J Sci Food Agric 1997, 73, 513È516

Mechanism of Browning in Fresh HighbushBlueberry Fruit corymbosum(Vaccinium L).Partial Purification and Characterisation ofBlueberry Polyphenol OxidaseFarid Kader, Bernard Rovel,* Michel Girardin and Maurice Metche

Laboratoire de Biochimie Applique� e, Ecole Nationale Supe� rieure 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 5 August 1996 ; accepted 9 October 1996)

Abstract : The most efficient method for polyphenol oxidase (PPO) extractionfrom Highbush blueberry fruits was the preparation of an acetone powder. Noactivity was detected after direct extraction with phosphate bu†er (pH 6É5) anddetergents such as Triton X-100. PPO has been puriÐed 19-fold, by ultraÐltra-tion, ammonium sulphate precipitation and hydrophobic chromatography.Native-PAGE of the puriÐed fraction revealed the presence of two isoforms.PPO has an observed optimum pH at 4É0, followed by a shoulder at pH 5É0.Ca†eic acid is the best substrate (100%), followed by chlorogenic acid (60%) andpyrocatechol (32É5%). No activity was detected towards catechin, protocatechuicacid, resorcinol and monophenols.

Key words : V accinium corymbosum, polyphenol oxidase, extraction, puriÐcation,characterisation.

INTRODUCTION

Polyphenol oxidase (EC 1.10.3.1) is largely responsiblefor enzymatic browning in fruits and vegetables. Theuse of chromatographic methods particularly hydro-phobic and affinity chromatographies (Janovitz-Klappet al 1989 ; Richard-Forget et al 1994) have often beenapplied successfully to the puriÐcation of PPO.

The occurrence of PPO in blueberry fruits has beenreported by Wagenknecht et al (1960). However, noinformation has been published on the puriÐcation andthe physicochemical properties of this enzyme.

MATERIALS AND METHODS

Fruit sampling and storage

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

* To whom correspondence should be addressed.

were cleaned, packaged in polyethylene containers (4¡C)and transported to the laboratory and stocked at 4¡C.

Enzyme extraction

A 150 g sample of fruit was homogenised with 200 mlof ice-cold acetone/water/Triton X-100 (80 : 19 : 1, v/v)([25¡C) in a Braun Mx32 blender. The resulting slurrywas left at [25¡C during 1 h, then Ðltered through aporous glass Ðlter (100È160 km). The residue waswashed three times with 250 ml of acetone ([25¡C).The resulting acetone powder was dried (2 h) at roomtemperature.

A 20 g sample of the acetone powder was suspendedin 300 ml of 0É1 M phosphate bu†er pH 7É4 containing0É01 M cystein and stirred for 2 h (4¡C). The suspensionwas centrifuged (12 100] g, 20 min, 4¡C). The pro-cedure was repeated two more times. The supernatantswere Ðltered through a glass Ðlter and concentrated byultraÐltration under nitrogen through a cellulose tri-acetate membrane (cut o† 20 kDa, Sartorius). Theretentate (E1) was used for the puriÐcation.

513J Sci Food Agric 0022-5142/97/$09.00 1997 SCI. Printed in Great Britain(

514 F Kader et al

Enzyme puriÐcation

Ammonium sulphate was added to E1 (30% molarsaturation). After 2 h at 0¡C, the solution was centri-fuged (41 400] g, 40 min, 0¡C). The pellet was dis-carded and ammonium sulphate was added to thesupernatant (to 80% molar saturation). After 2 h at 0¡Cand centrifugation, the resulting pellet was homo-geneised in 10 ml of 50 mM phosphate bu†er pH \ 6É5,containing 1 M and 1 M KCl and dialysed(NH4)2SO4overnight against the same bu†er.

