Redalyc.Alternative Processing of Port-Wine Using Pectolytic Enzymes

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Ciencia y Tecnología Alimentaria ISSN: 1135-8122 [email protected] Sociedad Mexicana de Nutrición y Tecnología de Alimentos México

Rogerson, F.S.S; Vale, E.; Grande, H.J.; Silva, M.C.M. Alternative processing of port-wine using pectolytic enzymes Ciencia y Tecnología Alimentaria, vol. 2, núm. 5, julio, 2000, pp. 222-227 Sociedad Mexicana de Nutrición y Tecnología de Alimentos Reynosa, México

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ALTERNATIVE PROCESSING OF PORT-WINE USING PECTOLYTIC ENZYMES

PROCESADO ALTERNATIVO DEL VINO DE OPORTO USANDO ENZIMAS PECTOLÍTICOS

PROCESADO ALTERNATIVO DO VIÑO DE OPORTO USANDO ENZIMAS PECTOLÍTICOS

Rogerson, F.S.S*1; Vale, E.3; Grande, H.J.2; Silva, M.C.M.3

1* Departmento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto. Portugal. E-Mail: [email protected] 2 Licentec, Bernadottelaan 15, P.O.Box 8323, 3503 RH Utrecht, The Netherlands. 3 Escola Superior de Biotecnologia/Universidade Católica. Rua Dr.Antonio Bernardino de Almeida, 4200 Porto. Portugal.

Recibido: 24 de Octubre de 1999; recibida versión revisada: 9 de Marzo de 2000; aceptado: 15 de Marzo de 2000 Received: 24 October 1999; rreceived in revised form: 9 March 2000; accepted: 15 March 2000

Abstract The objective of the present study was to investigate the application of a commercial pectolytic enzyme preparation “Ultrazym” during grape maceration for the alternative processing of 7 single varietal Port-Wines (Tinta Barroca, Mourisco Tinto, Tinta Roriz, Rufete, Tinta da Barca, Tinta Santarém and Touriga Nacional). The effect of enzymatic treatment resulted in improvements in: i) juice yield; ii) colour extraction; iii) wine filterability. Juice yields increased by up to 6%, wine colour intensified by up to 40%, and wine filtration speed was considerably more rapid in the enzyme treated wines. Ó 2000 Altaga. All rights reserved.

Keywords: Wine, pectolytic enzyme.

Resumen El objetivo de este trabajo fue investigar la aplicación de un preparado enzimático comercial durante la maceración de la uva para realizar un procesado alternativo de 7 variedades monovarietales de vino de Oporto (Tinta Barroca, Mourisco Tinto, Tinta Roriz, Rufete, Tinta da Barca, Tinta Santarém y Touriga Nacional). El efecto del tratamiento enzimático resultó en una mejora: i) del rendimiento en jugo; ii) de la extracción de colorantes; iii) filtrabilidad del vino. El Rendimiento en jugo se incrementó hasta un 6%, la intensidad del color del vino hasta un 40% y la velocidad de filtración fue considerablemente más rápida en los vinos preparados con enzimas. Ó 2000 Altaga. Todos los derechos reservados.

Palabras clave: Vino, enzimas pectolíticos.

Resumo O obxetivo deste traballo foi investiga-la aplicación dun preparado enzimático comercial durante a maceración da uva para realizar un procesado alternativo de 7 variedades monovarietais de viño de Porto (Tinta Barroca, Mourisco Tinto, Tinta Roriz, Rufete, Tinta da Barca, Tinta Santarém e Touriga Nacional). O efecto do tratamento enzimático resultóu nunha mellora: i) do rendemento en xugo; ii) da extracción de colorantes; iii) filtrabilidade do viño. O Rendemento en xugo incrementóu-se hasta un 6%, a intensidade do color do viño hasta un 40% e a velocidadd de filtración foi considerablemente más rápida nos viños preparados con enzimas. Ó 2000 Altaga. Todolos dereitos reservados.

