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Food Chemistry 117 (2009) 122–126

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Effect of various anti-browning agents on phenolic compounds profile of fresh


lettuce (L. sativa)
Arzu Altunkaya, Vural Gökmen *
Hacettepe University, Department of Food Engineering, 06800 Beytepe, Ankara, Turkey

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

Article history: High performance liquid chromatography (HPLC) was used to characterise phenolic profile and to select
Received 29 December 2008 the most effective anti-browning compound(s) on fresh lettuce. Four anti-browning agents, ascorbic acid,
Received in revised form 12 February 2009 cysteine, citric acid and oxalic acid were tested for their effectiveness on preventing loss of phenolic com-
Accepted 23 March 2009
pounds in lettuce during processing and storage. Aliquots of the reaction mixture were withdrawn at dif-
ferent times varying from 0 to 24 h, and directly analysed by HPLC. Protocatechuic acid, chlorogenic acid,
ferulic acid, caffeic acid, p-coumaric acid and phloridzin were identified in fresh lettuce. Degradation of
Keywords:
phenolic compounds followed a first-order kinetic pattern. The effect of anti-browning agents on first-
Lettuce (L. sativa)
Browning
order degradation rates of phenolic compounds was determined. Lettuce treated with oxalic acid and
Phenolic compounds ascorbic acid maintained a higher level of phenolic compounds than citric acid and cysteine. Interest-
Enzymatic oxidation ingly, cysteine had no positive effect for the prevention of oxidation of phenolic compounds even though
Inhibitors it prevented browning in lettuce.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Among natural antioxidants, plant polyphenols play a very impor-


tant role. Most of the beneficial health effects of flavonoids are
Minimally processed lettuce has become an important area of attributed to their antioxidant and chelating abilities; the protec-
potential growth in the rapidly expanding fresh-cut produce indus- tive effects can be ascribed to their capacity to transfer electron-
try because of its fresh-like character and convenience. A major free radicals, chelate metal catalysts, activate antioxidant enzymes,
challenge faced by the produce industry is to manipulate the qual- reduce a-tocopherol radicals and inhibit oxidases (Heimler, Isolani,
ity of fresh-cut produce so that the shelf-life is long enough to en- Vignolini, Tombelli, & Romani, 2007).
sure efficient marketing (Gonzalez-Aguilar et al., 2005). Fresh-cut Being deprived of their natural protecting matrices, the pheno-
produce deteriorates faster than intact produce because of internal lic compounds are exposed to oxygen during processing and may
and external browning of the cut surface. Browning detracts from therefore be oxidised to quinones. Various classes of phenolic com-
the appearance of the slices and reduces their marketability. Phys- pounds, showing a great diversity of structures such as catechins,
ical damage during the peeling and cutting process also causes an hydroxycinnamic acid derivatives and anthocyanins have been
increase in respiration rates, biochemical changes and microbial found to contribute to non-enzymatic and enzymatic browning
spoilage, which often result in degradation of colour, texture, nutri- of foods (Pati et al., 2006).
ent and flavour of the produce (Buta, Moline, Spaulding, & Wang, Basically, enzymatic browning can be defined as an initial
1999). enzymatic oxidation of phenolic compounds into slightly coloured
Phenolic compounds are plant secondary metabolites synthes- o-quinones, catalysed by polyphenol oxidase (PPO). Although PPOs
ised mostly through the phenylproponaid pathway and are in- are localised in plastids, their phenolic substrates are mainly lo-
volved in the defence of plants against invading pathogens. cated in the vacuole so that enzymatic browning only occurs when
Therefore, they are widely distributed in plant-derived foods this sub-cellular compartmentation is lost (Rigal, Gauillard, & Rich-
significantly affecting their stability, colour, flavour, taste, nutri- ard-Forget, 2000).
tional and aesthetic value. In fact, plant secondary metabolites ac- Various approaches to control the extent of browning have been
count for most antioxidant properties (Pati, Losito, Palmisano, & investigated. In general, enzymatic browning can be avoided by
Zambonin, 2006). Today, it is believed that regular consumption thermal inactivation of PPO, but heat can cause unwanted soften-
of dietary antioxidants may reduce the risk of several diseases. ing of the tissues. Instead of blanching, chemical additives have
been used to prevent enzymatic browning (Tortoe, Orchard, &
* Corresponding author. Tel.: +90 312 297 71 08; fax: +90 312 299 21 23. Beezer, 2006). Reducing agents, antioxidants, and enzymatic inhib-
E-mail address: vgokmen@hacettepe.edu.tr (V. Gökmen). itors prevent browning by chemically reducing the o-quinones to

