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International Journal of Food Studies IJFS April 2014 Volume 3 pages 8292

The relationship between antibrowning, anti-radical andreducing capacity of Brassica and Allium extracts

Bustos Mariela Ca, Agudelo-Laverde Lina Ma, Mazzobre Florenciaa, and BueraPilara*

a Dpto. de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Inte. Guirandes100, C1428EGA Buenos Aires, Argentina

*Corresponding [email protected]

Tel: +54 11 4576 3366

Received: 30 September 2013; Published online: 18 April 2014

Abstract

Aqueous vegetable extracts from Allium and Brassica families were assayed for antibrowning capacityand related to their anti-radical and reducing power activities. The treatment of mushrooms andavocado slices, with white cabbage, cauliflower, garlic and scallion extracts, reduced color changesduring storage at 4 oC and -18 oC. Storage temperature and the type of extract employed influencedchange of color variables. The contribution of polyphenols on measured antioxidant activity of extractswas also discussed. Allium antibrowning properties were closely related to antioxidant capacity, whilethe Brassica extracts were less effective. Treatment with Allium extracts extended the storage time offrozen and refrigerated mushrooms and avocado slices, in comparison with untreated samples.

Keywords: Anti-browning; Antioxidant capacity; Allium; Brassica

1 Introduction

Browning is one of the main factors affecting con-sumers acceptability or rejection of fresh prod-ucts such as avocado and mushrooms. Whilstinfluenced by storage conditions and composi-tion, browning of fresh fruits and vegetables ismostly due to enzymatic reactions. Sulphiteshave been employed for decades to control en-zymatic and non-enzymatic browning, and havethus been considered as universal browning in-hibitors. Since the use of sulphites has beenbanned for fresh fruits and vegetables (Gendel,2012), there is a need to find simple and naturaltreatments to control browning (Rico, Martin-Diana, Barat, & Barry-Ryan, 2007).In addition, an increased consumers demandfor minimally processed vegetables has promotedmany studies focused on the control of browning

using inhibitors of natural origin (Kim, Kim, &Park, 2005; Thorat, 2013).Research on potential inhibitory compoundsfrom edible vegetables is increasing (Kim etal., 2005) since they are non-toxic and haveno known adverse side effects. Vegetablesfrom Brassica (known also as crucifers) andAllium families have been reported as poten-tial browning inhibitors (Zocca, Lomolino, &Lante, 2010; Cabello-Hurtado, Gicquel, & Es-nault, 2012). They have the further advantageof being commonly grown and consumed world-wide. They also have been reported to possessrelevant antioxidant and anti-carcinogenic prop-erties (Leelarungrayub, Rattanapanone, Cha-narat, & Gebicki, 2006), which makes them evenmore interesting to study.Antioxidants can deactivate radicals by two

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82 Bustos et al.

major mechanisms: Hydrogen Atom Transfer(HAT) and Single Electron Transfer (SET).HAT-based methods measure the classical abilityof an antioxidant to quench free radicals by hy-drogen donation and SET-based methods detectthe ability of a potential antioxidant to transferone electron to reduce any compound, i.e. theirreducing capacity. In vegetables, SET and HATmechanisms almost always occur together, withthe balance determined by antioxidant structureand pH (Prior, Wu, & Schaich, 2005). As a con-sequence, determination of the capacity for ex-tracts from vegetables to avoid or retard oxida-tion by both types of reactions is critical in orderto characterize the appropriate potential of theextracts.Avocado and mushrooms have a short shelf lifecompared to most fruits and vegetables. Themain problem associated with preserving fresh-cut avocado fruit is the high browning rate ofthe cut surfaces caused by oxidation of phe-nols into quinones, catalyzed by polyphenoloxi-dase (PPO) enzyme, that subsequently polymer-ize into brown pigments. The intact mushroomsalso lose their commercial value within a fewdays, due to senescence, water loss and brown-ing, which is attributed to activation of tyrosi-nase, an enzyme belonging to the PPO family,and/or spontaneous phenol oxidation (Jolivet,Arpin, Wichers, & Pellon, 1998). Concomitantbacterial activity favors the browning develop-ment of both vegetables. The genus Bacillusspp. is frequently involved in avocado deterio-ration (Soliva, Elez, Sebastian, & Martn, 2000),while Pseudomonas spp. and Flavobacteriumspp. are the two main groups that predominateduring postharvest mushrooms storage (Singh,Langowski, Wani, & Saengerlaub, 2010).The present study was conducted to analyze therelationship between anti-browning capacity ofBrassica and Allium vegetable extracts on avo-cado and mushroom slices, and their antioxidantactivity mediated by different mechanisms.