The dialysed solution was applied to a Phenyl Sep-harose CL-4B column (20] 1É1 cm) previously equili-brated with the same bu†er (Fig 1). PPO activity wasassayed with chlorogenic acid as substrate in McIlvainebu†er, pH \ 4É0. The active PPO fractions were pooledand concentrated by ultraÐltration. The retentate waslabelled E2.

PPO activity and protein determination

The assays were performed using 850 kl of McIlvainebu†er pH 3É5, 100 kl of 1 mM chlorogenic acid in thesame bu†er and 50 kl of the diluted enzyme extract(5É3 kg protein). The increase in absorbance at 400 nmwas measured (25¡C). One unit of enzyme activity wasdeÐned as the amount of enzyme which caused anabsorbance increase of 0É001 unit min~1 (initial rate).

The optimum pH of the PPO activity was determinedas described above with the partially puriÐed enzyme(E2) in McIlvaine bu†er ranging from 3É0 to 6É5.

Protein concentration was determined usingbicinchoninic acid (Smith et al 1985). Bovine serumalbumin was used as a standard.

Fig 1. Hydrophobic chromatography of Highbush blueberryPPO on phenyl Sepharose CL-4B absorbance(È)È)280 nm; PPO activity (U ml~1) and (È È È) elution(È+È)proÐle, 50 mM phosphate bu†er pH \ 6É5, containing

and KCl in stepwise decreasing concentrations.(NH4)2SO4The arrow indicated the elution with ethylene glycol (50%, byvol). The Ñow rate was 1 ml min~1, and 3É0 ml fraction were

collected.

Electrophoresis

Native PAGE were performed with Phastsystem(Pharmacia) using Phastgel (8È25% gradient), pH 8É6 at250 V, 10 mA (3 W) at 15¡C for 30 min. Proteins werestained with Coomassie blue R-250. PPO activity wasdetected by incubating the gel in a reaction mixturecontaining 5 mM ca†eic acid in McIlvaine bu†er pH 4É0.

RESULTS AND DISCUSSION

Extraction and puriÐcation of blueberry PPO

The main problem in the isolation of PPO from planttissues is the occurrence of phenols and their oxidisedproducts which react with PPO resulting in its inac-tivation (Loomis 1974). The use of ascorbic acid(Janovitz-Klapp et al 1989), PVP, PVPP, hydrophobicanionic exchange resins and polyethylene glycol in theextracting media, increases the PPO activity (Vamos-Vigyazo 1981 ; Nicolas et al 1994). PPO has beendescribed as membrane-bound protein and can requirefor solubilisation the use of detergents such as TitronX-100 (Janovitz-Klapp et al 1989), Titron X-114(Sanchez-Ferrer et al 1989), Tween 80 (Mowlah et al1982). We have used procedures involving these con-siderations but a low PPO activity was detected evenafter precipitation with ammonium sulphate. In fact, thepreparation of an acetone powder was found to be thebetter procedure to extract the blueberry PPO.

Hydrophobic chromatography puriÐcation

The elution proÐle of blueberry PPO from phenyl sep-harose CL4B column is presented in the Fig. 1. All thePPO activity was eluted in a single peak with a lowstrength elution bu†er. The elution patterns for blue-berry PPO were reproducible. Blueberry PPO was puri-Ðed 19-fold on the increase of speciÐc activity, with atotal yield close to 50%.

Electrophoresis

The e†ectiveness of the puriÐcation procedure describedhere was assessed by native PAGE (Fig 2). The stainingof gel for proteins using Coomassie Blue R-250, showsthree bands (I, II and III) (Fig 2A). When the proteinbands were stained with ca†eic acid (reagent of PPOactivity) only two bands were revealed and(PPO1

(Fig 2B). Therefore, blueberry PPO presents twoPPO2)isoenzymes, one band II) shows a slight(PPO2 ,response to the test for proteins.

