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B R O W N I N G O F W H I T E W I N E S N D N C C E L E R T E D T E S T

F O R B R O W N I N G C P C I T Y

V L S i n g l e t o n a n d T E K r a m l i n g a

D e p a r t m e n t o f Vi t i c u l t u r e a n d E n o l o g y, U n i v e r s i t y o f C a l i f o r n i a , D a v i s 95 6 16 . ~ P re s -en t addres s E . J . Gal lo W inery , Mo des to , Ca l i forn ia 95353.

T h e C a l i f o r n i a Wi n e A d v i s o r y B o a r d i s t h a n k e d f o r p a r t o f t h e s u p p o r t f o r t h i sw o r k , a n d a s e p a r a t e p o r t i o n o f t h e r e s e a r c h w a s s u p p o r t e d b y t h e K W V w h i l e V L S w a so n s a b b a t i c a l l e a v e a t t h e R e s e a r c h I ns t i t u t e f o r O e n o l o g y a n d Vi t i c u l t u r e , S t e l l e n b o s c h ,S o u t h A f r i c a .

A c c e p t e d f o r p u b l i c a t i o n O c t o b e r 4, 1 9 7 6.

A B S T R A C T

A s t a n d a r d i z e d t e s t f o r t h e b r o w n i n g c a p a c i t yo f w h i t e w i n e s w a s d e v e l o p e d. A p o r t i o n o f t h ew i n e i s t r e a t e d w i t h 1 g o f b e n t o n i t e p e r 1 0 0 m lu n d e r n i t r o g e n s o t h a t t h e d e v e l op e d b r o w n p i g -m e n t w i ll s t a y i n s o l u ti o n . P o r t i o n s o f th e t r e a t e d

w i n e a r e i n c u b a t e d i n s e a le d t u b e s w i t h 2 5 o rm o r e h e a d s p a c e w e l l s p a r g e d w i t h o x y g e n a n ds e p a r a t e l y w i t h n i t r o g e n f o r 5 d a y s a t 5 5 C . T h e

a b s o r b a n c e a t 4 2 0 n m r e m a i n s n e a r l y u n c h a n g ef o r t h e N 2 - s p a r g e d s a m p l e s a n d u n d e r o x y g em e a s u r e s w i n e s ' r e l a t i v e c a p a c i t y t o b r o w n o xd a t i v e l y. E v i d e n c e i s p r e s e n t e d t h a t a w i n e w ep r o t e c t e d f r o m o x i d a t i o n i s l a r g e l y p r e v e n t e d f r o

b r o w n i n g w h e r e a s a w i n e t r e a t e d b y a d d i n g d - c a r ec h i n i s c o n s i d e r a b l y i n c r e a s e d i n i t s c a p a c i t y tb r o w n b y t h is t e s t .

T h e r e h a v e b e e n a s i z a b l e n u m b e r o f s t u d i e su p o n t he b r o w n i n g o f w h i t e w i n e s . M o s t h a v e u se da r b i t r a r i l y c h o s e n c o n d i t i o n s t o p r o d u c e a n d m e a -s u r e t h e b r o w n , w i t h o u t s t u d y i n g t h e v a r i a b l e s .

B e rg ( 1 ) u s e d 20 0 m l o f w i n e i n a s c r e w - c a p p e db o t t l e l a rg e e n o u g h t o p r o v i d e a i r e q u i v a l e n t t o 6 0 0

m l 0 ., /1 . S a m p l e s w e r e h e l d 7 d a y s a t 4 9 C b e f o r ef i l t e r i n g ( i f n e c e s s a r y ) a n d c o m p a r i n g a b s o r b a n c ea t 42 5 n m w i t h t h e o ri g i n a l va l ue . W r i g h t ( 2 )u s e d a b s o r b a n c e a t 4 7 0 n m a f t e r s t o r a g e o f v e r-m o u t h s a m p l e s f o r 4 m o n t h s a t 2 , 2 0 , o r 3 0 C t oe s t i m a t e t h e r e l a t i v e b r o w n i n g o f d i f f e r e n t w i n e s .D e Vi l li e r s ( 3 ) u s e d a p o r t i o n o f e a c h w i n e i n as e a l e d f u l l f l a s k a t 4 C a s t h e c o n t r o l a n d m e a s u r e db r o w n i n g a t 4 2 5 n m a t 1 0 - d a y i n t e r v a l s a f t e r s t o r -i n g 1 5 0 m l o f w i n e w i t h 2 0 0 m l o f a i r a t 4 9 C .F u l l e r a n d B e rg ( 4 ) h e a t e d 2 0 m l o f w i n e p l u s 1 2m l o f a i r i n c l o s e d g l a s s t u b e s u n t i l i n c r e m e n t o fa b s o r b a n c e a t 4 4 0 n m r e a c h e d 0 . 1 5 0 i n a 1 2 - m mc o l o r i m e t e r t u b e . C a p u t i a n d P e t e r s o n ( 5 ) c o m -