2 Materials and Methods

2.1 Mushroom and avocadosamples preparation

Mushroom (Agaricus bisporus) and avocado(Persea Americana Mill; var. Hass) were cho-sen for this research because they are highly sus-ceptible to enzymatic browning. Selected piecesof uniform size and color of both products, atcommercial maturity, were purchased at a localmarket and immediately processed.

2.2 Allium and Brassica extractspreparation

Fully mature Allium (garlic, onion and scallion)and Brassica (white cabbage, cauliflower andBrussels sprouts) vegetables were produced onfarms near Buenos Aires, Argentina, during 2012and purchased at a local market. Selected veg-etables were washed, peeled if necessary, and cutinto pieces. The pieces were homogenized andextracted under constant agitation, in phosphatebuffer solution pH 6.0, at 50 oC for 1 hour. Theratio of vegetable- buffer was 1:2. The extractswere sterilized at 121 oC for 5 minutes to avoidfurther enzyme activity, and then they were cen-trifuged at 1600 g for 30 minutes at 4 oC and fil-tered on filter paper (20-25 m, Whatman ECN-512-1026). Trehalose was added to the liquid ex-tracts at a final concentration of 15 % w/v in or-der to obtain a physically adequate dry matrix.Aliquots (40 ml) of the extracts were distributedin plastic trays (1 cm height) and frozen at -20 oCfor 48 hours, further cooled under liquid nitrogenand freeze dried (ALPHA 1-4 LD2 Martin ChristGefriertrocknungsanlagen GMB, Germany).

2.3 Antibrowning treatment andstorage

Cap mushroom and avocado were cut transver-sally into slices (2.5 cm or 1.5 cm diameter and0.3 cm thickness, respectively) and were treatedin 25 ml of 10% w/v dipping antibrowning ex-tract for 5 minutes and drained. The excess so-lution was blotted with an absorbent paper and

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Anti-browning and antioxidant capacity of vegetables extracts 83

slices were placed in rubber o-rings (2.5 cm inter-nal diameter) between two glass plates hermeti-cally sealed (to avoid water loss) according to themethod reported by Acevedo, Briones, Buera,and Aguilera (2008) and stored at 4 oC or -18 oC.Control sample was dipped in phosphate bufferpH 6.0.

2.4 Color measurement

Changes in color of the treated cap mushroomand avocado slices were determined by imageanalysis using a computer vision system (CVS)according to Agudelo-Laverde, Schebor, andBuera (2013). The lighting system included aD65 lamp inside a grey chamber (N7 in theMunsell color space). A high-resolution (10.1mega-pixel) digital camera, an EOS 40D (CanonInc., Japan) was used, with an EF-S 60mm f2.8macro lens (Canon Inc., Japan).Avocado and mushrooms samples in glass plateswere placed in the grey box (white background)and images acquired at different times duringthe whole storage period (96 and 240 hours for4 oC and -18 oC storage, respectively). Thedigital camera was operated in manual mode,with the lens aperture at f 146.3 and speed

18 s (no

zoom, no flash) to achieve high uniformity andrepeatability.Color images were obtained in Lab values usingAdobe Photoshop CS4 software (Adobe SystemsInc., San Jose, CA) and then were converted tothe standard CIELAB space. From the CIELABcoordinates L* (luminosity), a* (red/greencoordinate) and b* (yellow/blue coordinate) thetotal color change (E) has been calculatedaccording to the following equations:

E = (L2 + a2 + b2)0.5 (1)

The corresponding samples at time zero weretaken as references.In order to determine the effectiveness of selectedaqueous extracts, an Antibrowning Index (ABI)was calculated as follows:

ABI = (Xcontrol*Xtreated)/Xcontrol (2)Where X* is the total color parameter changeduring storage of control sample or treated slices.

