Effects of pectolytic enzymes on selected phenolic compounds in strawberry and raspberry juices

Food Research International, Vol. 30, No. 10, pp. 811±817, 1997 # 1998 Canadian Institute of Food Science and Technology Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0963-9969(98)00050-7 0963-9969/98/$19.00+0.00

E€ects of pectolytic enzymes on selected phenolic compounds in strawberry and raspberry juices A. Versari,a* S. Biesenbruch,b D. Barbanti,a P. J. Farnellc & S. Galassid a

Dipartimento di Biotecnologie Agrarie ed Ambientali, UniversitaÁ di Ancona, Via Brecce Bianche, 60131, Ancona, Italy b Leatherhead Food Research Association, Randalls Road, Leatherhead, Surrey, KT22 7RY, UK c Laboratory of the Government Chemist, Queens Road, Teddington, Middlesex, TW11 0LY, UK d Istituto di Industrie Agrarie, UniversitaÁ di Bologna, Via San Giacomo 7, 40126, Bologna, Italy To evaluate the e€ect of commercial pectolytic enzymes on the content of phenolic compounds (anthocyanins, ¯avonols and ellagic acids), strawberry and raspberry juices under enzymatic pectinase treatment were analyzed using High Performance Liquid Chromatography (HPLC) with Spectral Array Detection (SAD). Each fruit had a distinctive phenolic pattern which enabled identi®cation and characterization. The stability of phenolic compounds during processing was monitored at 2, 4 and 6 h. The use of commercial pectinases modi®ed the composition of phenolic compounds in relation of the fruits and the time considered. At 6 h, a loss of anthocyanins (ÿ20%) present in raspberry juice was observed when Pectinex1 BE 3-L, Rohapect1 B1L, Rohament1 MAX and Pectinex2 3XL were used. This result con®rmed the hypothesis of a b-glycosidase activity present into commercial pectinase. On the other hand, in strawberry treated samples the ellagic acids concentration always increased (up to 19 mg lÿ1 with Pectinase2 LM, at 6 h) while ¯avonols content decreased (ÿ35% with Pectinase2 LM, at 6 h). Rohapect1 MB was the only enzyme able to considerably increase the content of total quercetin derivatives in raspberry (36 mg lÿ1). # 1998 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved Keywords: HPLC/SAD, pectolytic enzymes, phenolic compounds, quality control, red fruits

forms (Henning, 1981; Mazza and Miniati, 1993) which di€er in physico-chemical characteristics in plant as well as in animal systems. Natural occurring ¯avonols and anthocyanins can be used as markers to evaluate the authenticity of fruit products (Siewek et al., 1984; TomaÂs-Lorente et al., 1992; Wrolstad et al., 1995) even if the composition of foods and beverages is a€ected by the process and storage conditions (Macheix et al., 1990). The pectolytic enzymes used for processing the red fruits improve the yield of extraction (Schmitt, 1988), but under certain conditions some enzyme preparations may hydrolyze the anthocyanins in raspberry and cranberry juices (Wrolstad et al., 1994) and in cherry nectars (Blom and Juul, 1982). The anthocyanin, through hydrolytic removal of glycosidase substituents, may be converted into monoglycosides and/or aglycones (Jiang et al., 1990). The hydrolysis of anthocyanins a€ects the

INTRODUCTION Plant phenolics have di€erent chemical, biological and pharmacological properties (Herrmann, 1976; KuÈhnau, 1976; Hollman et al., 1996). In particular, ¯avonoids such as anthocyanins, quercetin, kaempferol and ellagic acid may inhibit various stages in the cancer process (Maas et al., 1991a; Hertog et al., 1992a; Castonguay et al., 1994; Saija, 1994). Anthocyanins, ¯avonols (quercetin and kampferol derivatives) and ellagic acid derivatives found in strawberry and raspberry fruits exist under several glycosylation, methylation, methoxylation and acylation *To whom correspondence should be addressed at Biotechnology Research Institute, National Research Council, 6100 avenue Royalmount, Montreal (QC), H4P 2R2, Canada. Fax: +1-514-496-6144; e-mail: [email protected] 811