o a r e d n i t r o g e n - s t r i p p e d a n d o x y g e n - s a t u r a t e d b o t -t l e d w i n e a f t e r 1 0 d a y s a t 4 0 C o r 1 0 0 d a y s a t r o o mt e m p e r a t u r e . D u P le s s is a n d U y s ( 7 ) h e a t e d s a m p l e si n 3 0 - m l s c r e w - c a p p e d b o t t l e s , l e a v i n g 2 m l a i r s p a c ea n d c e n t r i f u g i n g i f n e c es s a r y, a n d d e t e r m i n e d a b -s o r b a n c e p e r i o d i c a l l y i n l - c m c e l ls a t 4 2 5 n m . S a p i sa n d R i b 6 r e a u - G a y o n ( 8 ) s t o r e d w i n e s w i t h ex c e sso x y g e n i n s e a l e d b o t t l e s f o r 7 a n d 2 1 d a y s a t 4 0 C

b e f o r e m e a s u r i n g b r o w n i n g a t 4 4 0 n m . P e r i e t a( 9 ) e s t i m a t e d ( 4 50 n m ) e n z y m a t i c b r o w n i n g a5 C a n d n o n e n z y m i c b r o w n i n g a t 5 0 C i n 1 0 0 mof w ine in 500-ml con ica l f l a s ks fo r up to 15 day

B e r g a n d A k i y o s h i ( 1 0 ) s t u d i e d t h e p a r a m e t e ro f b r o w n i n g o f w h i t e w i n e s in so m e d e ta i l . T h e

s h o w e d t h a t i n c r e a s e s i n o x y g e n u p t o b u t n ob e y o n d 6 0 m l / 1 of w i n e g a v e g r e a t e r b r o w n i n g , d i d a ls o h i g h e r p H o r t e m p e r a t u r e . T h e y c o n c lu d et h a t e n z y m i c o x i d a t i o n w a s n o t s i g n i f i c a n t i n t hb r o w n i n g o f w h i t e w i n e .

I t a p p e a r e d t o u s d e s i r a b l e t o i n v e s t i g a t e f u r t h et h e c o n d i t i o n s f o r a r e p r o d u c i b l e a c c e l e r a t e d t e s t d e t e r m i n e t h e c a p a c i t y o f a w i n e t o b r o w n . I f s u ca t e s t c o u l d q u a n t i t a t e d i f f e r e n c e s a m o n g w i n e s a nc l a r i f y b r o w n i n g r e a c t i o n c o n d i t i o n s , i t s h o u l d bv e r y u s e fu l . S t a n d a r d i z a t i o n o f t e s t c o n d i ti o nw o u l d a l s o b e v e r y h e l p f u l i n c o m p a r i n g v a r i o uw o r k e r ' s r e s u l t s .

M AT E R I A L S A N D M E T H O D S

W h i t e w i n e s w i t h a l c o h ol le v e ls o f t a b l e w i n ew e r e p r e p a r e d b y s t a n d a r d m e t h o d s e i t he r a t D a vo r S t e l l e n b o s c h a n d u s e d i n t h e s e e x p e r i m e n t s . As o r b a n c e w a s d e t e r m i n e d a t 4 2 0 n m w i t h e i t h e r S p e c t r o n i c 20 ( D a v i s ) o r a S p e k k e r p h o t o m e t e( S t e l l e n b o s c h ) i n 1 8 - m m - I D te s t t u b e s . T h e s a m p l

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158--BROWNING OF WHITE WINES

to be tested for browning were placed in the testtubes (7-10 ml) sparge d with N2 or 02 gas, fir mlysealed with either a rubber stopper held in placeby electrician's plastic tape under tension or byrubber-lined screwcaps, and heated in racks in aregulat ed oven. The samples were removed period-ically, cooled to room temperature, absorbancemeasured, and ret urn ed to the oven.

