J Sci Food Agric 1997, 74, 31È34

Mechanism of Browning in Fresh HighbushBlueberry Fruit corymbosum(Vaccinium L).Role of Blueberry Polyphenol Oxidase,Chlorogenic Acid and AnthocyaninsFarid Kader, Bernard Rovel,* Michel Girardin and Maurice Metche

Laboratoire de Biochimie Applique� e, Ecole Nationale Superieure dÏAgronomie et des IndustriesAlimentaires, 2 Avenue de la Foreü t de Haye, BP 172-54505 Vandoeuvre-les-Nancy Cedex, France

(Received 2 January 1996 ; revised version received 24 April 1996 ; accepted 9 October 1996)

Abstract : Browning reactions which occurred after crushing fresh blueberries(V accinium corymbosum L) were followed by determination of chlorogenic acidcolour density, polymer colour and the level of polymers. A polyphenol oxidase(PPO) activity plays a dominant role in enzymatic browning. Peroxidase (POD)does not contribute to brown colour formation. The loss of 29% of the colour ina model system containing PPO and blueberry anthocyanins indicated that PPOcould act directly on these pigments. However, the rate of anthocyanin degrada-tion was stimulated by the addition of chlorogenic acid.

Key words : V accinium corymbosum, anthocyanin, degradation, chlorogenic acid,polyphenol oxidase (PPO), browning.

INTRODUCTION

Many studies have been devoted to the enzymaticbrowning occurring in fruits and vegetables during postharvest handling, processing and storage. Both PPOand POD have been implicated in enzymatic browningof plant tissues (Vamos-Vigyazo 1981). But the oxida-tive browning of fruits is due mainly to polyphenoloxidase (EC 1.10.3.1). PPO catalyses the oxidation ofendogenous phenols into quinones that subsequentlypolymerised into brown pigments (Lee 1991). In thepresence of anthocyanins, the quinones can oxidisethese pigments to brown polymers (Mathew and Parpia1971).

Peroxidases (EC 1.11.1.7) are iron-containingenzymes capable of oxidising phenolics to quinones inthe presence of hydrogen peroxide. Peroxidases arefound in most plant tissues but the low level of hydro-gen peroxide is the factor which limited the peroxidaseactivity (Nicolas et al 1994).

The aim of this study was to determine the contribu-tion of the PPO, chlorogenic acid and anthocyanins tobrowning.

* To whom correspondence should be addressed.

MATERIALS AND METHODS

Fruit sampling and treatment prior to browning analysis

Ripe blueberries of the “CovilleÏ variety “Bluets deswere used for our experiments.VosgesÏ}

250 ml of cold McIlvaine bu†er pH 3É5 was added to500 g of fresh blueberries and homogenised in a blender(Braun Mx32) at high speed for 2 min at 4¡C. Thehomogenate was placed in a beaker and air wasbubbled through the mash in order to speed up thereaction rate. At time, 0, 10, 20, 30 and 60 min twosamples (1 and 2) were taken and treated as describedbelow. For each time, sample 1 was used for the deter-mination of chlorogenic acid and sample 2 for colourmeasurements.

Extraction and quantiÐcation of chlorogenic acid byHPLC

Seventy grams of sample 1 were mixed with 80 ml ofacetone/water (80 : 20, by vol) ([20¡C). The resultingmixture was centrifuged for 20 min at 12 100 ] g at0¡C. The pellet was discarded and the supernatant

31J Sci Food Agric 0022-5142/97/$17.50 1997 SCI. Printed in Great Britain(

32 F Kader et al

Fig 1. Change during browning of chlorogenic acid content expressed in mg kg~1 colour density (È2È), polymeric(ÈLÈ);colour percent of polymer(È+È); (È|È).

(acetoneÈwater) was extracted three times with diethyloxide (2 : 1, by vol). The aqueous phase (A) was storedat 4¡C. The acetoneÈdiethyl oxide phase which con-tained carotenoids, chlorophylls and phenolic com-pounds, was collected and evaporated under vacuum.The residue was dissolved in 25 ml of distilled waterand the resulting solution was extracted twice with pet-roleum ether 60È85¡C (2 : 1, by vol) in order to elimi-nate chlorophylls and carotenoids. The aqueous phaseswere combined and pooled to the phase A and waslabelled A1.