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colour quality of products; in fact, the aglycones are unstable and are easily converted to polymeric brown or colorless compounds (Francis, 1989). In addition, the variation of the original anthocyanins composition could lead to problems in establishing the authenticity of red fruit juices. On the other hand, the use of pectolytic enzymes in raspberry fruit processing lowers the ¯avonol content while the ellagic acid derivatives amount varies on the base of the extraction techniques (Rommel and Wrolstad, 1993a,b). High-performance liquid chromatography (HPLC) is a suitable techniques for the analysis of phenolic compounds (Dhingra and Davis, 1988; Goi€on et al., 1991; Hertog et al., 1992b), but their identi®cation is often complicated by the lack of speci®c standards. However, the use of Spectral Array Detection (SAD) provides an useful aid in the HPLC analyses of phenolic compounds (Law and Das, 1987; Hong and Wrolstad, 1990; Bartolome et al., 1993). Moreover, the glycosidase activities of enzyme preparations used in fruit processing can be measured by anthocyanin HPLC analysis (Wightman and Wrolstad, 1995). Our objective was to investigate the e€ect of seven commercial pectolytic enzymes on the composition of anthocyanins, ¯avonols and ellagic acid derivatives of strawberry and red raspberry juices. This work was undertaken, taking into account that there is great interest about the natural phenols of foods as potential bene®cial human health, and that the phenolic composition is useful for determining the authenticity of strawberry and red raspberry juices. MATERIALS AND METHODS Sample preparation Strawberry and red raspberry fruits were purchased from the market. The juice extraction process was carried out in a laboratory as shown in the ¯ow diagram (Fig. 1). The whole fresh strawberry and raspberry fruits (2 kg each) were processed (domestic juice extractor, Mod. 864 Moulinex, France) and homogenized (domestic blender, Mod. 1738 Braun, Germany) into mash. The mash was pasteurized (I), at 85 C for 2 min, (microwave oven), then cooled 20 min into a waterbath and homogenized 30 sec again. Samples (50 g) of strawberry and red raspberry mash were poured in a 250 ml glass conical ¯asks, then added with pectolytic enzyme solutions (treated samples), hence mixed thoroughly and ®nally held for up to 6 h at 45 C. Control samples (pasteurized untreated mash) for both fruits were also run at 45 C without enzyme addition. At 2, 4 and 6 h, respectively, a control sample and one sample for each enzyme used were pasteurized (II), at 85 C for 2 min, and then cooled into a waterbath. The juices were centrifuged at 27216g for 15 min (Ultracentrifuge MSE

Fig. 1. Flow diagram for juice processing with and without pectolytic enzymes.

