biochemical changes in cut vs. intact lamb’s lettuce (valerianella olitoria) leaves during storage

Original article

Biochemical changes in cut vs. intact lamb’s lettuce (Valerianella

olitoria) leaves during storage

Antonio Ferrante,* Livia Martinetti & Tommaso Maggiore

Department Produzione Vegetale, University of Milano, Milan, Italy

(Received 28 July 2008; Accepted in revised form 25 November 2008)

Summary Consumers are oriented towards fresh-cut vegetables that provide phytonutrients useful for preventing

stress-related diseases. The aim of this work was to evaluate the cut operations on the quality changes of

lamb’s lettuce (Valerianella olitoria L.) cv. Trofy during storage at 4 �C for 8 days. Results showed that

chlorophyll and carotenoids reduction was observed after 8 days of storage. In both treatments, total

carotenoids after 8 days decreased from 20 to 16 mg g)1 FW. Free and total phenols increased with storage

in both treatments. Total phenols were 23% higher in control (32 lmol g)1 FW) compared to cut leaves

(25 lmol g)1 FW) after 8 days of storage. Anthocyanins increased after 8 days and reached 30 mg 100 g)1

FW without significant difference between treatments. Ascorbic acid (AsA) and dehydroascorbic (DHA)

acid increased in cut leaves compared to control. After 1 day AsA concentration was 3 300 nmol g)1 FW in

cut leaves, while in control leaves was 1 500 nmol g)1 FW. Analogously AsA + DHA was higher in cut

leaves, 4 100 nmol g)1 FW, while in control leaves the mean was 3 000 nmol g)1 FW. After 5 days of

storage the values of AsA returned to initial values, while AsA + DHA were lower.

Keywords Anthocyanins, ascorbic acid, fluorescence, lipid peroxidation, minimally processed, phenols, ready to use.

Introduction

In the recent years, the intensification of human lifeactivities and the development of new technologies inprocessing foods led the producers to offer new productsto satisfy consumer needs. The lifestyle of people hasbeen changing and the human nutrition has beenorienting towards ready-to-eat foods such as pre-cookedand minimally processed vegetables or fruits. In partic-ular, fresh-cut or minimally processed fruits and vege-tables play an important role in the human diet tocontrast the high caloric diets rich in lipids and sugars(Thompson et al., 1999; Brecht et al., 2004). Processingoperations acting to reduce the size of different fruits orvegetables to obtain ready-to-use produces, inducesevere mechanical damages, which are often accompa-nied by oxidative stresses (Rico et al., 2007).The internal quality of vegetables is represented by

vitamins, antioxidants, minerals and functional elementssuch as carotenoids and polyphenols that may preventsome diseases (Brecht et al., 2004; Artes & Allende,2005; Rico et al., 2007). Furthermore, external qualitymay be sub-divided into non-visible and visible compo-

nents. The latter is essentially referred to the visualappearance (yellowing, morphological defects andmechanical damages). The leafy vegetables’ colour isvery important, because of its attractiveness functiontowards consumers (Bolin & Huxsoll, 1991). Colourchanges are the most common postharvest disordersthat may compromise the economic value of produces.The green colour loss of leafy vegetables depends onmany factors, such as browning appearance on cutsurface or chlorophyll and carotenoids degradation(Ferrante et al., 2004). The chlorophyll loss is the lastevent of senescence and is not a good parameter formonitoring quality losses of vegetables during storage.However, chlorophyll a fluorescence has been satisfac-tory used for monitoring the leaf health status in lamb’slettuce (Ferrante & Maggiore, 2007). During storage allparameters related to chlorophyll a fluorescence areaffected and they may be used as markers for evaluatingstorage time and temperature. Chlorophyll a fluores-cence was also used for estimating the storage potentialof iceberg lettuce. Data collected at harvest time werecorrelated with storage quality of lettuce stored at 6 �Cand results obtained were promising (Schofield et al.,2005). Wounds in several species of Acanthaceae andGesneriaceae reduced chlorophyll fluorescence intensity(Yang et al., 2002). Therefore chlorophyll a fluorescence

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e-mail: [email protected]