INTRODUCTION stored for 9 months at -20oC, were then defrosted, destemmed, weighed and divided. The first half (blank) Over the past few decades, increasing interest has and second half (added “Ultrazym”, 30mg/L) were crushed been shown in the application of enzymes in wine-making. with the addition of SO2 (30mg/L). The crushed grapes, Enzymatic treatments of grapes, musts and wine have contained in 2.5L glass flasks, were left macerating for 8 multiple purposes, since they influence wine clarification hours (20oC), after which the Saccharomyces cerevisiae and filtration, juice yield, colour and aroma extraction and yeast, Firmivin, was inoculated (200mg/L). At specific wine stability (Ough et al., 1975; Villettaz, 1984; Zamorani, gravity (S.G.) 1.045, the fermenting mash was pressed 1989; Canal-Llauberes, 1989 &1990; Villettaz and (cone press) and the resulting juice was filtered (Whatman Dubourdieu, 1991). Nº1 filter paper) and yield determined. The semi-fermented must was then fortified with alcohol (99.8% v/v, Merck) Industrial pectolytic enzyme preparations have to give calculated alcoholic strength of 20% v/v. The been widely used for many years, most being derived from resulting Port-Wines were bottled and allowed to mature cultures of Aspergillus niger which is a species accepted for two months. The micro-vinification procedure is as G.R.A.S. (Generally Recognised As Safe) (Canal- represented in Figure 1. Llauberes, 1993). Besides the main pectolytic activities (pectinmethylesterase, polygalacturonase, pectin lyase & Analytical methods pectate lyase), industrial pectinase preparations also contain hemicellulolytic, cellulolytic and side-activities Juice volume including glycosidases (Cordonnier et al., 1989) and Port-Wine juice volume was measured before the proteases (Schmitt et al., 1989). addition of the fortification alcohol, but after the removal of both the skins (pressing) and insoluble solids (filtration). Pectolytic enzyme preparations are valuable processing tools for many wine and grape processing Colour applications, including: mash treatment for juice extraction Colour measurements were made after 2 months (Ough & Crowell, 1979; Canal-Llauberes, 1989); juice wine maturation, using a Pye-Unicam spectrophotometer clarification and wine filtration (Brown & Ough, 1981; according to the method of Jackson et al. (1978). Canal-Llauberes, 1989); colour extraction (Ough et al., 1975; Castino & Ubigli, 1979) and aroma liberation Measurements included: Wine colour (W.C.) at (Cordonnier et al., 1989; Rogerson et al., 1995). wine pH (l520nm, 1mm) and total pigment colour (W.C.A.) measured in 1.0M HCl (1% solution, l520nm, Recent investigations (Birks & Sarmento, 1991; 10mm). Bakker et al., 1996) compared the two most typically used maceration techniques for Port-Wine production (open tank Total Phenolics treading & static tanks with pumped over juice), however Total phenolics were measured in 1.0M HCl (1% nothing appears to have been studied, looking at the solution, l280nm, 10mm), and calculated according to the application of pectolytic enzymes for alternative processing method of Somers & Evans (1977) using the formulae of Port grapes, musts and wines. Therefore the objective (A280nm –4). of this work was to investigate the influence of a pectolytic enzyme preparation on juice yield, colour release and wine filterability, during the production of several single varietal Port-Wines.

GRAPES MATERIALS AND METHODS Defrost Destem 1/2 1/2 Crush + SO (30 mg/L) Crush 2 The A. niger enzyme preparation was “Ultrazym + Ultrazym (30mg/L) + SO2 (30mg/L) 100G” from Novo Nordisk. The inoculation yeast was Maceration Maceration o Saccharomyces cerevisiae , “Firmivin”, from Gist- 8 Hrs, 20 o C 8 Hrs, 20 C

Brocades. Yeast 200mg/L Yeast 200mg/L Fermentation 25 º C Fermentation 25 º C until S.G. 1.045 until S.G. 1.045 The grape cultivars (Touriga Nacional, Tinta Press, Filter Press, Filter Roriz, Tinta Barroca, Mourisco Tinto, Tinta da Barca, & Fortify & Fortify Bottle & Bottle & Tinta Santarém and Rufete; 1992 vintage) were from Tua Storage Storage (20 º C) (20 º C) in the Douro valley (Portugal). All reagents used were o research grade. PORT WINE PORT WINE (BLANK) (Ultrazym)