R E S U LT S A N D D I S C U S S I O N

Oxygen content : For oxidative browning to pro-ceed as rapidly as possible, excess oxygen must beavailable thro ugho ut the reaction period. For thisreason 02 was used in the headspace rather than air.To be sure that 02 transfer into the solution was notl imiting the reaction rate, wine samples werebrowned with and without agitat ion. Agitation wasin a tissue-culture rotor, a 2-foot-diameter wheelturn ing at 1 rpm at about 60 from vertical , versusthe stat ic samples vert ical in a rack. Experi mentswere conducted also upon the minimum headspace

of 02 which gave maximum browning rate and pro-duced maximum brown pigment levels af ter pro-longed tre atm ent. Typical data are shown in Table1; this and other tests gave no significant differ-ences in brown color level in relation to agitationor 02 headspace as large as or larger than 5-ml/22-ml-capaci ty tube (23 %). Thus, the oxygen con-tent and transfer rate into the wine do not appearto be in any danger of l imiting the amount or rateof the reaction under the conditions in use here.

Wavelength The absorbance was measured at420 nm. This is commonly measur ed in red wine,along with 520 nm absorbance, to estimate brownversus red shades of color and is thus a usual value

for an enologist. Some brown ed wines exhibit ashoulder or broad maximum near 445 nm, thoughnot all do so, and such higher wavelengths would bemore subject to interference from possible pinkingof leucoanthocy anidins. Owing to the general facttha t brown s are spillover visible color from more

intense absorbance in the ultraviolet, many wave-lengths in the 400-480-nm range have been usedto measure br owning in various products. Never-theless, 420 nm is preferred for wine for the reasonsgiven and to provide standardization.

Sugar temp erature and am ino acids : he effectsof sugar and different temperatures upon browningof a Chardo nnay wine are shown in Table 2. Near-

ly identical data were obtained with a ThompsonSeedless wine. These data showed that a t 52C orbelow, glucose content was not an appreciable fac-tor in accelerated browning, whereas at 80C extrabrowning was caused by the presence of sugar.Fur ther tes t ing wi th in te rmedia te tempera tu resgave oxidative browning absorbance at 14 days in

Table 2. Effect of temperature, oxygen, and glucose onbrowning of a Chardonnay wine.

Absorbance (420 nm) a t i n d i c a t e d days

No glucose 10 glucose

C 0 2 8 14 0 2 8 14

Nitrogen-sparged

31 .18 0 .12 8 .13 3 .165

52 .181 .14 6 .16 0 .210

80 .18 3 .420 .375 .520

Oxygen-sparged

31 .18 0 .18 5 .21 2 .330

52 .178 .230 .380 .530

80 .180 .430 .690 .900

.180 . 127 .13 5 .190

.176 .1 38 .154 .210

.181 .255 .9 00 1.50

.180 .18 2 .205 .310

.171 .224 .3 60 .530

.183 .420 1.50 2.10

Table 3. Effect of amino acid addition on the abilit y of aw h i t e wine to brown under nitrogen at 52C.

Table 1. Effect of headspace oxygen and roller tubea g i t a t i o n on browning of a white table wine (18 x 150-mmt e s t tubes, 52C).

Amino acid added

AbsorbanceAmo unt at 420 nm aadded(mg /I) 26 days 44 days

Headspace

Absorbance at 420 nm

Agitated 0 days 3 days 7 days

5 ml no .133 .270 .460

10 ml no .133 .310 .450

15 ml no .133 .280 .460

5 ml yes .133 .290 .460

10 ml yes .133 .300 .480

15 ml yes .133 .300 .480

Control - - .278 .355

L-arginine monohy droch lorid e 250 .295 .360

L-g lut ami c acid 250 .292 .362

Glyc ine 250 .270 .368

L-histidine monoh ydroc hlor ide 250 .290 .390monohydrate

L-p rol ine 500 .310 .380

L-h ydr oxy pro lin e 500 .278 .365

L-t hre oni ne 250 .330 .365

Absorbance at 0 days for all samples was .175.

