EfFect of sulphur dioxide on colour and composition during ageing

Transcription

1 Vitis 33, (1994) Moel wine solutions : ffect of sulphur ioxie on colour an composition uring ageing by ANNA PICINI, BAKKR ari P BRID C~ ~e[,~f~:` Institute of Foo Research, Reaing aboratory, Reaing, Berkshire, Unite Kingom S u m m a r y : The effect of sulphur ioxie an acetalehye on the interaction between malviin 3-glucosie, the main anthocyanin in re wine mae from Vitis vinifera grapes, an (+)-catechin was examine In all moel wines the molar losses of malviin 3-glucosie were significantly greater than the losses of catechin The concentration of malviin 3-glucosie ecrease fastest in the presence of acetalehye while the most rapi loss of catechin occurre in the moel containing malviin 3-glucosie an catechin only In the presence of sulphur ioxie, these losses still occurre, but much more slowly, inicating that conensation reactions may take place even in the presence of sulphur ioxie Polymerisation was most prominent in the moel containing malviin 3-glucosie, catechin an acetalehye Concurrent with the losses in anthocyanin an catechin, qualitative an quantitative changes in visible colour were also observe Changes in colour monitore by measuring hue angle an chroma are also reporte K e y w o r s : moel wines, anthocyanins, acetalehye, sulphur ioxie, catechin, HPC, colour measurements, ageing Introuction ne of its most important quality inicators of a re wine is its colour During maturation, re wine colour changes from bright re to a reish-brown tint, ue to the anthocyanins extracte from the grape skins uring fermentation forming polymeric pigments, by mechanisms summarise an iscusse previously (BAKKR et al 1993) Sulphur ioxie is wiely use in wine making as an antioxiant an an inhibitor of unesirable microbial growth ; if ae prior to fermentation, it can increase the extraction of colour from grape skins when making re wines, an stabilise the colour when ae at bottling (BURRUGHS 1974) However, this is achieve at the expense of reversible bleaching of the monomeric anthocyanin colour of re wine ue to the formation of anthocyanin-4-bisulphite, a stable colourless compoun (URD 1964 ; TIMBRAK an BRID 1967) Furthermore, Sulphur ioxie bins strongly to acetalehye (BURRUGHS an SPARKS 1973), hence the presence of sulphur ioxie woul be expecte to influence the colour changes uring maturation The effect of sulphur ioxie on wine polymerisation reactions has not been stuie hitherto The aim of this work is to exten the stuy of polymerisation in moel re wine solutions an examine the effect of sulphur ioxie on these reactions We stuie interactions between malviin 3-glucosie, the main anthocyanin in re wine mae from ~tis vinifera grapes, an (+)-catechin ; the role of acetalehye an sulphur ioxie was also investigate High performance liqui chromatography (HPC) was use to measure the anthocyanin an catechin concentrations Colour changes were monitore using spectrophotometey an tristimulus measurements Materials an methos Sample preparation : Anthocyaninswere extracte from the skins of various Portuguese grape varieties, with methanol an formic aci (97 :3, v/v), an malviin 3-glucosie was separate an purifie by preparative HPC (BAKKR et al 1993) Reagents were analytical grae : (+)-catechin (Sigma), acetalehye (Alrich) an soium metabisulphite (Merck) Solvents were HPC grae M o e 1 s o 1 u t i o n s : Reactants were issolve in a sterilise moel wine meium of potassium bitartrate (Merck) buffer (002 M, ph = 3 7), containing ethanol (10 % v/v) The reactants were use in amounts calculate to give the following final concentrations in reaction vials (3 ml sample in a 7 ml vial) : malviin 3-glucosie 0 25 mm (012 mg/ml), (+)-catechin 0 4 mm (0 12 mg/ml), acetalehye 0 9 mm (004 mg/ml) an sulphur ioxie 0 9 mm (0 05 mg/ml) Five ifferent experimental mixtures were prepare, filtere (045 ~tm sterile membrane filters) an ispense into 5 sets of vials as follows : (A) malviin 3-glucosie (control), (AC) malviin 3-glucosie an (+)-catechin, (ACA) malviin 3-glucosie an (+)-catechin an acetalehye, (ACS) malviin 3-glucosie an (+)-catechin an sulphur ioxie, (ACAS) malviin 3-glucosie an (+)-catechin an acetalehye an sulphur ioxie All samples were allowe to react in the ark at ambient temperature, an were analyse in uplicate from separate vials over a perio of 95 Colour measurements : APhilipsPU8740 spectrophotometer an 2 mm path length glass cells were use for CIAB 76 colour measurements (BAKKR et al Corresponence to : Dr KI BAKKR, Institute of Foo Research, Reaing aboratory, arley Gate, Whiteknights Roa, Reaing, Berkshire RG6 2F, Unite Kingom