0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.03.085
A. Altunkaya, V. Gökmen / Food Chemistry 117 (2009) 122–126 123

colourless diphenols. Acidulants, such as citric, oxalic, malic, or As a result of enzymatic oxidation, a sharp decrease in the con-
phosphoric acid, can also inhibit PPO activity by reducing pH centration of phenolic compounds was observed in the control let-
and/or chelating copper in a food product (Ibrahim, Osman, Saari, tuce. Loss of individual phenolic compounds identified in lettuce
& Abdul-Rahman, 2004). was expressed in terms of the first order degradation rate constant
In agricultural products, phenolic compounds and PPO may re- (k). The results are summarised in Table 1. The results are in agree-
act more or less during processing or storage resulting in a decrease ment with the results obtained by others (Nourian, Ramaswamy, &
of phenolic content. Phenolic compounds have a significant role in Kushalappa, 2003). Phenolic compounds and PPO originally exist
oxidation processes as antioxidants and as substrates in browning separately in different organelles in plant cells. When the plant is
reactions (Robards, Prenzler, Tucker, Swatsitang, & Slover, 1999). damaged, phenolic compounds and PPO come into contact and re-
In both roles, the key process is oxidation. Anti-browning agents act and parts of the phenolic compounds are isomerized (Rigal
were used to prevent or delay oxidation in different ways, but their et al., 2000).
effect on the phenol profile of fresh-cut produce is unclear. In this Adding of anti-browning agents is one of the typical methods
work, high-performance liquid chromatography (HPLC) has been used to inhibit the browning reactions. Treatments with AA, cys-
used to investigate how various inhibitors influence the polyphenol teine, CA and OA were more effective in delaying polyphenol oxi-
profile of lettuce during enzymatic oxidation processes. dation in lettuce during storage. Fig. 2 illustrates relative changes
in chlorogenic acid concentration in the presence or absence of
2. Material and methods various inhibitors. It was observed that the effectiveness of anti-
browning compounds in reducing polyphenols content diminished
2.1. Material with the length of storage (except for cysteine). These results are in
agreement with our previous findings (Altunkaya & Gökmen,
Lettuce was obtained from a local market in Ankara. Oxalic acid 2008).
(OA), ascorbic acid (AA) and cysteine were purchased from Merck,
and citric acid (CA) from Carlo Erba. Caffeic acid, chlorogenic acid 3.1. Effect of ascorbic acid on lettuce enzymatic oxidation
and gallic acid were purchased from Acros. Protocatechuic acid,
chlorogenic acid, ferulic acid and caffeic acid were obtained from The concentrations of phenolic compounds identified in the let-
Sigma. tuce extract have been monitored up to 24 h in the presence of AA.
Aliquots of the sample were withdrawn after 0, 1, 3, 6 and 24 h for
2.2. HPLC analysis of phenolic compounds in lettuce HPLC analyses. AA showed effective protection against oxidation.
Moreover, the rate of degradation of all identified phenolic com-
Three grams of lettuce were homogenised in 9 ml of distilled pounds was decreased slowly in lettuce when 0.05% of AA was
water, AA (0.5%), cysteine (0.05%), CA (0.5%) and OA (0.5%) solu- added (Table 1).
tions, respectively. Aliquots were taken from the slurry at 0, 0.25, AA is an important nutrient and very likely to decompose dur-
0.5, 1, 2, 4, 6 and 24 h. The slurry was centrifuged at 15,000 g for ing storage. It was reported that temperature, pH, oxygen concen-
15 min. The supernatant was used for HPLC analysis. tration, metals (iron, copper) and light are important parameters
Chromatographic analyses were performed on an Agilent 1200 for the destruction of AA (Jung, Kim, & Kim, 1995). Degradation
