel discsthus obtained using a cork borer and scraping off theuppermost 2 mm of peel (upper skin, epidermis, anda small part of the sub-epidermis). Then, the discswere homogenised and extracted twice with 20 mL ofhexane. Hexane layers were collected, filtered throughanhydrous sodium sulphate, and the solvent was evap-orated in a vacuum rotary evaporator. Pulp sam-ples were homogenised using the remaining apple fruit(5 g).

Dry mass of the apple fruit was determined gravi-metrically. An appropriate part of the apple fruit waspartially homogenised and weighed. After drying at60◦C for 4 h, samples were cooled to laboratory tem-perature and weighed to constant mass. All quanti-tative analyses were related to dry mass of the applefruit.

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Thin layer chromatography of apple lipids

Preparative thin layer chromatography (TLC) wasused for the separation of neutral lipids from aliphaticchains cleft from the wax surface on top of the applepeel. A sample of an appropriate amount (150–200 µg)of dry lipid fraction was applied onto TLC silicagel on aluminium sheets (Sigma–Aldrich, Germany).Neutral lipids were separated using hexane/diethylether/acetic acid (ϕr = 8 : 2 : 1). Individual lipidfractions were scraped off the plate and dissolved inheptane.

Analysis of fatty acids in lipid fractions

After separation, particular fractions of lipids wereindividually converted to FA methyl esters and de-tected by GC-FID and GC-MS systems (GC 6890NApparatus, Agilent Technologies Inc., USA). Fattyacid methyl esters were prepared from heptane ex-tracts using a modified procedure reported by Christo-pherson and Glass (1969). To 1 mL of lipid heptaneextract (10–30 mg mL−1), 0.1 mL of transesterifica-tion agent (1 mol L−1 sodium methanolate in benzene)and internal standard (margaric acid) were added. Af-ter 20 min methanolic HCl was added and the upperlayer of FA methyl esters was collected and analysedby GC-FID.

Individual aliquots of lipid extract (1 mL) wereinjected in split/splitless mode onto a DB-23 capil-lary column (60 m length 0.25 mm internal diameter;0.25 mm film thickness). The injector and detectortemperatures were set at 230◦C and 250◦C, respec-tively. The oven temperature was set at 150 ◦C for 3min, and increased to 175◦C at a rate of 7◦C min−1,maintained for 5 min, then raised to 230◦C at a rateof 10◦C min−1, and held at 230◦C for 40 min. The lin-ear flow-rate of H2 used as carrier gas was 58 cm s−1,and the electronic pressure control set at the constantflow mode. A calibration mixture of FA standards wasused for identification of FAs. An internal standardwas used for quantification. Individual FAs amount to100 g of apple dry mass. GC-FID analyses were per-formed in duplicate and the average values were usedfor data evaluation.

Statistical and surface microflora analysis

Statistical evaluation was carried out using theTWO-WAY ANOVA software version. 15.1.1.0 (Mini-Tab Inc., USA).

Surface microflora were determined using Eviro-check� kit; Contact TVC, for determination of to-tal viable counts of bacteria and fungi colonies, Con-tact YM(R), for determination of counts of yeasts andfungi colonies. The results were expressed as colony-forming units (cfu). These tests can be used for theanalysis of liquid material as well as for surface test-

ing. Light surface microscopy was used for a detailedanalysis of apple surface layers.

Results and discussion

In this work, three apple varieties frequently usedin the Czech Republic and Central Europe were stud-ied. The lipid and FA composition of surface layersduring the long-term storage under different storageconditions was analysed. To date, no similar studiesfocused on changes in the FA profiles in stored ap-ples have been published. Apples were stored usingtwo types of controlled atmosphere: (i) RA – normaloxygen content, darkness, and reduced temperature(6◦C); (ii) FAN – darkness, ULO (1 % oxygen, 0.5 %carbon dioxide) and low temperature (1◦C). The oxy-gen content in the atmosphere is an important fac-tor in the ripening processes and may affect complexchanges in the fruit along with surface lipid composi-tion, sensitivity to post-harvest infection and to gen-eral quality of the stored fruits (Prabha et al., 1988;Wu et al., 2007).

Immediately after harvesting, the lipid composi-tion and surface microflora of apple fruits were anal-ysed as initial values. Long-term changes were evalu-ated after 4 months and 6 months of storage at RAand FAN, respectively. After 4 months, significant vi-sual changes on the apple surface (wrinkled peel, signsof infection) were observed in Champion apples storedin RA, while only mild changes were exhibited in FAN.Champion apples in RA exhibited dramatic visual aswell as sensory changes after 6 months because of theshort storage period of this variety. Under FAN condi-tions, relatively less damage to the apple fruit was ob-served; nevertheless, longer storage could not be rec-ommended.