Red Grape Micro-Vinifications

The grapes, picked at commercial maturity, were Figure 1. Processes of micro-vinifications performed in this study (S.G. taken back to the University in Porto where they were = Specific Gravity) . 223 Cienc. Tecnol. Aliment. Vol. 2, No. 5, pp. 222-227, 2000 ISSN 1135-8122 ©2000 ALTAGA

Cross-Flow Micro-Filtration Maceration gives red wine its essential colour and tannic Micro-filtration analysis was performed on the structure, being particularly important for grape processing fortified wines after two months bottle storage, using a for the production of great red wines intended for ageing. laboratory scale Carbosep® 40 (TECH-SEP, Grupe Rhône- Apart from traditional maceration, alternative extraction Poulenc), equipped with a tubular membrane (zirconion technologies include thermovinification (Dubourdieu, oxide contained in a carbon support) with pore size 0.14mm 1986) and enzymic methods (Villettaz and Dubourdieu, and filtration area of 0.008m2. The membrane was initially 1991). washed for 10 minutes with wine which was discarded. Each filtration was carried out using 120mL of wine, using Thermovinification (typically 70-80ºC) has been a transmembranal pressure of 1.7 bar. The filtration shown as beneficial for increasing colour (anthocyanin equipment was allowed to filter the wine, using a closed release), however it also increases pectin levels and circuit during 30 minutes after which the circuit was opened denatures endogenous grape pectinases, thereby hindering and collection of 50mL of filtered wine was timed. On clarification and filtration. Enzyme colour extraction has completion of filtration, the membrane was alternately been proposed as alternative technology to be used either washed with sodium hydroxide (0.1M) and nitric acid with or without thermovinification (Villettaz & Dubourdieu, (0.1M) at 80ºC until the flow-rate of water was measured 1991). as 850L/hr/m2 at 4 bar and 25ºC. The principle components responsible for red grape and wine colour are the anthocyanins which are RESULTS AND DISCUSSION thought, in the case of Pinot Noir grapes, to exist in anthocyanoplasts located in vacuoles present in the skins Juice Yield of the fruit (Pecket and Small, 1980; Merlin et al., 1985). Documented trials, using pectolytic enzymes for maceration purposes, have resulted in increased juice yields The application of pectolytic enzymes would for both red and white grapes (Ough et al., 1975; Ough facilitate break up of the grape cell wall enabling more rapid and Crowell, 1979; Villettaz and Dubourdieu, 1991; Haight release of anthocyanins from the anthocyanoplasts, and and Gump, 1994). would also aid juice and wine clarification by breaking- down the released grape pectins. The present study showed that grapes treated with the pectolytic enzyme preparation “Ultrazym” resulted in Two measurements are commonly used to give no change in juice yield with 2 cultivars (Tinta da Barca representations of actual perceived wine colour and total and Touriga Nacional), however increased levels were red pigment colour. Red wine colour (W.C.) is due to the observed with the other 5, with percentage increases red and violet coloured forms of anthocyanins, along with respectively greatest for Rufete (6.2%), Mourisco (4.5%) various polymeric pigments (Jackson et al., 1978) but is and Barroca (4%) (Figure 2). influenced by both pH and SO2. Total Wine Colour (W.C.A.) is obtained after acidification to pH<1 converting The principle activities present in the pectolytic all the colourless anthocyanins and polymerics into their “Ultrazym” preparation are pectintranseliminase, coloured cationic forms. polygalacturonase, pectinesterase and arabinanases (Novo Nordisk, 1991). The application of the pectolytic enzyme preparation «Ultrazym», during ambient temperature grape Pectinase enzymes are known to work on the maceration for Port production, resulted in wine colour pectic substances which occur as structural polysaccharides intensification, with increases in both W.C. (up to 40%) in the middle lamella and primary cell wall (Haight & and W.C.A. (up to 55%) for all 7 cultivars examined (Table Gump, 1994). The presence of macerating side-activities 1 and Figure 2). Lower temperature extraction technology in the pectinase preparation, such as cellulases and for intensifying colour in Port-Wine production would be hemicellulases would result in a more complete break- more favourable than the thermovinification method, since down of the polysaccharide structure, causing greater savings in energy costs and improved retention of solubilisation of the middle lamella and improved juice volatile aroma compounds would result. extraction, clarification and filtration (Haight & Gump, 1994). The side-activity, b-D-glucopyranosidase, present in the pectolytic enzyme preparation may have a negative Colour Extraction effect on wine colour. This activity may strip the When assessing wines, colour and appearance play anythocyanins of their glycoside glucose, producing the an important role, being heavily relied upon for making corresponding anthocyanidin, which is unstable and judgements associated with both buying and quality control. spontaneously breaks-down into colourless derivatives An examination of the effect of colour on the assessment (Huang, 1956). However the concentration of glucose of Ports, concluded that appearance attributes, dominate typically found in red Port-wine (around 30-60g/L; Alves the assessment of both aroma and flavour (Williams et al., and Ferreira, 1988) is known to strongly inhibit b-D- 1984). Indeed, the colour of Port-Wine is considered the glucopyranosidase activities of A.niger origin (Dugelay most important component of quality (Bakker et al., 1986). 1993; Rogerson et al, 1995; Rogerson 1997).