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B R O W N I N G O F W H I T E W I N E S - - 1 5

C h a r d o n n a y p l u s 1 0 g l u c os e t h a t a t 3 1 C w a s1 .0 6 t i m e s t h e a b s o r b a n c e o f t h e s a m e w i n e w i t h o u tg lucos e , 1 . 00 t im es a t 5 2C , 1 . 24 t im es a t 65C ,1 .3 9 t i m e s a t 7 2 C , a n d 2 . 8 8 t i m e s a t 8 0 C . I t t h u sa p p e a r s t h a t s u g a r i s o f n e g l i g ib l e e f f e c t a t 5 5 Co r b e l ow b u t i s i n c r e a s i n g l y i m p o r t a n t a s t e m p e r a -t u r e r i s e s, p a r t i c u l a r l y a t 8 0 C o r h i g h e r. T h i sc a r a m e l i z a t i o n e f f e c t i s e v i d e n t a l s o u n d e r n i t r o g e n

( Ta b l e 2 ) . T h e a d d i t i o n o f a m i n o a c i d s u n d e ro x i d a t i v e c o nd i t io n s o r u n d e r n i t r o g e n ( Ta b l e 3 )g a v e l i t t l e o r n o i n c r e a s e i n b r o w n i n g i n a d r yt a b l e w i n e o v e r t h a t i n t h e c o n t r o l s .

B e n t o n i t e t r e a t m e n t P r o t e i n s o r p o ly p e p t i d e se v i de n t ly i n t e r f e r e i n b r o w n i n g m e a s u r e m e n t s b yr e a c t i n g w i t h a n d p r e c i p i t a t i n g t h e b r o w n p i g m e n t .T h a t i s p r e v e n t e d b y a d d i n g a r e l a t i v e l y h e a v y d o s eo f b e n t o n i t e . To a v o i d d i l u ti o n p r o b l e m s t h e b e n -t o n i t e w a s a d d e d d r y t o t h e w i n e s a m p l e , m i x e di n u n t i l t h o r o u g h l y w e t t e d , a n d t h e w i n e h e l d u n d e rN 2 a t r o o m t e m p e r a t u r e f o r a b o u t 2 h o u r s w i t ho c c a si o n a l s h a k i n g . T h e w i n e w a s t h e n f r e e d o fb e n t o n i t e b y c e n t r i f u g a t i o n a n d f i l t r a t i o n t h r o u g ha m e m b r a n e ( 0 . 4 5 - m i c r o n ) f i l t e r . F i g . 1 s h o w st h a t w i t h o u t b e n t o n i t e t h e m a x i m u m b r o w n c o l o ri s l e s s a n d i n c r e a s e s r a p i d l y a s t h e b e n t o n i t e l e v e li s i n c r e a s e d t o 0. 3 84 g / 1 0 0 m l ( 3 2 l b / 1 0 0 0 g a l ) b u tve ry l i t t l e beyon d . Leve l s a s h ig h as 3 .84 , 7 . 68 , and1 5 .3 6 g o f b e n t o n i t e / 1 0 0 m l o f w i n e g a v e l i t t le o rn o f u r t h e r i n c r e a s e i n b r o w n i n g r a t e o r e x t e n t i na n y w i n e t e s t e d e x c e p t a s t h e p H b e g a n t o b e i n -c r e a s e d s l i g h t l y a t t h e s e e x t r e m e l e v e ls . T h e b e n -

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Fi g. 1 . T h e e f f e c t on b r o w n i n g c a u s e d b yp r i o r t r e a t m e n to f a w h i t e w i n e w i t h d i f f e r e n t l e v e l s o f b e n t o n i t e ,a - - 0 ben-ton i t e , b = 8 Ib /1000 ga l (0 .1 g /10 0 m l ) , c = 16 Ib /1000 ga l(0 .19 g /100 m l ) , d = 32 Ib /1000 g a l 0 . 3 8 g / 1 0 0 m l ) , e = 6 4Ib /1000 g a l (0 .77 g /10 0 m l ) , f = 128 Ib /1000 ga l (1 .54 g /100ml ) .

t o n i t e t r e t m e n t s d i d n o t f f e c t t h e t o t l p h e n o lc o n t e n t a p p r e c i a b l y, e . g. , a n u n t r e a t e d w i n e w i t1 63 m g G A E / 1 t o t a l p h e n o l a s sa y e d 1 60 m g G A E /a f t e r 3 . 8 4 g / 1 0 0 m l o f b e n t o n i t e a n d 1 6 8 mG A E / 1 a f t e r 7 .6 8 g . F u r t h e r t e s t s in d i c a t e d t h a3 . 8 4 g o f b e n t o n i t e p e r 1 0 0 m l m a y l o w e r w h i tw i n e t o t a l p h e n o l a b o u t 5 , a n d t h a t 7 . 68 g / 1 0 0 mu s u a l l y l o w e r e d it a b o u t 1 0 . C o n s i d e r i n g t h a