2 3 2 ANNA PICINI, BAKKR ari P BRID 1993) Colour measurements of samples containing malviin 3-glucosie an (+)-catechin an sulphur ioxie were mae immeiately after the aition of 500 mg/1 acetalehye, to ensure that the base colour was measure, rather than the colour of the anthocyanins bleache by sulphur ioxie T o t a l p i g m e n t s : Total pigments an total phenols were etermine in triplicate by spectrophotometry using 10 mm path length UV cells Samples were ilute as require with 01 M HCl an absorbances were measure at 520 an 280 nm respectively The total pigment absorbance measurement was expresse as concentration (c) of malviin 3-glucosie (molar mass 529) using the molar absorptivity value of 28,000 at 520 nm (NIKTIC- AKSIC an HRAZDINA 1972) Hence, c(mg/1) = 1889 absorbance Anthocyanin an (+)-catechin analy s i s : A Hewlett-Packar 1090 M HPC with an auto injector (25 ltl) an a ioe array etector recoring at 280 an 520 nm was use to measure the anthocyanin an (+)- catechin concentrations using malviin 3-glucosie chlorie an (+)-catechin as external stanars (BAKKR et al 1993) Sulphur ioxie analysis : Free an total sulphur ioxie were etermine using a Dionex 4500 ion chromatograph (Dionex Corporation, Sunnyvale, CA, USA) Immeiately before analysis samples were ilute as require in buffer : for total SZ a ph 9 buffer was use (20 mm NaZHP04) an for free SZ a ph 2 buffer was use (10 mm HZS4) Samples (50 ltl) were analyse using a Dionex Ionpac IC-AS1 column, with 10 mm HZS4 as eluent at 1 ml/min flow Detection was by pulse amperometry, using a Pt electroe at +0 7 V Quantification was one using an external stanar prepare from soium metabisulphite in ph 9 buffer Acetalehye analysis : Free acetalehye was etermine by heaspace gas chromatography using a Perkin lmer 8700 moel, equippe with a HS-101 heaspace sampler Separation was accomplishe on a capillary CP-WAX 57CB (50 m x 032 mm i ) column (Chrompak) The oven was hel at 45 C for 1 min, followe by a 30 C/min increase to 250 C, an hel 5 min at this temperature ther parameters were : flame ionization etector 300 C, injector temperature 300 C, carrier gas (N z) flow rate 1 4 ml/min The etermination was calibrate using an external acetalehye stanar Total acetalehye was etermine by an enzymatic metho (Boehringer Mannheim GmbH, foo analysis enzymatic kit) Statistical analysis : inear regression analysis of variance was one using SPSS Results an iscussion C o 1 o u r c h a n g e s : During ageing the hue angle of all moels increase, inicating that they ha all evelope some browning (Fig 1) The browning reactions starte slowly in all moels ; after about 20 of storage there were only small ifferences between the samples, with the exception of ACS ; however, there were large ifferences in the extent of browning by the en of the experiment Moel AC browne most, while the least browning was observe in this sample when sulphur ioxie was also present (ACS) The formation of yellow xanthylium pigments, note by TURD an SMRS (1970) an later in the context oflong-term wine reactions by TIMBRAK an BRID (1976), is the probable explanation of the browning reactions occurring in mixture AC It is interesting to note therefore, that this reaction is apparently retare in the presence of sulphur ioxie (ACS) Moel ACA showe only a very small increase in browning uring the first 50, after which time this moel ha browne less than control A This very slow browning in the presence of acetalehye can be explaine by the superimpose blueing effect of acetalehye - meiate reactions with anthocyanin an catechin Previously, BAKKR et al (1993) observe in a moel containing 0 5 % v/v acetalehye, a ecrease in hue angle of 25 inicating blueing uring the first 25 of storage, after which a slow increase in hue angle was observe inicating the onset of browning ; at the en of the experiment (140 ) the authors reporte that the hue angle was the same as that of the starting mixture Uner the current experimental conitions, moel ACA i not show a rapi increase in browning until after 50 storage At this time the concentration of acetalehye ha ecrease to about 10 mg/1 (Fig 2), an it is probable that this concentration became a limiting factor, since there was still 50 mg/1 free malviin 3-glucosie (Fig 3) remaining in this moel There was only a small ifference in browning pattern between samples ACA an ACAS, even though equimolar concentrations of sulphur ioxie an acetalehye were