E€ects of pectolytic enzymes on selected phenolic compounds Europa 24 M, Kontron Instr, UK) and the supernatant was ®ltered through a 0.22 mm cellulose±acetate membrane (F-0139, Sigma Chemical Co, Dorset, UK) before direct injection into the HPLC system. Pectolytic enzymes The following commercial pectolytic enzymes were evaluated for processing the soft fruits: Pectinex1 BE 3L (Novo Nordisk Ferment Ltd, Dittingen, Switzerland), Rohapect1 MB, Rohapect1 B1L, Rohament1 MAX (RoÈhm, Darmstad, Germany), Grindamyl Pectinase2 LM, Grindamyl Pectinase2 LX, Pectinex2 3XL (Danisco, Brabrand, Denmark). Enzymes were used at the average dosage recommended by the manufacturers, resulting in a range of 3±30 g 100 kgÿ1 mash. HPLC analyses Strawberry and raspberry juices were analyzed using a Dionex DX 500 chromatographic system (Dionex Corporation, Sunnyvale, CA, USA) equipped with a pump (GP 40 LC), an autosampler with `push loop' automatic injection system (AS 3500), a spectral array detector (UVIS 206), a temperature control module (AS 3500) and a Peaknet software acquisition data station. Anthocyanins were analyzed by gradient elution as previously described (Versari et al., 1997a) using a Nucleosil 120 C18-3ODS (Hichrom Ltd, Berkshire, UK) reversed phase column (2503.9 mm, 5 mm). The visible spectra of anthocyanins were performed at 370± 600 nm; two wavelengths, 505 and 520 nm, were selected for monitoring pelargonidin and cyanidin derivatives, respectively. Flavonols and ellagic acid derivatives were analyzed as previously described (Versari et al., 1997b) using a Hicarbosphere 3ODS (Hichrom Ltd) reversed phase column (1504.6 mm, 5 mm). The UV spectra of these compounds were performed at 200±370 nm; two wavelengths, 280 and 355 nm, were selected for monitoring both class of compounds. Standards Cyanidin-3-glucoside, pelargonidin-3-glucoside (Extrasynthese, Genay, France), quercetin-3-glucoside, quercetin-3-rutinoside, quercetin-3-rhamnoside, kaempferol (Sigma Chemical Co) and ellagic acid (Lancaster Chemical Ltd, Lancs, UK) were used as standards for peaks identi®cation and quanti®cation. Peaks identi®cation was based on both the retention time (Rt) and the UV-visible spectra recorded during the HPLC analyses; these ®ndings were also compared to the literature values (Henning, 1981; Rommel and Wrolstad, 1993a,b; Boyles and Wrolstad, 1993; Bakker et al., 1994; Versari et al., 1997a). The calibration curves of cyanidin-3-glucoside and pelargonidin-3-glucoside were

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used for the quanti®cation of anthocyanins. Due to the low solubility and the degradation demonstrated by ellagic acid standard (Rommel and Wrolstad, 1993b) the quercetin-3-rutinoside was used for the quanti®cation of all the other phenolic compounds. Statistical analysis Experimental design for the enzyme assay was randomized with enzymes as treatments. Data sets were analyzed by a linear ®tting model using Statistica 5.0 software (StatSoft, OK, USA). The repeatability of the experiment was tested by triplicate treatment of both strawberry and raspberry mashes with one selected enzyme (Pectinex1 BE 3-L). The controls for both fruits were also replicated three times. While the analytical variation limits the conclusion that can be drawn for treatments e€ects, the quantitative estimates are still useful for evaluating trends and providing a perspective of the amounts of ¯avonoids in strawberry and raspberry juices. RESULTS AND DISCUSSION HPLC analyses HPLC pro®le of strawberry juice [Fig. 2(A)] showed three anthocyanins which were identi®ed, in order of elution, as: pelargonidin-3-glucoside (Pg-3-glu), cyanidin-3-glucoside (Cy-3-glu) and pelargonidin-3-rutinoside (Pg-3-rut). Moreover, three quercetin derivatives (peaks Nos 1, 4 and 5, respectively), ellagic acid (peak No. 3) and one ellagic acid derivative (peaks No. 2) were also identi®ed in strawberry juices [Fig. 2(B)]. According to Henning (1981) we identi®ed the main quercetin derivatives [Fig. 2(B)] as quercetin-3-glucuronide (peak No. 4), and peak No. 5 as quercetin-3-glucoside. On the other hand, raspberry juice [Fig. 3(A)] showed two main pigments, identi®ed as: cyanidin-3-sophoroside (Cy-3-sopho) and cyanidin glucoside. In addition, four main quercetin derivatives (peaks Nos. 1, 2, 6 and 7, respectively), ellagic acid (peak No. 5) and two ellagic acid derivatives (peaks Nos 3 and 4, respectively) were detected in raspberry juices [Fig. 3(B)]. According to Rommel and Wrolstad (1993a) we identify the main quercetin derivatives [Fig. 3(B)] as quercetin-3-glucuronide (peak No. 6), and peak No. 7 as quercetin-3-glucoside. Even if additional peaks were present in the HPLC pro®le of both strawberry and raspberry juices [Figs 2(B) and 3(B)] the coelution of peaks limited the identi®cation of these compounds [e.g. peak No. 6, Fig. 2(B)]. Moreover, taking into account that the HPLC limit of detection (signal/noise=3) for the quercetin-3-rutinoside was 0.4 mg lÿ1, the quanti®cation of minor peaks was not possible.