A1 (100 ml) was acidiÐed to pH 1É5 with 2 M HCl.The solution was extracted four times with ethyl acetate(1 : 1, by vol). The ethyl acetate phases containing chlo-rogenic acid were combined and evaporated undervacuum. The residue obtained was dissolved in meth-anol (4É0 ml).

A 50 kl sample of the methanolic extract wereanalysed by HPLC (Merck L-6200 pump and photo-diode array detector L-3000) with a Lichrosorb 100RP-18 column (250] 4 mm id, 5 km) using a mobilephase consisting of water/acetic acid (95 : 5, by vol)(solvent A) and methanol (solvent B). Elution was per-formed at a Ñow rate of 0É6 ml min~1. The conditionswere 20 to 30% B in A in 15 min followed by 30 to 50%B in A in 30 min. Detection was achieved at 325 nm.Each sample was run in triplicate.

Determination of the colour parameters

Fifteen grams of sample 2 were mixed with 12 ml ofacidiÐed methanol (pH 2É0). The resulting mixture wascentrifuged for 20 min at 12 100] g at 4¡C. The pellet

was discarded and the supernatant was used for thedeterminations.

Colour density (CD) and polymeric colour (PC) weredeterminated by the procedure described by Wrolstad(1976).

PPO activity measurement and protein determination

The enzymatic extract has been prepared as describedby Kader et al (1997).

InÑuence of chlorogenic acid on the rate of anthocyaninsdegradation

Extraction of anthocyaninsBlueberry anthocyanins were extracted as described byKader et al (1996). Chlorogenic acid was eliminated byfour successive extractions with ethyl acetate (1 : 1, byvol). The aqueous phase (anthocyanin extract) was usedas anthocyanin source. Anthocyanin concentration wasdeterminated using the pH di†erential method (pH 4É5and 1É0) (Wrolstad 1976) and was expressed asmalvidin-3-glucoside.

Assay for anthocyanin degradationThe reaction mixture contained 800 kl of McIlvainebu†er pH 3É5 ; 100 kl of 1 mM chlorogenic acid in thesame bu†er, 50 kl of anthocyanin extract (0É6 mg ml~1)and 50 kl of the enzyme preparation (16 kg of protein).The anthocyanin degradation was initiated by PPOaddition and followed at 520 nm.

Mechanism of browning in fresh Highbush blueberry fruit 33

RESULTS AND DISCUSSION

Role of PPO and chlorogenic acid on browningphenomenon

When fresh blueberries are homogenised, the colour ofthe resulting mash turns intensely brown, within30 min. The brown colour was more intense at thesurface which indicates that this phenomenon is oxygendependent.

The level of chlorogenic acid decreased drasticallyduring the Ðrst 10 min, reached a minimum at about30 min and then maintained a stable low level. In20 min 90% of the chlorogenic acid has disappeared(Fig 1). This decrease can be associated with an enzy-matic phenomenon. The addition of catalase (693 unitsg~1 of slurry) at the beginning of the experiment did notmodify the kinetics of chlorogenic acid degradationshowing that peroxidase does not play a role and thatonly PPO should be involved. Chlorogenic acid was themajor cinnamic derivative found in blueberries (Kaderet al 1996).

Figure 1 shows that polymeric colour (PC) increasedrapidly during the Ðrst 10 min, reached a maximum atabout 30 min and decreased gradually until the end ofthe treatment because the polymers became insoluble(Nicolas et al 1993). The decrease of colour density (CD)paralleled the decrease of chlorogenic acid showing thatanthocyanin degradation is dependent on the oxidationof chlorogenic acid. In addition, the optimum activity ofPPO was found to be at pH 4É0 (Kader et al 1997)which is close to the pH of blueberry juice pH 3É1(Kader et al 1994). Ca†eic acid and chlorogenic acid arethe best substrates for blueberry PPO (Kader et al1997).