After 4-months storage, apples of the Golden De-licious variety (apples with middle-term storage pe-riod) exhibited relatively good quality in both RA andFAN atmospheres; high degree of maturation (yellow-red colour, increased sweetness) was observed in RA.After 6-months storage, changes in taste and mouldinfection sensitivity were demonstrated especially inRA (less than in Champion apples); negative changesin taste were also observed.

The best quality was observed after long-term stor-age of Granny Smith apples (long storage-period vari-ety); after 4-months period no changes in fruit qualitywere observed in RA as well as FAN; after 6 months,only mild differences in sweetness and taste were ob-served in apples stored in RA and FAN, respectively.

Thin layer chromatography of lipids

Thin layer chromatography in semi-preparativemode was used for separation of aliphatic chainsbound onto surface waxes from neutral lipids to ob-tain more reproducible results of FA analysis. Fig. 1

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Fig. 1. TLC of apple lipids (Golden Delicious variety); mobile phase hexan/diethylether (ϕr = 7 : 3) (a), mobile phasehexan/diethylether (ϕr = 4 : 1) (b); 1 – Golden Delicious (fruit skin, hexane extract), 2 – Golden Delicious (fruit pulp,hexane extract), 3 – monoacylglycerol, 4 – diacylglycerol, 5 – triacylglycerol, 6 – amaranth oil, 7 – Golden Delicious (skinextract, chloroform/methanol (ϕr = 2 : 1)), 8 – Golden Delicious (pulp extract, chloroform/methanol (ϕr = 2 : 1)).

shows an example of neutral lipid separation from ap-ple skin and pulp (Golden Delicious). For prepara-tive purposes, lipids from samples extracted to hep-tane were applied onto a TLC plate in a line form toobtain higher yields of lipid fractions. Lipids were fur-ther processed as described in detail in Methods andused in the analysis of fatty acids by GC-FID.

Analysis of fatty acids in apple peel

After harvesting, FA analysis was performed on allthree apple varieties. Content and distribution of in-dividual FAs in the fruits analysed are demonstratedin Fig. 2.

Palmitic acid, stearic acid, linoleic acid, and oleicacids (Fig. 2) were identified as the major FAs in ap-ple surface layers. This finding is in agreement withrecently published data that palmitic acid and linoleicacid were the dominant fatty acids, constituting 70–80 % of the total FAs in the whole apple fruit (Wu etal., 2007).

The content and distribution of FAs was charac-teristic for individual varieties; some differences couldbe observed particularly in major FA (palmitic acid,stearic acid, and oleic acid); fewer differences were ob-served in linoleic acid content. The ratio of saturatedand unsaturated FAs also differed, the saturation de-gree decreased from Granny Smith to Champion andthe highest content of unsaturated FA was determinedin the Golden Delicious variety.

Fig. 2. Content and distribution of FA in individual apple va-rieties prior to storage: – Champion, – Golden De-licious, – Granny Smith: 1 – lauric acid, 2 – myristicacid, 3 – pentadecanoic acid, 4 – anteiso-pentadecanoicacid, 5 – palmitic acid, 6 – palmitoleic acid, 7 – mar-garic acid, 8 – stearic acid, 9 – oleic acid, 10 – vaccenicacid, 11 – linoleic acid, 12 – alpha-linolenic acid, 13 –arachidic acid, 14 – gondoic acid, 15 – behenic acid, 16– lignoceric acid.

In general, only minor changes in fatty acids con-tent were observed in the flesh of apples over 4 monthsof storage. This effect is probably caused by a depositin the deeper layers of fruits and reduced contact withthe storage atmosphere.

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Fig. 3. Changes in FAs content and distribution in Champion apple peel stored in RA (a) and and FAN (b) atmosphere: –four months, – six months: 1 – lauric acid, 2 –myristic acid, 3 – pentadecanoic acid, 4 – anteiso-pentadecanoic acid, 5 –palmitic acid, 6 – palmitoleic acid, 7 – stearic acid, 8 – oleic acid, 9 – vaccenic acid, 10 – linoleic acid, 11 – alpha-linolenicacid, 12 – arachidic acid, 13 – gondoic acid, 14 – behenic acid, 15 – lignoceric acid.