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Fig. 2. HPLC pro®les of strawberry. (A) Anthocyanins, peaks identi®cation: (1) Pg-3-glu; (2) Cy-3-glu and (3) Pg-3-rut. (B) Flavonols and ellagic acid derivatives, peaks identi®cation: (1) quercetin derivative; (2) ellagic acid; (3) ellagic acid derivatives; (4) quercetin-3-glucuronide; (5) quercetin-3-glucoside; (6) unknown (coelution). (Abs) absorbance.

The composition of ¯avonols and ellagic acid were not further characterized in both red fruits, but they may be ¯avonols and ellagic acid derivatives with varying methylation, glycosylation or methoxylation pattern (Maas et al., 1991a; Rommel and Wrolstad, 1993a,b). The anthocyanins pattern of both strawberry and raspberry juices [(Fig. 2(A) and (B)] provided an e€ective model to measure b-glucosidase activity present in commercial pectolytic enzymes. Pectolytic enzymes processing An enzyme/juice reaction time of 2 h was used to simulate commercial processing practices. Treatment extended for 4 and 6 h were also planned in order to test the stability of ¯avonoids and to measure glycosidase activity. For both fruits, the content (mg lÿ1) of each individual class of compounds (anthocyanins, ¯avonols, ellagic acids) at 2, 4 and 6 h of reaction time, was compared to the control. Strawberry The total content of anthocyanin detected in strawberry juices (Table 1) was similar for all the enzymes tested. At 2 h no di€erences between control and treated samples were detected. The values (range) of the individual pigments were as follow: Cy-3-glu 19±20 mg lÿ1 (Pectinex1 3XL and control, respectively), Pg-3-glu 267±273 mg lÿ1 (control and Rohament1 MB) and Pg-3-rut 28±30 mg lÿ1 (Rohament1 MAX and control). At 6 h the di€erences between control and enzyme treatments occurred

Fig. 3. HPLC pro®les of raspberry. (A) Anthocyanins, peaks identi®cation: (1) Cy-3-sopho; (2) Cy-3-glu. (B) Flavonols and ellagic acid derivatives, peaks identi®cation: (1) quercetin derivative; (2) quercetin derivative; (3) ellagic acid derivative; (4) ellagic acid; (5) ellagic acid derivatives; (6) quercetin-3glucuronide; (7) quercetin-3-glucoside; (8, 9 and 10) unknown.

in the amount of individual pigments (Table 1) were close to the variability (CV) resulted from the repeatability test (Cy-3-glu=10%; Pg-3-glu=11%; Pg-3rut=9%). The high variability of the experiment may have obscured enzyme induced pigment destruction. However, these results agreed with those of Wightman and Wrolstad (1996) who reported that only one pectolytic enzyme (over 23 tested at recommended dosages) produced signi®cant b-glucosidase activity on Cy-3-glu. It is important to point out that literature on anthocyanins destruction by glycosidase activity often refers to higher than recommended enzyme dosage (Jiang et al., 1990) or to hydrolytic activity di€erent than b-glucosidase (Wightman and Wrolstad, 1996). The content of quercetin derivatives identi®ed in strawberry treated juices (Table 1) varied as a function of the enzyme used and the time of treatment. During the treatment, Pectinex1 BE 3L, Pectinex1 B1L and Rohament1 MAX showed a moderate increasing trend (range: 38±46 mg lÿ1) while Rohapect1 MB, Pectinase2 LX and Pectinex1 3XL produced a stationary level of quercetin derivatives (range: 34±42 mg lÿ1). The ¯avonols content varied within the range of the repeatability test. However, after 6 hs of treatment the enzyme Pectinase2 LM hydrolyzed the peak No. 4 (Table 1). Consequently, the HPLC pro®le was modi®ed accordingly (data not shown) and the quercetin derivatives content decreased of 35% (27 mg lÿ1) when compared to the control (43 mg lÿ1). This variation was higher than the repeatability test (CV=13%). Since there is a lack of literature on HPLC analyses of individual strawberry