Mechanism of browning in fresh Highbush blueberry fruit 515

Fig 2. Native PAGE of the puriÐed enzyme: (A) the gel wasstained with Coomassie brillant blue ; and (B) the gel wasstained for PPO activity with 5 mM ca†eic acid in McIlvaine

bu†er (pH 4É0) for 20 min at room temperature (22¡C).

The enzyme eluted by hydrophobic conditions wasnot free from contaminating proteins (Fig 2A, band III)which explained the puriÐcation factor obtained.

Substrate speciÐcity and optimum pH of PPO

PPO activities using di†erent substrates are shown in

TABLE 1Substrate speciÐcity of Highbush blueberry polyphenol

oxidase

Substrate Concentration Relative(mM) activity

comparedwith that of

caffeicacid (%)

Caffeic acid 5 100Chlorogenic acid 5 60Pyrocatechol 5 32É5(])-Catechin 5 0Protocatechuic acid 5 0Resorcinol 5 0p-Coumaric acid 10 0p-Cresol 10 0Tyrosine 10 0

Table 1. Although ca†eic acid has the highest value,these results do not indicate that ca†eic acid is the bestsubstrate for this enzyme, for which purpose the ratio

could be a better parameter to measure theVm/Kmenzyme efficiency at low substrate concentration(Baritaux et al 1991). Moreover, we have shown thatchlorogenic acid is the main hydroxycinnamic deriv-atives in Highbush blueberry and no ca†eic acid hasbeen detected in fruits (Kader et al 1996).

The optimum activity of the enzyme was found to beat pH 4É0 with a shoulder at pH 5É0, about 80% of themaximum activity, and a rapid decrease in activitybetween pH 5É0 and 5É5. This behaviour is explained bythe presence of the two isoforms demonstrated bynative PAGE (Fig 2). This enzyme is very active at lowpH which is similar to the fruits pH (3É0È3É2) (Kader etal 1994).

ACKNOWLEDGEMENT

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

REFERENCES

Baritaux O, Amiot M J, Richard H, Nicolas J J 1991 Enzy-matic browning of basil (Ocimum basilicum L.) studies onphenolic compounds and polyphenol oxidase. Sci. Aliments11 49È62.

Janovitz-Klapp A, Richard F C, Nicolas J J 1989 Poly-phenoloxidase from apple, partial puriÐcation and someproperties. Phytochemistry 28 2903È2907.

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

Kader F, Rovel B, Girardin M, Metche, M 1996 Fractiona-tion and identiÐcation of the phenolic compounds of High-bush blueberry (V accinium corymbosum L). Food Chem 5535È40.

Loomis W D 1974 Overcoming problems of phenolics andquinones in the isolation of plant enzymes and organelles.In : Methods in Enzymology (Vol 31 Part A: Biomembranes),ed Colowick S P & Kaplan N A. Academic Press, NewYork, USA, pp 528È544.

Mowlah G, Takano K, Kamoi I, Obara T 1982 Character-ization of banana polyphenoloxidase. Fractions withrespect to electrophoretic and gel Ðltration behavior.L ebensm W iss T echnol 15 207È210.

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.

Richard-Forget F C, Goupy P M, Nicolas J J 1994 Newapproaches for separating and purifying apple polyphenoloxidase isoenzymes : hydrophobic, metal chelate and affinitychromatography. J Chromatogr 667 141È153.

516 F Kader et al

Sanchez-Ferrer A, Villalba J, Garcia-Carmona F 1989 TritonX-114, as a tool for purifying spinach polyphenoloxidase.Phytochemistry 28 1321È1325.

Smith P K, Krohn R I, Hermanson G T, Mallia A K, GartnerF H, Provenzano M D, Fujimoto E K, Goeke N M, OlsonB J, Klenk D C 1985 Measurement of protein usingbicinchoninic acid. Anal Biochem 150 76È85.

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

Wagenknecht A C, Scheiner D M, Van Buren J P 1960Anthocyanase activity and its possible relation to scald insour cherries. Food T echnol 14 47È49.