20 Juice % Increase W.C. W.C.A. 10 Phenolics

-10 Rufete Tinta Roriz Tinta Barroca Tinta da Barca Mourisco Tinto Tinta Santarém Touriga Nacional

Figure 2. Effect of pectolytic enzyme treatment on juice yield and colour parameters of single varietal Port-wines. Legends: W. C. = Wine Colour; W.C.A. = Total Pigment Colour.

Table 1.- Effect of the use of the pectolytic enzyme preparation Table 2.- Effect of the use of the enzyme preparation "Ultrazym 100" "Ultrazym 100" on colour and phenolic parameters in micro-vinified on times taken for the cross-flow micro-filtration of 50ml port-wine port-wine (1992 vintage and 2 months maturation). aliquots.

Cultivar BLK/ENZ W.C. W.C.A. Total Phenolics Cultivar Enz/Blk Time Temperature (seconds) (ºC) Tinta Barroca Blk 2.26 7.37 23.6 Tinta Barroca Blk 310 40.0

Tinta Barroca Enz 3.19 8.69 33.9 Tinta Barroca Enz 188 39.0

Mourisco Tinto Blk 0.59 0.91 8.8 Mourisco Tinto Blk 254 41.0

Mourisco Tinto Enz 0.67 1.41 12.0 Mourisco Tinto Enz 207 40.5 Tinta Roriz Blk 0.99 2.53 11.9 Tinta Roriz Blk 143 38.0

Tinta Roriz Enz 1.28 3.54 14.0 Tinta Roriz Enz 143 38.0

Rufete Blk 2.23 4.65 27.2 Rufete Blk 223 39.0

Rufete Enz 2.77 6.06 31.4 Rufete Enz 204 38.5

Tinta da Barca Blk 4.49 14.95 38.4 Tinta da Barca Blk 214 38.0

Tinta da Barca Enz 4.97 15.45 39.8 Tinta da Barca Enz 199 38.0

Tinta Santarém Blk 0.72 1.82 11.3 Tinta Santarém Blk 254 37.0

Tinta Santarém Enz 0.85 2.02 10.4 Tinta Santarém Enz 163 37.0

Touriga Nacional Blk 5.96 16.87 48.5 Touriga Nacional Blk 305 42.5

Touriga Nacional Enz 7.46 20.81 61.8 Touriga Nacional Enz 282 42.5

Legend: Blk = Blank; Enz = Enzyme. Legend: Blk = Blank; Enz = Enzyme.