International Journal of Food Science and Technology 2009, 44, 1050–10561050

doi:10.1111/j.1365-2621.2008.01891.x

� 2009 Institute of Food Science and Technology

measurements may be used for evaluating the stressinduced by cut operation in the fresh-cut or minimallyprocessed vegetables.Wounds generated during fresh-cut preparation act

as trigger for many physiological disorders that involveethylene production, phenolic accumulations, vitaminC losses, chlorophyll and carotenoid degradation(Bergquist et al., 2007; Heimler et al., 2007). Thevitamin C is considered the sum of ascorbic acid(AsA) and dehydroascorbic acid (DHA). Betweenthese two forms the AsA is the most perishablecomponent of leaf quality (Lee & Kader, 2000). Inwhole fruits or fresh cut AsA and DHA may havedifferent behaviour, but a general increase of DHAwas observed in several fruits during storage (Agaret al., 1999; Gil et al., 2006). The aim of this work wasto evaluate the effect of cutting on Valerianella qualitychanges during storage at temperature that is com-monly used in the processing chain of fresh-cutvegetables. Lamb’s lettuce has high ascorbate contentin contrast with the most studied lettuce, which haslower content and rapidly decline during postharvestlife. Therefore, our study is focused on the role ofascorbate and DHA on the leaf cut response. Finally,the last objective of the work was to evaluate thequality changes and senescence process during storageusing a no invasive method such as the chlorophyll afluorescence. In particular, the chlorophyll parameterswere measured to verify if wounds increased leafdeterioration and quality losses.

Materials and methods

Plant material and treatments

Lamb’s lettuce (Valerianella olitoria L. [Valerianellalocusta (L.) Laterr.]) cv. Trofy was grown in hydroponicsystem with floating polystyrol tray (named floatingsystem) on the nutrient solution. Plants were harvestedat commercial stage (10–12 cm tall with three to fourtrue leaves). The growing period ranged from 36 to41 days depending from the season. Leaves were washedwith 20 mg L)1 sodium hypochlorite for 5 min andrinsed with distilled water, then slightly dried and placedcut (randomly five to six pieces per leaf) or intact(control) in sealed plastic bags (high-density polyethyl-ene film) for storage simulation at 4 �C in darkness. Thestorage was performed for 8 days using five bags pertreatment.

Chlorophyll, carotenoid content and chlorophyll afluorescence measurements

Leaf pigments were extracted using methanol 99.9% assolvent. Samples were kept in dark cold room at 4 �Cfor 24 h. Absorbance readings of extracts were taken at

665.2, 652.4 and 470 nm. Chlorophyll and total carot-enoids were calculated by Lichtenthaler formula (1987).Chlorophyll a fluorescence transients were determined

on dark adapted leaves kept for 30 min at roomtemperature, using a portable Handy PEA (Hansatech,Norfolk, UK). The measurements were taken on the leafsurface (4 mm diameter) exposed to an excitation lightintensity [ultra-bright red light emitting devices (LEDs)with a peak at 650 nm] of 3 000 lm m)2 s)1

(600 W m)2) emitted by three diodes. Leaf fluorescencedetection was measured by fast response PIN photodi-ode with RG9 long pass filter (Technical manual,Hansatech). The parameters measured were Fo, Fm,Fv ⁄Fm and Performance Index (PI). The Fo representsthe fluorescence level when the plastoquinone electronacceptor pool (Qa) is fully oxidised. Fm represents thefluorescence level when Qa is transiently fully reduced.The Fv ⁄Fm ratio represents the maximum quantumefficiency of photosystem II, the Fv is the variablefluorescence (Fm–Fo). Finally the PI represents a veryuseful term providing information concerning relative‘vitality’.

Estimation of lipid peroxidation

Lipid peroxidation was determined by thiobarbituricacid reactive substances (TBARS) content according toHeath & Packer (1968). One gram of tissue sample washomogenised in 3 mL of trichloroacetic acid 0.1% andcentrifuged at 10 000 r.p.m. for 5 min. The supernatantwas collected and 1 mL was mixed with 4 mL of 20%trichloroacetic acid and 0.5% thiobarbituric acid. Themixture was heated at 95 �C (30 min), quickly cooledand centrifuged at 10 000 r.p.m. for 5 min. The super-natant was used to determine TBARS concentration at532 nm and 600 nm.