HPLC system consisting of a photodiode array detector, quaternary of AA has been also implicated with colour changes as well as re-
pump, autosampler and column oven. Phenolic compounds were duced nutritional quality.
separated on a Waters Atlantis C18 column (250 mm  4.6 mm, AA is a reducing compound but it does not directly inhibit PPO.
5 lm) using a linear gradient elution programme with a mobile Following enzymatic oxidation, AA reduces the o-quinone formed
phase containing solvent A (formic acid/H2O, 1:99, v/v) and solvent by the enzyme to the original diphenol to limit browning through
B (methanol) at a flow rate of 0.8 ml/min. The solvent gradient was a process known as ‘‘reaction deactivation”. Unfortunately, AA of-
programmed as follows: linear gradient elution from 10% B to 60% fers only temporary inhibition of browning (Altunkaya & Gökmen,
B, 0–15 min; isocratic elution of 60% B, 15–20 min; linear gradient 2008). During oxidation, it appears that AA is most likely converted
elution from 60% B to 10% B, 20–25 min; isocratic elution of 10% B, to dehydroascorbic acid (DHAA) and further degraded to 2,3 dike-
25–30 min. The chromatograms were recorded at 280 nm by mon- to-gluconic acid (Gökmen, Kahraman, Demir, & Acar, 2000). AA
itoring spectra within a wavelength range of 190–400 nm. Identifi- content of lettuce treated with 0.5% AA have been previously
cation of phenolic acids was accomplished by comparing the shown to follow a similar pattern (Gonzalez-Aguilar et al., 2005).
retention time and absorption spectra of peaks in samples to those AA acts as a protector of pigments preserving them from chem-
of standard compounds. The quantitation of phenolic compounds ical and biochemical oxidation by competing amides and amine–
was based on calibration curves built for each of the compounds carbonyl interactions that result in browning. An alternative mode
identified in the samples. of action may be suppression of free radical formation involved in
the browning reaction. There is also evidence that AA has polyphe-
nol-protective and -enhancing activities, probably due to the
3. Results and discussion reduction of oxidised phenols and regeneration of polyphenols. A
cooperative action between AA and polyphenols could be benefi-
The profile of phenolic compounds in fresh lettuce extract was cial in enhancing the ability of the latter to rescue cells from dam-
determined by HPLC analysis. Relative changes in the concentra- age induced by an oxidative stress (Gonzalez-Aguilar et al., 2005).
tion of individual phenolic compounds in lettuce with time as AA can react either with the radicals generated in chemical
influenced by the addition of 0.5% AA, 0.05% cysteine, 0.5% CA ways, or it can regenerate polyphenols from their oxidised forms
and 0.5% OA at 25 °C were compared to that of the control. Proto- due to its antioxidant potential. In order to obtain synergy via
catechuic acid, chlorogenic acid, caffeic acid, p-coumaric acid, feru- regeneration reactions between AA and phenolic compounds,
lic acid and phloridzin were identified by matching the retention these two reactions must compete efficiently with the other reac-
time of the compounds determined in the lettuce extract to those tions in which AA is oxidised (Beer, Joubert, Gelderblom, & Manley,
of pure standards (Fig. 1). The chemical structures of the peaks 2005). Based on standard reduction potentials, it has been pre-
identified in the lettuce extract were further confirmed by compar- dicted and confirmed by kinetic studies that ascorbate regenerates
ing their UV spectra to those of pure standards. plant phenolic compounds in homogeneous solutions from their
124 A. Altunkaya, V. Gökmen / Food Chemistry 117 (2009) 122–126