The color parameter (X) used to calculate ABIwas selected on the basis of the highest degreeof change, so that it could better represent thebrowning of each matrix.

2.5 Antioxidant properties

Total polyphenol content of selected Bras-sica and Allium extracts was determined bythe Folin-Ciocalteau method, using gallic acidas a calibration standard (Prior et al., 2005).Vegetable extracts 10% w/v (50 L) or gallicacid standard solutions were mixed with 800L deionized water, 125 L sodium carbon-ate 20 %w/v and 125 L of 1:2 dilution ofFolinCiocalteu phenol reagents. After 40 minin darkness the absorbance at 765 nm was mea-sured. The concentration of total polyphenolswas expressed as mg gallic acid per 100 ml ofextract at 10% w/v.

Antioxidant activity

Radical scavenging activity was measuredby the ABTS method according to Re et al.(1999). The ABTS+ solution was diluted 1:2,with distilled water, to an absorbance of 0.700 at734 nm. After addition of 50 l of the sample to3.0 mL of diluted ABTS+ solution, absorbancereadings were taken for 30 minutes using a spec-trophotometer (UV-visible Jasco V630, JascoCorporation, Japan). A bi-exponential fit wasapplied using ORIGIN 8.0 software (Origin-LabCorporation, Northhampton, MA, USA). ABTSradical scavenging rate was calculated by thefollowing equation:

ABTS radical scavenging rate (%) = 100[1 (As /A0)](3)

Where A0 means the absorbance at time 0 with-out sample addition and As means the sampleabsorbance at stationary state, calculated by bi-exponential fitting.Ferric reducing ability of aqueous extracts(10% w/v) was determined by the FRAP assayaccording to Pulido, Bravo, and Saura-Calixto(2000) using gallic acid as a standard. Briefly,900 L of FRAP reagent prepared freshly andwarmed at 37 oC, was mixed with 90 L of dis-tilled water and 30 L of test sample or water for

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84 Bustos et al.

the reagent blank. The reading at the absorp-tion maximum (595nm) was taken using a spec-trophotometer (UV-visible Jasco V630, JascoCorporation, Japan) equipped with a thermosta-tized auto-cell-holder. Temperature was main-tained at 37 oC for up to 30 min.

2.6 Statistical analysis

Experiments were performed three times, withthree sample replicates each time. The resultswere analyzed by adjustment to a model, withfixed effects for a classification factor with sevenlevels (treatments and control). The model in-cluded a variance function to account for thepresence of an increasing variability pattern re-lated to medium levels of response variable. Theadjustment was carried out using an Infostat(Di Rienzo et al., 2012) by implementation ofthe gls function from the nlme library (J. Pin-heiro, Bates, DebRoy, & Sarkar, 2012) of R (RCore Team, 2012). The variance function appliedwas a function of the implementation of powervariance varPower() from the nlme library. Re-sults of the analysis were compared by the DGCmeans-comparison test (Di Rienzo, Guzman, &Casanoves, 2002), with a degree of significance ofp=0.05. Pearson correlation coefficients were cal-culated in order to find any relationship betweenantioxidant activity and polyphenol content inthe extracts of vegetables.

3 Results and discussion

3.1 Browning progress evaluation

Statistical analysis based on the proposed mixedmodel showed a significant time-dependentsource strength change pattern of the analyzedcolor variables of the mushrooms and avocadoslices for all applied treatments. In Figure 1 and2, L* values of cap mushrooms and avocado slicesin the absence and presence of Brassica (Fig. 1)or Allium (Fig. 2) extracts were plotted as afunction of storage time at 4 and -18 oC. Ta-ble 3.1 summarizes the total change of yellow-ness (b*) and redness (a*) extensively usedto characterize the chromatic variation.