d i f f e r e n t l o t s of b e n t o n i t e m a y d i f f e r c o n s i d e r a b l yt h e t r e a t m e n t s h o u l d b e a s h i g h a s p o s s i b l e t o a l l of o r l e s s e f f e c t i v e b e n t o n i t e . F i g . 1 s h o w s t h a t l e v eo f 0 . 38 , 0 . 77 , and 1 . 54 g /100 ml g ive s imi la r r e s u l ta n d t h e o t h e r t e s t s s h o w e d t h a t t h e s e s h o u l d h a vl i t t l e o r n o e f f e c t o n t o t a l p h e n o l c o n t e n t o r p HF o r r o u t i n e u s e , 1 g o f b e n t o n i t e p e r 1 0 0 m l ow i n e i s r e c o m m e n d e d .

R e p r o d u c i b i l i t y : I f n o b e n t o n i t e i s u s e d , s o m ew i n e s p r o d u c e p r e c i p i t a t e s of b r o w n e d m a t e r i a l a nt h e d e v e l o p m e n t o f b r o w n i n s o l u t i o n i s d e c r e a s e( F i g . 1 ) . I f h e a v i l y b e n t o n i t e d , t h e s e w i n e s p r od u c e b r o w n p i g m e n t a t a u n i f o r m r a p i d r a t e ( F i g1 ) b u t e v e n t u a l l y r e a c h a m a x i m u m o f s o lu b le b r o wp i g m e n t . I n so m e s a m p l e s t h e b r o w n p i g m e n t th eb e g i n s t o d e c r e a s e b y p o l y m e r i z a t i o n a n d p r e c i p i t at io n . T h e m a x i m u m b r o w n i s a r e p r o d u c i b l e v a l ui f e i t h e r n o p r e c i p i t a t i o n o c c u r s o r t h e b r o w n p r ec i p i t a t e i s d is s o l v e d b y a c c u r a t e d i l u t i o n w i t h 6 0a q u e o u s e t h a n o l a t p H 3 .3 . Ta b l e 4 s h o w s t h a t r ep r o d u c i b l e v a l u e s w e r e o b t a i n e d i n t r i p l i c a t e t r i a lw i t h t w o d i f f e r e n t w i n e s.

Wi t h w i n e s h a v i n g c o n s i d e r a b l y d i f f e r e n t b r o w ni n g r a t e s t h e t i m e t o r e a c h m a x i m u m b r o w n i n t h e st e s t s ca n b e d i f f e r e n t ( F i g . 2 ) b e s i d e s r e q u i r i nt w o w e e k s o r m o r e f o r a t e st . D u r i n g t h e f i r s t d ao r t w o o f h e a t i n g o f a h e a v i l y b e n t o n i t e d w h i tw i n e i n t h e p r e s e n c e o f o x y g e n t h e w i n e m a y e v ed e c r e a s e i n b r o w n , m a y r e m a i n u n c h a n g e d , o r , m oc o m m o n l y, m a y b r o w n , t h o u g h a t a d i f f e r e n t r a tt h a n l a t e r ( F i g . 2 ) . F r o m a b o u t 3 t o 11 d a y s , h o we v e r, t h e r a t e o f b r o w n a b s o r b a n c e i n c r e a s e

T a b l e 4 . R e p r o d u c i b i l i t y o f t h e a c c e l e ra t e d m e t h o d u s e di n d u c e b r o w n i n g i n w h i t e w i n e s ( 0 .7 7 g b e n t o n i t e / 1 0 0 m52C) .

A b s o r b a n c e a t 4 20 n m a

S a m p l e 0 days 21 days 27 days

W h i t e Ries l ing .167 1 .30 1 .32W h i t e Rie slin g .165 1.30 1.31

W h i t e Ri esl ing .171 1.28 1.31

Ch a rd on nay .240 1.19 1.18

Cha rdon nay .241 1 .20 1 .20

Cha rdon nay .228 1 .21 1 .20

a For 21 and 27 da ys t h e a b s o r b a n c e v a l u e i s c a l c u l a t e df r o m t h e a b s o r b a n c e r e a d i n g o f o n e p a r t s a m p l e d i l u t e dw i t ht h r e e p a r t s o f 6 0 a q u e o u s e t h a n o l a d j u s t e d to p H 3 .3 .

A m . J . E n o l . Vi t i c . , Vo l . 2 7 , N o . 4 , 1 9 7 6

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1 6 0 - - B R O W N I N G O F W H I T E W I N E S

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