ae in the latter moel Sulphur ioxie has a high af- Fig 1 : Changes in hue angle (egrees) for all moels A(~) : malviin 3-glucosie (control) ;AC (~) : malviin 3-glucosie an (+)- catechin ; ACA (o) ; malviin 3-glucosie an (+)-catechin an acetalehye ; ACS (o) : malviin 3-glucosie an (+)-catechin an sulphur ioxie ; ACAS (~) : malviin 3-glucosie an (+)-catechin an acetalehye an sulphur ioxie Fig 2 : Changes in total acetalehye concentration in moels ACA an ACAS ACA, ACAS : see Fig 1 Fig 3 : Changes in malviin 3-glucosie concentration for all samples The following equations were calculate using stepwise linear regression ; the stanar error is shown in brackets an the multiple R is given at the en of the equation A : Y = X (2 76-4) (1 19-2) ; 0995 ; AC : Y = X (2 64-4) (1 14-2) ; 0966 ; ACA : Y = X (2 20-4) (8 62-3) ; ; ACS : Y = X (1 48-4) (6 46-3) ; 0933 ; ACAS : Y = X (2S-4) (8 85-3); 0969 A, AC, ACA, ACS, ACAS : see Fig 1 Fig 4 : Changes in chroma for moels A, AC an ACA A, AC, ACA : see Fig 1 Fig 5 : Slopes calculate for ecrease in anthocyanin an catechin concentrations expresse in mmol/, with 95 % confience regions A, AC, ACA, ACS, ACAS : see Fig 1 Fig 6 (right column) : Decrease in total pigment measurement an anthocyanin concentration etermine by HPC, both expresse in mg/l A, AC, ACA, ACS, ACAS : see Fig l

3 ffect of S Z on wine composition uring ageing N I _W 01 C Q T W W w _7 l M I c_ ~C WT CN 01 N V ays T m Anthocyanins Catechins I v N v w 0 V1 0 5 _ o o_ 5 a ac aca acs acas ac aca acs acas ays

4 34 ANNA PICINI, BAKKR an P BRID finity for acetalehye at this ph (BURRUGHS ari SPARKS 1973), an hence shoul bin the available acetalehye, leaving very little free to react Thus moel ACAS woul have been expecte to have a browning pattern similar to moel AC, if exactly equimolar concentrations of acetalehye an sulphur ioxie ha been ae However, the average free acetalehye in ACAS was 7 9 mg/1, whereas in the sample not containing sulphur ioxie (ACA) all the acetalehye is available for reactions with the flavonoi components The chroma (Fig 4) of control A showe a steay ecrease, while moel AC showe a more graual ecrease uring the first 50 of storage, followe by an increase in chroma, in agreement with earlier finings (BAKKR et al 1993) In contrast to these earlier observations mae in the presence of much higher concentrations of acetalehye, when an increase in chroma was observe uring the first 7 of storage, inicating an increase in the amount of colour expresse by the mixture, we now note a slow ecrease in chroma uring the entire storage perio of ACA These observations accor well with the fact that no new peaks were observe by HPC, just a graual ecline in the concentration of anthocyanins an catechin Acetalehye an sulphur ioxie c h a n g e s : The total acetalehye concentration in moels ACA an ACAS ecrease uring the first 80 of the experiment, from 35 mg/1 to less than 5 mg/1 (Fig 2) The variation between the measuring points is ue to inter-vial variation The effect of sulphur ioxie on the loss of acetalehye cannot be etermine from these ata The free acetalehye concentration in the ACAS moel i not change uring the course of the experiment, remaining at an average of 7 9 mg/1 The sulphur ioxie concentration ecrease slowly in both ACS an ACAS, an no obvious effect of acetalehye on the rate of loss was observe Anthocyanin an catechin losses : n storage, all solutions exhibite a logarithmic ecrease in total anthocyanin concentrations (Fig 3) The linear loss of anthocyanins in all four moel mixtures inicates a first orer reaction with respect to this loss, confirming the fin- 1rigS f BARANW$KI ari NAG (1983) In moels AC an ACA the rate of loss was greater than in control A, while sulphur ioxie ha a slowing effect on this process in both cases, although in ACAS the rate of loss was only marginally less than in control A The reaction rates k (ay') are shown in the Table an compare with rates reporte previously (BAKKR et al 1993 ; BARANWSKI an NAG 1983) Using a 3 6-fol molar excess of acetalehye, the observe increase in reaction rate for the moel ACA was less than when the molar excess was 5-fol Surprisingly, even in the presence of a 3 6-fol molar aition of sulphur ioxie (ACAS), the anthocyanin concentration still ecrease, although at a much slower rate The slowing effect of sulphur ioxie on the rate of anthocyanin loss is shown even better comparing AC an ACS reaction rates The apparent equilibrium constant for acetalehye with sulphur ioxie at ph 4 is 1 4 x 10-6 (BURRUGHS an SPARKS 