Effect of storage at refrigeration andsubzero temperatures in untreatedcontrol slices

In the present study, the lightness (L*) of freshuntreated A. Bisporus cap (control) was 87.1,similar to the data reported by Czapski andSzudyga (2000). The chromatic attributes a*and b* were -0.6 and 10.0, respectively, indicat-ing redness lack and slight yellowness.Throughout the storage time at both analyzedtemperatures, cap mushrooms became darker(L* values of -6.0 (Fig. 1A) and 21.4 (Fig.1C) at 4 and -18oC, respectively) and turnedtheir initial whiteness into brownish color (b* in-creased) (Table 3.1). Refrigerated slices, in theabsence of extracts, presented a significant lu-minosity decrease after 24 h (Fig. 1 A) whileb* increased 4.9 units and redness (a*) did notchange during the whole storage.When the storage temperature was -18 oC, con-trol mushrooms showed a dramatic decrease inL* and an increase in b* value, with changeswhich were 56.7% and 26.5% higher than thoseobserved in samples stored at 4 oC, respectively(Fig. 1A and 1C). Contrary to refrigeratedslices, frozen mushrooms presented an increasein a* parameter, according to the high degree ofbrowning observed, indicating that mushroomswere more sensitive to freezing temperatures.The effect of temperature observed on luminos-ity changes was also reflected in the E value(25.3).Avocado slices without treatment presented ini-tially the following color coordinates: L*=78,b*=35.1, and a*=-5.3, in agreement with thedata obtained by Soliva et al. (2000) for avocadopuree. Browning of avocado slices was character-ized by a decrease in luminosity and yellowness.No changes in luminosity were observed duringthe first 48 h of storage at 4 oC (Fig. 1B), butb* decreased significantly (Table 3.1). On theother hand, browning of avocado was clearly in-hibited at freezing temperature, and only a fewbrowning points (Fig. 3) were observed in thesurface (L*=-4.4, Fig. 1D). After 48 h of stor-age a slight decrease in L* was observed, but nofurther changes were recorded.In control frozen avocado slices the browning in-hibition by low temperatures prevailed over the

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Anti-browning and antioxidant capacity of vegetables extracts 85

Figure 1: Lightness of mushrooms (A, C) and avocado (B, D) stored at 4 oC (A, B) or -18 oC (C, D). Slices untreated (open circle and dashed line) and treated with Brassica extracts: white cabbage (N),cauliflower (), and Brussels sprouts ().

Table 1: Yellowness (b* )and redness (a*) changes in mushroom and avocado slices over the durationof storage

Color Sample Control White Cauliflower Brussels Garlic Onion Scallionparameter cabbage sprouts

b* M4 4.9b 3.7a 3.3a 3.2a 3.2a 4.3b 3.6a

M-18 11.1e 3.1b 2.6b 5.7d 3.7c 5.0d 1.9a

A4 -3.3b 0.5d 1.1d -1.8c -1.6c -6.1a -5.6a

A-18 0.5d -1.8c -2.3c -1.9c -3.8b -5.5a -1.0c

a* M4 0.5a 0.9a 1.3a 2.9c 1.8b 1.9b 3.2c

M-18 7.6b 0.0a -0.4a -0.4a -0.3a -0.4a 0.6b

A4 2.8a 4.6b 5.3b 5.5b 2.9a 5.0b 2.3a

A-18 1.5b 1.7b -1.6a 1.4b 0.7b 1.5b 0.7b

M4: mushroom slices stored at 4 oC, M-18: mushroom slices stored at -18 oC, A4: avocado slices storedat 4 oC, A-18: avocado slices stored at -18 oC. The values with different superscripts in a line differsignificantly (p

86 Bustos et al.

deteriorative effect of ice formation, and the to-tal color change was very low in these samples(E=4.9).In vegetable tissues, polyphenols are locatedmainly in vacuoles and the PPO enzymes-systemis located in organelles (Ono et al., 2006). Thus,in normal conditions, polyphenols are not in con-tact with PPO and the reaction is not possi-ble. However, if due to ice formation the com-partments structure is broken, substrates andenzymes come into contact and the productionof quinones begins. The different response ofmushrooms and avocado to the browning reac-tion in frozen conditions can be thus attributedto the dissimilar relative sensitivity of both tis-sues towards the deleterious effects of ice crys-tals, in terms of the effectiveness of the counter-acting natural antioxidant mechanisms and thedegree of inhibition provided by decreasing tem-perature.