Fig 2. Variation in the absorbance of blueberry anthocyaninsat 520 nm during their degradation by blueberry PPO at pH3É5 in presence (È2È), absence of chlorogenic acid(È+È)and after thermal denaturation of the PPO in presence of

chlorogenic acid (È>È).

Degradation of blueberry anthocyanins by the crudeenzyme in the presence and absence of chlorogenic acid

In the absence of chlorogenic acid the rate of antho-cyanin discoloration (pH 3É5) was low (29%) after20 min of reaction (Fig 2). This activity could not beattributed to a contamination with phenolic compoundsof the anthocyanin extract which was prepared using aprocedure designed to eliminate phenolic compounds.The decrease of absorbance at 520 nm is associated to adecolorising system since no brown products have beendetected. Most of the red colour remained in the reac-tion mixture. PPO could act directly on anthocyanins,however they remain fairly poor substrates (Mathewand Parpia 1971).

The presence of chlorogenic acid in the reactionmixture drastically increased the rate of anthocyanindegradation (Fig 2). However, no degradation occurredwhen enzymatic extract was inactivated by heating andchlorogenic acid was also not oxidised. The quinoneformed from chlorogenic acid stimulated the antho-cyanin degradation (Raynal and Moutounet 1989 ;Cheynier et al 1994). Anthocyanins with o-diphenolic Bring were oxidised via the enzymatically generated o-quinone by coupled oxidation mechanism, whereasnon-o-diphenolic anthocyanins formed adducts with thesame o-quinone (Sarni et al 1995).

ACKNOWLEDGEMENT

Financial support was provided by the Association forthe Valorisation of the Economic Mountain (AVEM,Cleurie 88120 Vosges, France).

REFERENCES

Cheynier V, Souquet J M, Kontek A, Moutounet M 1994Anthocyanin degradation in oxidising grape musts. J SciFood Agric 66 283È288.

Kader F, Rovel B, Girardin M, Metche M 1994 Compositionof the fruit of Highbush blueberry (V accinium corymbosum,L.) grown in the East of France (Vosges). Sci Aliments 14281È290.

Kader F, Rovel B, Girardin M, Metche M 1996 Fractionationand identiÐcation of the phenolic compounds of Highbushblueberries (V accinium corymbosum, L.). Food Chem 55 35È40.

Kader F, Rovel B, Girardin M, Metche M 1997 Mechanism ofbrowning in fresh Highbush blueberry fruit (V acciniumcorymbosum, L.). Partial puriÐcation and characterization ofblueberry polyphenol oxidase. J Sci Food Agric 73 513È516.

34 F Kader et al

Lee C Y 1991 Browning reactionÈEnzymatic. In : T he Ency-clopedia of Food Science and T echnology, ed Hui Y N.Wiley, New York, USA, pp 223È230.

Mathew A G, Parpia H A B 1971 Food browning as a poly-phenol reaction. Adv Food Res 19 75È145.

Nicolas J J, Cheynier V, Fleuriet A, Rouet-Mayer M 1993 APolyphenols and enzymatic browning. In : PolyphenolicPhenomena, ed Scalbert A. INRA Editions, Paris, France,pp 165È175.

Nicolas J J, Richard-Forget F C, Goupy P M, Amiot M J,Aubert S Y 1994 Enzymatic browning reactions in appleand apple products. CRC Crit Rev Food Sci Nutr 34 109È157.

Raynal J, Moutounet M 1989 Intervention of phenolic com-pounds in plum technology. 2. Mechanisms of anthocyanindegradation. J Agric Food Chem 37 1051È1053.

Sarni P, Fulcrand H, Souillol V, Souquet J M, Cheynier V1995 Mechanisms of anthocyanin degradation in grapemust-like model solutions. J Sci Food Agric 69 385È391.

Vamos-Vigyazo L 1981 Polyphenol oxidase and peroxidase infruits and vegetables. CRC Crit Rev Food Sci Nutr 15 49È127.

Wrolstad R E 1976 Color and Pigment Analyses in Fruit Pro-ducts (Station Bulletin Vol 6). Oregon State University,Agricultural Experiment Station, OR, USA, pp 1È17.