Total and free phenols extraction and determination

Samples of 50 mg were placed in 3 mL of 80%methanol ⁄water for free phenols extraction. The sam-ples were vortexed for 1 min, subsequently heated to60 �C for 3 h. During incubation the samples werevortexed every 30 min. After incubation the sampleswere cooled at room temperature and centrifuged at5 000 r.p.m. for 1–2 min for removing solids. Totalphenols, instead, were extracted from 50 mg of sampleusing 3 mL of 1.2 m HCl in 80% methanol ⁄water andtreated as described for free phenols (Gorinstein et al.,2004).Phenols in the extracts were determined using the

Folin–Ciocalteu reagent (Sigma, Milan, Italy) (Single-ton et al., 1999). Readings of reactions containing200 mL extract and 0.5 mL Folin–Ciocalteu reagentplus water up to 10 mL were performed at 760 nm after2 h incubation at room temperature.

Effect of processing on Valerianella storage A. Ferrante et al. 1051

� 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009, 44, 1050–1056

Anthocyanins extraction and determination

Samples of the frozen tissue (100 mg) were ground in apre-chilled mortar and extracted into methanolic HCl(1%). Samples were incubated overnight at 4 �C indarkness. The concentration of anthocyanins wasexpressed in cyanidin-3-glucoside equivalents deter-mined spectrophotometrically at 535 nm using anextinction coefficient (e) of 29 600 (Cheng & Breen,1991).

Ascorbate and dehydroascorbate content

Samples (1 g) of leaves were homogenized in cold 5%trichloroacetic acid (25:6 ⁄w:v), using a cold mortar, andcentrifuged at 12 100 · g for 15 min. Ascorbic acid(AsA) and total ascorbate (AsA + DHA, ascor-bic + dehydroascorbic acid) were determined in thesupernatant following the method reported by Franciniet al. (2006). This assay is based on the reduction offerric to ferrous ion by ascorbic acid in acidic solutionfollowed by formation of a red chelate between ferrousion and 4,7-diphenyl-1,10-phenanthrolin (bathophe-nantroline) which absorbs at 534 nm. Total ascorbatewas determined through a reduction of DHA to AsA by3.89 mM dithiothreitol, and DHA levels were estimatedon the basis of the difference between total ascorbateand AsA values. A standard curve covering the range0–10 nmol AsA was used.

Statistical analysis

Every sampling time, five bags were opened for takingthe leaves to use for analyses. The data reported infigures and tables are means ± standard errors (SE).Sample were taken for each bag plus a mix composed byleaves collected from all bags (n = 6). Chlorophyll datawere subjected to two-way anova using NCSS 2000statistical analysis system software. Separation of meanswas determined by l.s.d. test.

Results and discussion

Total chlorophyll content of Valerianella, in bothtreatments control (no cut) or cut leaves, was measuredfor evaluating the colour changes (external quality)during storage at 4 �C. Results showed that chlorophyllcontent did not change until 5 days in both treatments,while significantly decreased ()41%) in cut leaves after8 days of storage compared to control (Fig. 1a). On thecontrary, chlorophyll content did not statisticallydecline in control Valerianella leaves during wholeexperimental period. Carotenoids did not decreaseduring the first 5 days of storage, while after 8 daysthe reduction was by 25% compared to initial values inboth treatments (Fig. 1b).

Chlorophyll contents and carotenoids are very impor-tant for their role on consumer health and attractiveness(Ferrante et al., 2004). Our previous study on leafyValerianella storage confirmed these results and showedthat at low temperature (4 �C) the chlorophyll reductionwas very slow and was dependent on the length ofstorage time (Ferrante & Maggiore, 2007). However, thechlorophyll losses during storage have been observed ina wide range of vegetables (Rico et al., 2007). Yellowing(senescence symptoms) or browning spots compromisethe commercial value and reduce the shelf life of leafyvegetables during storage.The thiobarbituric acid reactive substances (TBARS)

progressively increased during storage in both controland cut Valerianella leaves (Fig. 2), even if the lattershowed higher values. TBARS measured in fresh har-vested leafy vegetables were, in average, 5 nmol g)1 FW.After 8 days of storage TBARS values reached 41 and54 nmol g)1 FW, in control and cut leaves respectively(Fig. 2). Statistical differences between treatments wereonly observed after 8 days (Fig. 2). TBARS represent amarker of lipids peroxidation that has been used in

(a)

(b)

Figure 1 Total chlorophyll (a) and total carotenoids (b) in cut and

control Valerianella leaves stored at 4 �C. Values are means with

standard errors (n = 6). Data were subjected to two-way anova

analysis and differences among treatments were determined by l.s.d.

test. Different letters mean statistical differences at P < 0.05.