Effect of Brassica extracts

The antibrowning capacity of water extractsfrom white cabbage, cauliflower and Brusselssprouts was studied on cap mushrooms and avo-cado slices stored at 4 and -18 oC. Figure 1 showsthe capacity of Brassica extracts to delay the lu-minosity decrease.Refrigerated mushrooms slices, treated withBrassica extracts at 4 oC (Fig. 1A) presented, ingeneral, a higher L* value than control samplesand the b* coordinate value increased until 48h of storage with no significant further changes.The samples treated with Brussels sprout extractshowed a decrease in L* similar to that of un-treated slices (L*=-5.2). The samples withwhite cabbage extract maintained the luminos-ity of the original slices until 72 h, with a slightb* increase, while in cauliflower treated slices aslight decrease of L* (L*=-1.8, Fig. 1A) and anincrease of b* (Table 3.1) were observed. None ofthe samples showed a significant change of red-ness (a* coordinate).The antibrowning properties of crucifer extractswas even more evident on frozen mushrooms (-18oC), since all treated slices maintained luminos-ity during the analyzed period (Fig. 1C). Brus-sels sprout extracts generated an L* value 28.3%higher than the control, followed by white cab-

bage (26.5%) and cauliflower (25.3%) treatments,but no significant differences were observed be-tween treatments (Fig 1C). The frozen samplestreated with Brussels sprout extract presented aconsiderable increase of b*, while for the othercrucifer treatments the yellowness increase wasunaffected. In agreement with results obtainedat refrigeration storage, redness was unaffectedby dipping in Brassica extracts (Table 3.1). Sta-tistical analysis showed significant differences be-tween treated frozen mushrooms slices and theircontrols, with the L* value of white cabbage andcauliflower-treated samples the less affected bystorage time (p

Anti-browning and antioxidant capacity of vegetables extracts 87

Figure 2: Lightness of mushrooms (A, C) and avocado (B, D) stored at 4 oC (A, B) or -18 oC (C, D).Slices untreated (open circle and dashed line) and treated with Allium vegetables extracts: onion (N),garlic (), and scallion ().

(Fig. 2A) for those samples treated with onionextract. In addition, the b* parameter of thegarlic extract treated samples was significantlylower than the control during 4 oC storage (p

88 Bustos et al.

variables (data not shown). The change of a*value was small at both temperatures, indicatingslight modification of greenness (Table 3.1).

4 Limit of storage time andantibrowning index

In fresh mushrooms the L* value is best cor-related with the market value (Gormley, 1975),where L* values

Anti-browning and antioxidant capacity of vegetables extracts 89

Figure 3: Effect of Brassica and Allium extracts on browning of mushroom (M) and avocado (A) slicesstored at 4 oC and -18 oC, for 96 and 240 h respectively. Cauliflower (C), white cabbage (Wc), Brusselssprouts (Bs), garlic (A) and scallion (S) treatments are shown.

Negative and positive correlations were observedbetween avocado ABI at 4 oC and -18 oC for Al-lium (r=-0.99, p=0.0002) and Brassica (r=0.83,p=0.0422) extracts, respectively. That means,avocado browning was clearly retarded at frozentemperatures and Allium extracts were more ef-fective for refrigerated storage.