E€ects of pectolytic enzymes on selected phenolic compounds

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Table 1. Concentration (mg lÿ1) of phenolic compounds (anthocyanins, quercetins and ellagic acid derivatives) detected in strawberry and raspberry control and treated juices at di€erent time Compound (mg lÿ1) Time (h)

Anthocyanins

Quercetins

Ellagic acids

2

4

6

2

4

6

2

4

6

Fruit: Strawberry Control Pectinex1 BE 3-L Rohapect1 MB Rohapect1 B1L Pectinase2 LM Pectinase2 LX Rohament1 MAX Pectinex2 3XL

317(7) 321 (9) 323 320 316 323 304 296

298 (7) 316 (11) 308 297 307 301 279 298

300 (8) 286 (10) 289 291 301 286 273 264

38 (2) 38 (4) 40 35 38 36 42 34

40 (3) 46 (7) 35 34 29 36 44 41

43 (5) 47 (13) 42 46 27 35 45 33

6 (12) 8 (14) 11 8 11 8 14 8

7 (13) 14 (16) 9 7 15 13 15 8

8 (12) 17 (17) 17 17 19 16 17 7

Fruit: Raspberry Control Pectinex1 BE 3-L Rohapect1 MB Rohapect1 B1L Pectinase2 LM Pectinase2 LX Rohament1 MAX Rohament1 MAX

391 (5) 455 (4) 457 457 470 456 406 439

451 (6) 387 (7) 439 382 456 449 375 369

461 (5) 393 (8) 428 392 454 446 375 376

27 (4) 28 (6) 36 30 30 27 26 27

27 (3) 28 (9) 36 32 30 32 29 27

27 (3) 28 (7) 34 30 31 30 29 27

8 (14) 7 (21) 11 8 6 5 7 7

7 (14) 7 (18) 10 10 5 6 7 7

8 (15) 7 (20) 8 8 3 4 7 6

¯avonols after enzyme treatment, the glycosidase activity of Pectinase2 LM may only be hypothesized. The content of ellagic acid derivatives detected in strawberry treated juices (Table 1) was always higher than the values found in the control (6±8 mg lÿ1). The minimum value of ellagic acid derivatives for strawberry treated juices was 8 mg lÿ1 (Rohapect1 B1L, Pectinase2 LX and Pectinex2 3XL, after 2 h) and the maximum values was 19 mg lÿ1 (Pectolase2 LM, after 6 h). The ellagic acids derivatives content of treated juices (Table 1) showed a linear increase over the time of treatment for Pectinex1 BE 3L, Pectinase2 LM, Pectinase2 LX and Rohament1 MAX. In particular, the HPLC pro®le of strawberry ellagic acid derivatives was characterized by the increase of peak No. 3 (ellagic acid). These results could be explained by the hydrolytic release of the ellagic acid located in the cell-walls and in the achenes (Maas et al., 1991b). Red raspberry Treatment of raspberry juices with pectolytic enzymes (Table 1) modi®ed the level of individual pigment and the total anthocyanins content varied accordingly. However, the ratio of individual anthocyanins remained constant. In raspberry juices the Cy-3-sopho content was maximum for Pectinase2 LM after 2 h (316 mg lÿ1) and minimum for Pectinex2 3XL after 4 h (254 mg lÿ1). The range of Cy-3-glu was 126±154 mg lÿ1 (control after 2 h and Pectinase2 LM after 2 h, respectively). The concentration of pigments in the control (Table 1) increased with the time; the total anthocyanins content raised from 391 mg lÿ1 at 2 h, to 462 mg lÿ1 at 6 h. The pectolytic enzymes used could be divided into two groups: Rohapect1 MB, Pectinase2 LM and Pectinase2 LX showed