Effect of processing on Valerianella storage A. Ferrante et al.1052

International Journal of Food Science and Technology 2009, 44, 1050–1056 � 2009 Institute of Food Science and Technology

many studies for understanding the membrane destabil-isation in plants and animals (Garstka et al., 1994;Lukatkin et al., 1995). In plants the lipid peroxidationproduces hydroperoxide compounds that are catalysedby lipoxygenase enzyme, enzymes are released and comein contact with their substrates inducing physiologicaldisorders (Saltveit & Qin, 2008). In leafy vegetablesmembrane degradation is the main factor involved inthe quality losses and is responsible for leaf yellowingand tissue browning.Anthocyanins increased after 8 days of storage but no

differences were observed between treatments (Fig. 3a).Free phenols content almost doubled after 5 days inboth treatments, then remained constant until the end ofstorage (Fig. 3b), showing no statistical differencebetween the two treatments. Total phenols increasedfaster in cut leaves after 5 days compared to control andremained constant until 8 days of storage (Fig. 3c). Incontrol leaves, total phenols, instead, were almostconstant after 5 days and subsequently sharply in-creased by 23% compared to cut leaves after 8 days ofstorage (Fig. 3c). Similar results were obtained in fresh-cut lettuce cv. Lollo Rossa, which was less sensitive tobrowning and showed an increase of antioxidant abilityand phenolic compounds (Tavarini et al., 2007). Theincrease of phenols and anthocyanins is usually aconsequence of an activation of phenylalanine ammonialyase (PAL) activity. This enzyme belongs to cold genesand is induced by light, cold and wounds (Tavariniet al., 2007). The increase of phenolic compounds maycounteract the AsA losses keeping the antioxidant statusof leaves unchanged inhibiting the postharvest disorder.Total phenols showed similar trend in Valerianella

leaves stored at 4 �C or 10 �C (Ferrante & Maggiore,2007). The phenols oscillation during storage may bedue to the interconversion of free and bound phenols.Carotenoids, polyphenols (including anthocyanins) andAsA represent the most important antioxidants, whichcontribute the total antioxidant capacity of leafy vege-tables (Yamasaki, 1997; Brecht et al., 2004; Rico et al.,2007). In the human health the anthocyanins provide

Figure 2 Lipid peroxidation expressed as thiobarbituric acid reactive

substances (TBARS) equivalent nmol g)1 FW during storage of

Valerianella leaves whole or cut. Values are means with standard errors

(n = 6). Data were subjected to two-way anova analysis and

differences among treatments were determined by l.s.d. test. Different

letters mean statistical differences at P < 0.05.

(a)

(b)

(c)

Figure 3 Anthocyanins content expressed as cyanidin-3-glucoside (a)

Free phenols (b) and Total phenols (c) expressed as gallic acid

equivalents (lmol g)1 FW) during storage of Valerianella leaves

without cut (control) and cut. Values are means with standard errors

(n = 6). Data were subjected to two-way anova analysis and

differences among treatments were determined by l.s.d. test. Different

letters mean statistical differences at P < 0.05.



defence or prevention against cardiovascular disorders,advancing age-induced oxidative stress, inflammatoryresponses, and diverse degenerative diseases (Zafra-Stone et al., 2007).Total ascorbic acid (AsA + DHA) and ascorbic acid

(AsA) were higher (30% and 55% respectively) in cutleaves compared to control after 1 day of storage. Bothsignificantly declined, showing the same trend even if thevalues were always higher in cut leaves (Fig. 4a, b).After 5 days, AsA values, declined by 67% in cut leavesand by 44% in control leaves (Fig. 4b). Analogously as