5 Polyphenols, anti-radical andreducing capacity of Allium andBrassica extracts

Anti-radical and reducing power of the studiedextracts from vegetables were determined inorder to correlate the antioxidant activity andthe observed antibrowning capacity.One of the most studied antioxidant compounds

are polyphenols. Phenolic compounds are ableto scavenge reactive oxygen species due to theirelectron donating properties. Their antioxidanteffectiveness depends on their stability in dif-ferent systems, as well as on the number andlocation of hydroxyl groups (Rice-Evans, Miller,& Paganga, 1997).Table 5 shows the radical scavenging rate,reducing capacity and polyphenol content ofBrassica and Allium extracts. The highestpolyphenol content and antioxidant capacitywas observed in scallion extract and Brusselssprout extract. Cabbage extract had boththe lowest polyphenol content and antioxidantcapacity, while cauliflower extract, which hadsimilar polyphenol content to white cabbage,showed double the radical scavenging rate andferric reducing power (Table 5).

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90 Bustos et al.

Table 2: Limit of storage time and anti-browning index for mushroom and avocado slices.

ControlBrassica Allium

White Cauliflower Brussels Garlic Onion Scallioncabbage sprouts

M4 72(ws)/>96(c) >96* >96* >96* >96* 96*Limit of M-18 48(ws)/216(c) >240* >240* >240* >240* >240* >240*storage time A4 48 96 48 24 72 72 >96(h) A-18 >240 >240 >240 >240 >240 >240 >240

AntibrowningM4 - 0.42 0.70 0.13 0.92 -0.03 0.78

index (ABI)M-18 -2.57 0.88 0.84 0.90 0.92 0.99 0.95A4 - 0.48 0.40 0.19 0.51 0.55 0.76

A-18 0.76 1.15 0.81 0.73 1.11 1.05 0.91

M4: mushroom slices stored at 4 oC, M-18: mushroom slices stored at -18 oC, A4: avocado slices storedat 4 oC, A-18: avocado slices stored at -18 oC. ws: acceptable for wholesale, c: acceptable for consumers.* indicates acceptable for wholesale and consumers.

In Brassica extracts, polyphenol contentcorrelated with reducing capacity (r=0.95,p=0.0035). In Allium extracts, positive corre-lations were found between polyphenol contentand anti-radical (r=0.97, p=0.0016) and re-ducing (r=0.98, p=0.0004) capacities. In bothBrassica and Allium extracts, polyphenol con-tent correlated with antioxidant activity (r=0.91, p=0.0115).Cauliflower and white cabbage extracts hadsimilar polyphenol contents, although slicestreated with these extracts showed differencesin browning rate delay. Negative correlationswere observed between polyphenol content ofBrassica extracts and the antibrowning indexfor refrigerated mushrooms (r=-0.88, p=0.0211)and avocado (r=-0.96, p=0.0029) slices. Theseresults indicate that phenolic compounds ofBrassica extracts contribute only in part totheir antibrowning properties. Furthermore,the antibrowning index of crucifer extracts foravocado stored at 4 oC (r=-1.00, p=

Anti-browning and antioxidant capacity of vegetables extracts 91

Table 3: Radical scavenging rate, reducing power and polyphenol content in Brassica and Allium extracts

Vegetable ABTS radical FRAP Total polyphenolsextract scavenging rate (%) (mg GA/100 ml (mg GA/100 ml

of extract) of extract)

White cabbage 33a 0.18a 7.4a

Brassica Cauliflower 63d 0.31c 7.1a

Brussels sprouts 66d 0.64e 17.6c

Garlic 38b 0.23b 15.4b

Allium Onion 46c 0.19a 14.7b

Scallion 82e 0.42d 25.8d

The values with different superscripts in a column differ significantly (p

92 Bustos et al.

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IJFS April 2014 Volume 3 pages 8292

IntroductionMaterials and MethodsMushroom and avocado samples preparationAllium and Brassica extracts preparationAntibrowning treatment and storageColor measurementAntioxidant propertiesStatistical analysis

Results and discussionBrowning progress evaluation

Limit of storage time and antibrowning indexPolyphenols, anti-radical and reducing capacity of Allium and Brassica extractsConclusionsReferences