Fig. 4. Changes in FA content and distribution in surface peel of Granny Smith apple fruits stored in RA (a) and FAN (b)atmosphere: – four months, – six months: 1 – lauric acid, 2 – myristic acid, 3 – pentadecanoic acid, 4 – anteiso-pentadecanoic acid, 5 – palmitic acid, 6 – palmitoleic acid, 7 – stearic acid, 8 – oleic acid, 9 – vaccenic acid, 10 – linoleicacid, 11 – alpha-linolenic acid, 12 – arachidic acid, 13 – gondoic acid, 14 – behenic acid, 15 – lignoceric acid.

Changes in fatty acid content in apple peelduring storage

The content of stearic acid, oleic acid, linoleic acid,and alpha-linolenic acid in the peels of all varieties ofapples varied continuously with storage time. The ma-jor FA content in Champion apple peel ranges from1.4–3.4 g per 100 g of dry mass (d.m.), while the mi-nor FAs are present in amounts ranging from 0.020–0.291 g per 100 g of d.m. (Fig. 3a and 3b). The highestcontent of the major FA palmitic, stearic, and oleicacids was reached after the 4-months period in RA,whilst, in FAN atmosphere, a higher level of linoleicacid could be found and, in general, FAs with longerchains were present. After 6 months of storage in RA,a significant decrease in the total FA content was ob-

served. Whilst an increase in palmitic and stearic acids(about 40 %) was observed in RA, a dramatic de-crease (about 80 %) in oleic acid could be found un-der these conditions. A similar increase in saturationdegree was observed in FAN (about 25 % increase inpalmitic acid and 40 % increase in stearic acid) wasaccompanied by a dramatic decrease in oleic acid andlinoleic acid.

In FAN, the total amount of FAs remained rela-tively stable throughout the 6-months period and sat-uration degree was relatively lower than in RA. Thefatty acids profile in FAN after 6 months is similar tothe fatty acids RA profile after 4 months of storage.

In Granny Smith apples stored in RA for 4 months(Figs. 4a and 4b), a similar content of palmitic, stearic,and oleic acids was observed; the other FAs were

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Fig. 5. Changes in content and distribution of selected FAs in Golden Delicious apple fruits (hexane extract of apple peel andapple pulp) stored in regular RA and modified FAN atmospheres. Apple peel/FAN (a), apple peel/RA (b): – palmiticacid, – palmitoleic acid; apple peel/FAN (c), apple peel/RA (d), apple pulp/FAN (e), apple pulp/RA (f): – stearicacid, – oleic acid (c–f); – vaccenic acid, – linoleic acid, – alpha-linolenic acid.

present only rarely. After a 4-months period in FAN, asignificant increase in oleic acid and linoleic acids anda similar content of palmitic and stearic acids was ob-served. After 6 months in RA, a substantial increase

(2.5-times) in palmitic and stearic acids was observed;a significant increase in linoleic acid and other higherFAs could also be demonstrated. In FAN, stable valuesof saturated FAs and a significant decrease in higher

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Table 1. Comparison of FA content in skin and pulp of Golden Delicious apples during storage in different types of controlledatmosphere

RA (skin)a RA (pulp)a FAN (skin)a FAN (pulp)a

Fatty acids4 months 6 months 4 months 6 months 4 months 6 months 4 months 6 months

Lauric acid 0.221 0.039 0.005 0.005 0.107 0.023 0.028 0.001Myristic acid 0.280 0.118 0.010 0.017 0.112 0.066 0.042 0.001Anteiso-pentadecanoic acid 0.195 0.026 0.006 0.003 0.083 0.014 0.035 0.000Pentadecanoic acid 0.039 0.028 0.002 0.003 0.026 0.019 0.008 0.000Palmitic acid 1.395 2.833 0.144 0.389 1.004 1.780 0.325 0.041Palmitoleic acid 0.330 0.031 0.010 0.003 0.130 0.020 0.053 0.000Stearic acid 0.710 3.231 0.049 0.465 0.561 1.953 0.214 0.043Oleic acid 3.653 0.291 0.118 0.016 1.873 0.204 0.363 0.047Vaccenic acid 0.259 0.004 0.012 0.002 0.737 0.019 0.137 0.001Linoleic acid 0.978 0.150 0.293 0.016 0.942 0.341 0.399 0.035alpha-Linolenic acid 0.075 0.023 0.021 0.022 0.131 0.065 0.062 0.002Arachidic acid 0.096 0.061 0.012 0.008 0.155 0.047 0.044 0.001Gondoic acid 0.022 0.003 0.001 0.000 0.038 0.004 0.004 0.000Behenic acid 0.118 0.026 0.007 0.003 0.156 0.022 0.044 0.001Lignoceric acid 0.138 0.014 0.007 0.002 0.439 0.040 0.137 0.000

Total FA 8.509 6.878 0.697 0.954 6.494 4.617 1.895 0.173

a) In g per 100 g of d.m.