Enzyme treatment also resulted in wines with ACKNOWLEDGEMENTS increased levels of total phenolics (Table 1). The Authors express their thanks to Dr.Janser Filterability (Novo Nordisk), for providing the Aspergillus niger In nearly every case (exception Roriz), the wine enzyme preparation and to Dr.H.P.Reader (Cockburn treated with the enzyme was filtered much more rapidly Smithies Lda.) for providing the grapes from their than the control wine (Table 2). Most notable vineyards in Tua. This work was funded by “Programa improvements were obtained with the cultivars Mourisco, Ciencia” a grant from the Junta Nacional de Investigação Barroca and Tinta Santarém, probably due to the partial Científica e Tecnológica. hydrolysis of polymeric pectin and hemicellulolytic polymers present in the wine. REFERENCES Acidic pectic substances (pectins) have a rhamnogalacturonan backbone in which (1®4) linked a- Bakker, J.; Bridle, P.; Timberlake, C. F.; Arnold, G. M. D-galacturonan chains are interrupted at intervals with a- 1986. The colours, pigment and phenol contents of L-rhamnopyranosyl residues carrying neutral side chains young Port wines: effect of cultivar, season and site. (Saulnier & Brillouet, 1988). Pectic material can therefore Vitis 25, 40-52. be described as having two distinct regions, a linear Bakker, J.; Bellworthy, S . J.; Hogg, T. A.; Kirby, R. M.; «smooth» region composed of homogalacturonan units and Reader, H. P.; Rogerson, F. S. S.; Watkins, S. J.; a so-called «hairy» region, rich in neutral side-chains Barnett, J. A. 1996. Two methods of Port (Saulnier & Thibault, 1987). The neutral side-chains in vinification: comparison of changes during grape pectin have been identified to be hemicelluloses with fermentation and of characteristics of the wines. either arabinan, or arabinogalactan structures (Saulnier & Am. J. Enol. Vitic., 47 (1), 37-41. Brillouet, 1988). Birks, A. G.; Sarmento, M. M. 1991. Maceration for premium port wine production. Austral.NZ Wine The endo-polygalacturonase activity contained in Indust. J. 6, 278-280. the pectolytic preparation (Novo Nordisk, 1991) is Brillouet, J. M.; Bosso, C.; Moutounet, M. 1990. Isolation, important for breaking-down the so-called “smooth” region purification, and characterization of an of pectin, the polygalacturonan back-bone. Indeed an endo- arabinogalactan from a red wine. Am. J. Enol. Vitic. polygalacturonase activity has been shown to reduce pectin 41 (1), 29-36. solution viscosity by more than 50% by hydrolysing just Brown, M. R.; Ough, C. S. 1981. A comparison of activity 2-3% of the glycosidic bonds in the pectin chains (Villettaz and effects of two commercial pectic enzyme & Dubourdieu, 1991). preparations on white grape musts and wines. Am. J. Enol. Vitic. 32 (4), 272-276. The two glycosidase side-activities (a-L- Canal-Llauberes, R.M. 1989. Les enzymes industrielles arabinofuranosidase and b-D-galactopyranosidase) present dans la biotechnologie. Rev. Oenol. 53, 17-22. in the “Ultrazym” preparation (data not presented), would Canal-Llauberes, R. M. 1990. Utilization des enzymes play an essential role in the break-up of the hemicellulolytic dans les procédés d’extraction oenologie. Rev. Fr. fractions (arabinans and arabinogalactans), originating Oenol. 30 (122), 28-33. from the “hairy” regions of pectin, which are well known Canal-Llauberes, R. M. 1993. Enzymes in wine-making. constituents of Vitis vinifera varietal grapes and wines Chapter 17. In “ Wine microbiology and (Villettaz et al., 1981; Saulnier et al., 1987; Saulnier and biotechnology”; Fleet,G.H.,Ed.; Harwood Brillouet, 1988; Brillouet et al., 1990). Academic Publishers, 477-506. Castino, M.; Ubigli, M. 1979. Use of pectolytic enzymes The pectolytic enzyme preparation therefore preparation in manufacture of red wines. Riv. Vitic. permits faster, improved filtrations, which would Enol. 32 (2), 65-75. consequentially lengthen filter membrane life-times and Colagrande, O.; Silva, A.; Fumi, M. D. 1994. Recent increase total filtered volumes. applications of biotechnology in wine production. Biotechnol. Prog. 10, 2-18. Cordonnier, R. E.; Gunata,Y. Z.; Baumes, R. L.; Bayonove, CONCLUSIONS C. L. 1989. Recherche d’un matérial enzymatique adapté à l´hydrolyse des précursors d’arôme de The current study emphasizes the possible nature glycosidique du raisin. Conn.Vigne Vin 23, benefits from the application of pectolytic enzymes for 7-23. the alternative processing of Port-Wine. Improvements in Dubourdieu, D. 1986: Wine technology: current trends. juice yield, extracted colour and micro-filtration velocities Experientia 42, 914-921. were observed. The results emphasize the need for larger industrial trials.

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