Fig. 1. HPLC profile of phenolic compounds in fresh lettuce. Peaks identified are: (1) protocatechuic acid; (2) chlorogenic acid; (3) caffeic acid; (4) p-coumaric acid; (5) ferulic
acid; (6) phloridzin.

Table 1
First-order degradation rate constants of individual phenolic compounds identified in lettuce.

Phenolic compound Rate constants (1/min)


Control Ascorbic acid Cysteine Citric acid Oxalic acid
Protocatechuic acid 0.557 0.056 0.576 0.447 0.083
Chlorogenic acid 0.333 0.036 0.667 0.434 0.055
p-Coumaric acid 0.668 0.030 0.386 0.183 0.080
Ferulic acid 0.890 0.016 0.929 0.862 0.396
Phloridzin 0.985 0.027 0.113 0.167 0.068

due to this reason (Yamaguchi et al., 2003). As a consequence,


the assuring stability of AA during storage has been a major prob-
lem for preventing browning and phenol destruction.

3.2. Effect of cysteine on lettuce enzymatic oxidation

The concentrations of phenolic compounds were monitored in


lettuce in the presence of 0.05% cysteine. It was determined that
the degradation rates of individual phenolic compounds in the
presence of cysteine were similar to those obtained in the control
sample without anti-browning agent. It should be noted here that
the lettuce sample with 0.05% of cysteine showed no enzymatic
activity against catechol as determined by a spectrometer at
420 nm (data not shown).
The action of cysteine is complex. During enzymatic oxidation,
Fig. 2. Changes in chlorogenic acid concentration with time in the presence of it traps the o-quinone by forming cysteine adducts. These adducts
various inhibitors (h: control, e: 0.5% ascorbic acid, D: 0.05% cysteine, s: 0.5% citric
acid, : 0.5% oxalic acid).
are not substrate, but competitive inhibitors of PPO with a slightly
higher affinity than their polyphenol precursors (Richard-Forget,
Goupy, & Nicolas, 1992). In the presence of an excess amount of
thiols such as cysteine (cysteine/phenol > 1), phenolic compounds
oxidised form (Jovanovic, Steenken, Tosic, Marjanovic, & Simic, are fully converted to their corresponding cysteine adducts, and
1994). Polyphenols have a higher redox potential than ascorbate in theory they are protected against enzymatic browning. The
and consequently oxidise it to the ascorbyl radical (Bors, Heller, strong inhibition of polyphenol degradation by PPO in the presence
Michel, & Saran, 1990). of cysteine is explained primarily by the competitive inhibition of
Lettuce also contains high levels of ascorbate oxidase activity the enzyme, and by the secondary effect of the coupled oxidation
which oxidise AA to DHAA after the lettuce is cut and exposed to by o-quinone, leading to phenol regeneration (Richard-Forget
oxygen. The amount of AA could be decreased during oxidation et al., 1992).
A. Altunkaya, V. Gökmen / Food Chemistry 117 (2009) 122–126 125

Gorny, Hess-Pierce, Cifuentes, and Kader (2002) have demon- 4. Conclusion


strated that sulphites act as a reducing agent at a pH below 4. At
a pH above 4, quinones form colourless adducts with sulfites, cys- The degradation of individual phenolic compounds during
teine or glutathione. Nucleophilic attack of quinones by cysteine enzymatic oxidation followed a first-order kinetic model. The deg-
may be more effective at a neutral pH since the thiol group of cys- radation rate constants obtained for individual phenolic com-
teine has a pKa of 8.33. Because the vegetable tissue is at neutral pounds in the presence of cysteine and CA were found
pH values, cysteine alone is effective as an inhibitor of enzymatic significantly higher than that of AA and OA. Higher k values indi-
browning. cated more rapid degradation of phenolic compounds in lettuce.
A consideration of biochemical changes inside the lettuce sam- AA and OA could be recommended to prevent enzymatic browning
ples may be just as important as appearance in selecting treat- and loss of phenolic compounds.
ments to extend the shelf life of fresh-cut lettuce. It is important Among the anti-browning agents tested, OA and AA had a great
to mention that the concentrations of anti-browning agents that capability for the prevention of the degradation of phenolic com-
were used in the present study did not affect the sensorial charac- pounds in fresh-cut lettuce. Hence, it appears that they have strong
teristics of fresh-cut lettuce except for cysteine. Due to the excess potential for practical applications.
amount of cysteine, lettuce samples treated with cysteine became Degradation rates of phenolic compounds with cysteine were
yellow and did not turn pink. almost the same as the control. It allowed permanent protection
against enzymatic browning, but phenolic compounds could not
3.3. Effect of citric acid and on lettuce enzymatic oxidation be protected against oxidation using cysteine. Loss of phenolic
compounds is undesirable from a nutritional point of view.
CA was found to protect lettuce against browning. Moreover, Two of the important properties of phenolic compounds involve
the rate of degradation of individual phenolic compounds de- antioxidant activity and oxidative browning. They are both good
creased in lettuce within 24 h of storage when 0.05% of CA was browning substrates and good antioxidants. In both systems, the
added (Table 1). Acidification of samples appeared to have an potential oxidising agents are PPO and free radicals when phenolic
important function to stabilise phenolic compounds in lettuce. compounds act as substrates and antioxidants (Robards et al.,
CA has been reported extensively for its inhibitory effect on 1999).
PPO, so it is recommended as a potential anti-browning agent in Enzymatic browning in foods is reasonably well understood,
minimally processed fruits and vegetables (Ahvenaien, 1996). It but prevention of browning is not the only factor to take into ac-
lowers the pH and chelates the copper at the active site of the count to be sure of quality. Besides the anti-browning property
PPO. Its inhibitory effect could be related to the phenolase Cu-che- of inhibitors, protection of nutritional properties should be taken
lating power. Especially, at pH values below 4, the looser binding of into consideration to maintain a fresh like quality. As a conse-
copper at the active enzyme site causes the PPO activity to de- quence, formulations including additives have to be optimised to
crease further, permitting the CA to remove the copper (Ibrahim succeed in the control of enzymatic browning and loss of the nutri-
et al., 2004). The effect of CA on flavanols have been previously tional quality.
considered as both anti-browning and antioxidant agent (Wang,
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