a stationary high level of total anthocyanins over the time (range: 289±306 mg lÿ1). On the other hand, it was clear a decrease of total anthocyanins, after 6 h, when Pectinex1 BE 3-L (ÿ21%), Rohapect1 B1L (ÿ19%), Rohament1 MAX (ÿ20%) and Pectinex2 3XL (ÿ20%) were used. According to literature (Jiang et al., 1990; Wrolstad et al., 1994) these ®ndings could indicate the presence of b-glycosidase activity in the commercial pectinase enzymes. The pectolytic enzymes showed higher anthocyanins hydrolytic activity in raspberry then in strawberry juices. According to literature some hypotheses may be formulated: (i) many enzyme preparation increase pigment yield during juice extraction and this would tend to counteract the e€ects of anthocyanins destruction from glycosidase activity (Wightman and Wrolstad, 1995); (ii) the presence of competitive inhibition may retard anthocyanins hydrolysis (Aryan, 1987); (iii) it seems that enzyme anity is increased by the hydrophilic character of the aglycone (Pi€aut et al., 1994). The overall content of quercetin derivatives detected in raspberry juices (Table 1) ranged between 27 and 36 mg lÿ1. These values are similar to the results of Rommel and Wrolstad (1993a). The content of ¯avonol was almost stable over the time considered: no hydrolytic activity was detected in our experimental conditions. The enzyme Rohament1 MB (Table 1) produced a marked increase in total quercetin derivatives (36 mg lÿ1). The content of ellagic acid derivatives in raspberry juices (Table 1) ranged between 4 and 11 mg lÿ1 (Rohapect1 MB and Pectinase2 LM, after 2 h and 6 h, respectively). These values also resulted within the range reported by Rommel and Wrolstad (1993b) for di€erent

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raspberry cultivars. Two pectolytic preparation, Pectinase2 LM and Pectolase2 LX, decreased the overall content of ellagic acid derivatives (Table 1). These results may indicate the presence of hydrolytic activity as previously reported by Rommel and Wrolstad (1993b). CONCLUSIONS The HPLC ¯avonoids pro®les were characteristic for strawberry and red raspberry juices. The phenolic compounds could provide a useful marker in authenticity investigation; however, their high variability may limit their utility. Depectination a€ected the composition of juice in relation to the type of juice, the type of enzyme and the time considered. When pectolytic enzymes were used within recommended producers dosages only a little loss of anthocyanins in strawberry was detected. However, the use of some commercial pectinases decreased the total anthocyanins content in raspberry juices after 4±6 h of treatment. The results suggested that pigment loss could be minimal during early stages of juices processing but that may become substantial during later stages of treatment. The ¯avonols content was similar in strawberry and raspberry juices and was within the concentration range that has been shown to have anticarcinogenic e€ects in rodents. The use of Rohapect1 MB increased the content of total quercetin derivatives in raspberry. On the other hand, Pectolase2 LM decreased the ¯avonols content in strawberry juices. The concentration of ellagic acid derivatives in strawberry juices considerably increased after pectolytic enzymes treatments. Raspberry juices are a€ected by the commercial pectinases at a lower extent. The di€erent results observed when strawberry and raspberry were treated with pectolytic enzymes can be tentatively justi®ed on the basis of the current knowledge.

ACKNOWLEDGEMENTS This work was ®nancially supported by COMETT-MIT grant (Ancona, Italy) and by Leatherhead Food Research Association (Surrey, UK). The authors thank Ronald Wrolstad for his extensive and critical reading of this manuscript. REFERENCES Aryan, A. P., Wilson, B., Strauss, C. R. and Williams, P. J. (1987) The properties of glycosidases of Vitis vinifera and a comparison of their b-glucosidase activity with that of exogenus enzymes. An assessment of possible applications in enology. American Journal of Enology and Viticulture 38, 182±188.

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(Received 12 May 1998; accepted 13 June 1998)