FAs were observed, similar to the situation in RA after4 months of storage.

After a 4-months storage period, the major FAsin Golden Delicious fruits were higher derivatives ofstearic and oleic acids in RA as well as FAN. In FAN,about 30 % lower total content of FAs was observedand a higher mass ratio of linoleic acid to oleic acidthan with RA (Fig. 5). After 6-months storage periodin both atmospheres, a significant decrease in linoleicacid and a dramatic decrease in oleic acid accompa-nied with an increase in stearic acid was observed.A total decrease in 18 carbon acids (about 30 %)and increase in 16 carbon acids (40–50 %) was ob-served in both atmospheres. In FAN, a higher ratio ofhexadec-9-enoic (palmitoleic) acid to stearic acid canbe demonstrated.

Contents of unsaturated essential FAs differedin individual varieties and also different changes inlinoleic acid content were observed during storage. Thehighest linoleic acid content after the 4-months storageperiod was found in Granny Smith apples, followed byChampion variety and Golden Delicious. By contrast,Golden Delicious apples exhibited the lowest satura-tion degree (Fig. 2, Table 2). After 4-months storage inFAN, higher absolute values of unsaturated FA werefound in all varieties and a substantial decrease inlinoleic acid after 6-months period was observed whencompared with RA.

Similar differences in linoleic acid content were ob-served in a recent study in which eight apple vari-eties were analysed (Wu et al., 2007). The content oflinoleic acid as well as alpha-linolenic acid is of nu-tritional interest, since diets based on meat, starchsources, and fruits and vegetables are generally low inomega-3 fatty acids. The amounts of linoleic acid and

alpha-linolenic acid in plants are therefore significant,as they are essential fatty acids for humans and alsomust be obtained through the diet. As in our find-ings, this study also confirmed that the mass ratio ofsaturated-to-unsaturated fatty acids indicated an ex-cess of saturated fatty acids in each apple variety, theoptimal value of 3 : 7 or less (Song & Bangerth, 2003).

The data referred to above were obtained on freshapple samples. We may conclude, in addition, thatduring storage a further increase in saturation degreeof apple peel FAs occurs, accompanied by accumula-tion of FAs with shorter chain and with a total de-crease in FA content.

Fatty acids in apple pulp

Average values of FAs in skin and pulp of theGolden Delicious variety are demonstrated in Table 1.The pulp has a substantially lower FAs content thanthe skin. Major FAs found in apple pulp are palmitic,stearic, oleic, linoleic, and alpha-linolenic acids.

No changes in FAs levels were observed in the pulpof Golden Delicious apple fruits stored for 4 months inRA, while after 6 months the levels of myristic (80 %),pentadecanoic (100 %), palmitic (171 %), and stearicacid (840 %) increased; thus, an increased saturationdegree could be evaluated. In FAN, a dramatic de-crease in total FAs and mainly unsaturated 18 carbonFAs was observed (80–100 %) and a significant de-crease in oleic acid (86 %) and linoleic acids (95 %).The total degree of saturation in skin and pulp lipidcomposition increased with the storage period andoxygen content. The greatest differences in satura-tion degree were observed in Golden Delicious applesstored in RA, when saturation increase was about 2-

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Table 2. Approximate mass ratios of total saturated and total unsaturated FA in apple varieties analysed

Saturated-to-unsaturated fatty acids ratio

Atmosphere Champion Golden Delicious Granny Smith

4 months 6 months 4 months 6 months 4 months 6 months

RA 2.5 7.5 0.3 10.5 2.0 6.3FAN 0.8 6.0 0.4 5.0 0.7 6.0

times higher in comparison with FAN (Table 2).

Statistical analysis

Statistical analysis was performed according toTWO-WAY ANOVA. Data from Table 1 were eval-uated in two modes: (i) type of FA vs. time of storageand, (ii) type of FA vs. storage atmosphere. A signif-icant relation of type of FA and its content in appleskin (p < 0.05) and significant influence of combina-tion of storage time vs. FA content (p = 0.181) andtype of fatty acid vs. its content (p < 0.05) was found.Thus, the content of individual FAs changed signifi-cantly over the period of storage. Storage atmosphereexhibited no significant effect on the content of indi-vidual FAs (p = 0.418). The combination of type ofFA vs. its content (p = 0.000) and type of atmospherewas not statistically significant (p = 0.994).

In apple pulp, the storage time exhibited a signif-icant influence on individual FA content (p = 0.042)and a statistically significant relation between typeand content of individual FA was also found (p =0.004). The combination of both factors was not sig-nificant (p = 0.282). As was the case with apple skin,storage atmosphere exhibited no significant effect onFA content (p = 0.625); a significant relation wasfound between the type of FA and its content (p =0.042).