1973), while for rrialviin 3-glucosie with sulphur ioxie it is 6 x 10 -s (BURRUGHS, pers comm) Thus in the presence of both acetalehye an malviin 3-glucosie, sulphur ioxie woul mostly be boun to acetalehye, as can also be etermine from the lack of bleaching Although the anthocyanin was to some extent protecte against loss by sulphur ioxie, its egraation still occurre The losses of catechin in all four moels were also logarithmic with time In orer to compare the losses of anthocyanins with those of catechin (not shown), the re gression lines were calculate on the concentration expresse in mmol (Fig 5) The rate of loss of catechin was always significantly slower (p < 0 05) than for anthocyanins in corresponing moels This is attributable to the anthocyanin an catechin conensation which is superimpose on the egraation of anthocyanins alone, as inicate by the loss in moel A The extent to which both reactions occur cannot be ascertaine, but the ifferences in colour changes for the moels inicate that ifferent reactions occur Aing acetalehye increase the slope of anthocyanin loss, but ecrease the slope of catechin loss The implication appears to be that the conensation involving acetalehye, anthocyanin an catechin involves Table Reaction rates with respect to the isappearance of total anthocyanins in moel wine systems, calculate with respect to maliviin 3-glucosie Mixture k (ay -~) k (ay-t)t literature value` k (ay-~ ) Anthocyanin x x 10-a 6 31 x 10-3 Anthocyanins an catechin x x x 10-3 Anthocyanins, catechin an acetalehye x x x 10-3 Anthocyanins, catechin an sulphur ioxie 4 32 x 10-3 Anthocyanins, catechin, acetalehye 9 81 x 10-3 an sulphur ioxie t Bakker, Brile an Picinelli (1993), using a 400-fol molar excess ' Data calculate from Baranowski an Nagel (1983), using a 5-fol molar excess

5 ffect of S Z on wine composition uring ageing 35 relatively more anthocyanin molecules than the conensation occurring in the absence of acetalehye Comparison of total pigment m,ea-su rement with anthocyanin concentra-tio n : The total pigment absorbance measurement expresses the sum of the concentration of monomeric anthocyanins an the colour exhibite by polymerise material Since the molar absorptivity value of newly forme polymerise material is not known, the concentration from this absorbance measurement is expresse as malviin 3-glucosie, assuming the same molar absorptivity value for both The ifference between the total pigment ata an the anthocyanins etermine by HPC both expresse in concentration terms (Fig 6), gives an inication of the contribution of the coloure polymers to the overall colour ver the course of the experiment, the largest ifference was observe in the moel containing anthocyanins, catechin an acetalehye, e g after about 50, there was ouble the colour concentration as expresse by the total pigment measurement, i e 50 % of the colour was ue to polymers In contrast the ifference for the moel containing anthocyanins, catechin an sulphur ioxie remaine much smaller C o n c l u s i o n : In the absence of acetalehye sulphur ioxie reuces the rate of anthocyanin loss an slightly reuces polymerisation When acetalehye is also present these effects of sulphur ioxie are no longer observe Acknowlegements The authors wish to thank Inrr WAKING an DUNCAN IIDDRY for statistical analysis, the C for funing this project uner FAIR (project no 89053) an NAT's Scientific Committee, Spain, for financial support for Dr ANNA PICINI References BAKKR, ; Picuve~m, A ; Baior e, P ; 1993 : Moel wine solutions : Colour an composition changes uring ageing Vitis 32, BARANWSKI, S ; Nacs~, C W; 1983 : Kinetics of malviin 3-glucosie conensation in moel wine systems Foo Sci 48, , 429 BURRUGHS, F ; 1974: Browning control in fruit juices an wines Chem In (Sep ) ; SPARKS, A H ; 1973 : Sulphite bining power of wines an ciers I quilibrium constants for the issociation of carbonyl bisulphite compouns Sci Foo Agricult 24, URD, ; 1964 : Reactions involve in sulphite bleaching of anthocyanins Foo Sci 29, ; SM&RS, T C ; 1970 : The formation of xanthylium salts from procyaniins Phytochemistry 9, NixeTic-Aexs~c, G K ; HRAZDINA, G ; 1972 : Quantitative analysis of the anthocyanin content in grape juices an wine ebensm -Wiss Technol 5, T~Meexnx, C F ; BRID, P ; 1967 : Flavylium salts, anthocyaniins an anthocyanins II Reactions with sulphur ioxie Sci Foo Agricult 18, , - ; 1976 : Interactions between anthocyanins, phenolic compouns an acetalehye an their significance in re wines Amer nol Viticult 27, Receive May 12, 1993