AsA, the AsA + DHA decreased by 57% in cut leavesand by 53% in control leaves (Fig. 4a). The ratioAsA ⁄DHA was almost fourfold higher in cut leavesafter 1 day of storage. Subsequently the ratio valuesdeclined in cut leaves until 5 days of storage andincreased again after 8 days (Fig. 4c). In control leaves,the ratio AsA ⁄DHA decreased after 1 day and thenincreased again till 8 days of storage and significantdifferences were found only after 8 days of storage(Fig. 4c). The increase of AsA ⁄DHA ratio means thatDHA is reduced in AsA form. Changes of AsA + DHAduring storage depends from species and storage temper-ature (Degl’Innocenti et al., 2005). A rapid decrease ofAsA during storage has been observed in many fruits andvegetables (Lee &Kader, 2000). The increase of ASA andDHA levels immediately after cut operations is probablydue to protection response of tissue to wounds or tocontrast free radical formation. The AsA reductionduring storage was observed in many vegetables. Inspinach, after 3 days storage at room temperature theAsA content was the 10% of initial values (Wills et al.,1984). Similar results were found in spinach stored at airor modified packaging at 10 �C (Gil et al., 1999).The AsA is also used as antioxidant compound for

preventing tissue browning in sensitive leafy vegetables.AsA inhibits browning by its reducing power, inhibitionof polyphenol oxidase and by reducing the enzymati-cally formed quinones back to their parent o-diphenols(Altunkaya & Gokmen, 2008). AsA is also a powerfulfree radical scavenger and is a good substitute ofsulphites, which may give health problems to consumers(Artes & Allende, 2005).Chlorophyll a measurements and JIP indexes were

calculated on the intermediate steps of fluorescenceinduction curves. Values recorded did not show signif-icant differences between treatments during storage ofValerianella leaves (Fig. 5). The Fv ⁄Fm was lower in cutleaves compared to control leaves. This ratio declineduntil 5 days then remained constant (Fig. 5a). Analo-gous results were observed for Fm and PI values werealways lower in cut leaves even if no statistical differ-ences were observed (Fig. 5b, d). The Fo increased inboth treatments during storage without differencesbetween the two treatments (Fig. 5c).The Fv ⁄Fm ratio indicates the maximal efficiency of

PSII. Leaves of deciduous and evergreen trees that haveFv ⁄Fm values ranging from to 0.76 to 0.85 are consideredno stressed and healthy (Demmig & Bjorkman, 1987).Fv ⁄Fm ratio has been used for monitoring producequality during storage of flowers and vegetables (Meiret al., 1997; Lin&Jolliffe, 2000; Pompodakis et al., 2005).These results may be explained considering that

chlorophyll a fluorescence is measured in the middle ofleaves, where the effect of wounds is very limited. Thetrend of index variations were in according to previousexperiments (Ferrante & Maggiore, 2007).

(a)

(b)

(c)

Figure 4 Ascorbic acid (AsA) (a), total ascorbic acid (Ascorbic plus

dehydroascorbic acid, AsA + DHA) (b), AsA ⁄DHA ratio (c) mea-

sured during storage of Valerianella leaves whole (control) or cut.

Values are means with standard errors (n = 6). Data were subjected to

two-way anova analysis and differences among treatments were

determined by l.s.d. test. Different letters mean statistical differences at

P < 0.05.



Conclusion

Cut operations slightly affect the chlorophyll contentand chlorophyll a fluorescence in Valerianella leaves.Marked differences were found in ascorbate and dehy-droascorbate content. Wounds stimulate AsA + DHAbiosynthesis with an increase of AsA ⁄DHA ratio. TheAsA + DHA content is very unstable and is thefirst quality component to decrease during storage.Therefore, the highest value of vitamin C in Valerianellafor human health can be obtained if consumed within5 days of storage. However, the reduction of AsA +DHA is partly balanced by phenylpropanoids, whichalong with carotenoids and vitamin C represent themost part of antioxidant compounds in leafy vegetables.The AsA content might be used for monitoring the leafyvegetable quality during storage.

Acknowledgments

The present work was funded by MIUR with PRIN2006–2007 ‘Biochemical and molecular studies onmembrane degradation of fresh-cut leafy vegetables.(Analisi bio-molecolari sull’alterazione delle membranecellulari nei prodotti orticoli di quarta gamma)’.

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Figure 5 Chlorophyll a fluorescence indexes measured during storage

of Valerianella leaves whole or cut. Fo = fluorescence level when

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Fm = fluorescence level when Qa is transiently fully reduced;

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biochemical changes in cut vs. intact lamb’s lettuce (valerianella olitoria) leaves during storage

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