Data shown in Table 2 were tested in approxi-mate mode; the differences between individual vari-eties were neglected, hence storage time and atmo-sphere were the only factors tested for potential influ-ence of FA saturation mass ratio. Both time of storage(p = 0.000) and storage atmosphere (p = 0.048) exhib-ited a statistically significant influence on saturationdegree. Whilst over 4 months of storage no signifi-cant differences in saturation degree were found, overthe 6 months period statistically significant differenceswere evaluated. Modified atmosphere can significantlyreduce saturation degree in apple skin.

A particularly interesting finding was noted afterevaluation of the differences of total FAs content inapple skin and apple pulp during storage (Table 1).Whilst the total FA content in apple skin exhibited aslight decrease in RA as well as in FAN atmosphere,in apple pulp an increase in the total FA content and adramatic decrease in total FA in FAN were observed.Because of the generally low content of FAs in apple

Fig. 6. Changes in apple surface microflora (total counts ofyeasts and fungi) during storage under different con-ditions.

pulp, these changes could be associated with gradualtexture changes and sensory quality of apple fruits.

Microbial contamination

The amount of contaminating microorganisms onthe surface of apples increased gradually to fruitdegradation during the storage process (Fig. 6). In ap-ples stored in RA, a higher fungi level was found thanin the apples stored in ULO. By contrast, the ULO-stored apples were more contaminated with bacteria.This can be attributed to the fact that microorganismsare sensitive to oxygen content in the storage room.On the whole, the highest microbial load contamina-tion was found in the aged fruit stored in RA, probablydue to the increased invasiveness of fungi capable ofpenetrating through the damaged wax layer into theapple fruits that were already weakened. Apples storedin the normal atmosphere were more susceptible to in-fection. Susceptibility to microbial infection could beassociated, amongst others, with changes in the satu-ration degree in apple skin lipids. Very low saturationdegree in Golden Delicious apples stored for 4 monthsin RA corresponds to minimal microbial contamina-tion and, conversely, a dramatic increase in the num-ber of colony-forming units after 6 months of storagecould be related to a substantially higher saturationdegree (Table 2). Also with other varieties and underdifferent storage conditions, some connection betweensaturation degree and microbial colonisation could be

948 K. Duroňová et al./Chemical Papers 66 (10) 940–948 (2012)

observed (�Lata et al., 2005). Other factors with thepotential to influence microbial invasion are; (i) totaldecrease in FAs and (ii) potential damage to the sur-face lipid layer, which is associated with loss of relativehumidity and mechanical defects on apple skin surface.Microcracks in the wax-coat of apple peel can serve asentries for microorganisms into the apple fruit.

Conclusions

The present study elucidates changes in lipid con-tent and composition in the surface layers of applefruits of three popular local apple varieties: Champion,Golden Delicious, and Granny Smith during long-term storage in controlled atmosphere. The changesobserved depended on oxygen concentration, time ofstorage and apple varieties. The major FAs in applesurface layers of all three apple varieties studied werepalmitic, stearic, linoleic, and oleic acids. Accordingto statistical analysis, the content of individual fattyacids changed significantly over the storage period.The total degree of FA saturation was variety-specificand increased with the storage period and oxygenlevel. Under modified atmosphere conditions, a sta-tistically significant decrease in saturation degree inapple skin was confirmed in comparison with regularatmosphere.

The best quality was observed after long-term stor-age of Granny Smith apples (long storage-period va-riety); after a 4-months period, no changes in fruitquality were observed in RA as well as in FAN; after6 months only mild differences in sweetness and tastewere observed in apples stored in RA and in FAN, re-spectively. According to orientation, preliminary sen-sory analysis Golden Delicious apples (middle to longstorage period) were also relatively well protected, es-pecially in FAN.

The amount of contaminating microorganisms onthe surface of apples increased progressively duringstorage. Microbial contamination could be related tothe total decrease in FA content and also to changesin FA saturation degree during storage. On the whole,the highest microbial contamination was found in theaged fruits stored in RA, probably due to increasedinvasiveness of fungi that were capable of penetrat-ing through the damaged wax layer into the alreadyweakened apple fruits.

Acknowledgements. This work was financially supported byprojects “Centre for Materials Research” CZ.1.05/2.1.00/01.0012 of ERDF, 2B08057 of the Czech Ministry of Educationand QH 81056 of the Czech Ministry of Agriculture. Further,the work was supported by grant VEGA 1/0975/12 from theGrant Agency of the Ministry of Education, Slovakia.

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