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Effect of pitching yeast and wort preparation on flavor stability of beer
JOURNAL Or FERMENTATIONAND BIOENGINEERING VOI. 73, NO. 6, 456-460. 1992
Effect of Pitching Yeast and Wort Preparation on Flavor Stability of Beer HIROTAKA KANEDA,* YUKINOBU KANO, TAKATOSHI SEKINE, SHUICHI ISHII, KATSUHISA TAKAHASHI, ArrD SHOUHEI KOSHINO
Brewing Research Laboratories, Sapporo Breweries Ltd., 10, Okatohme, Yaizu-shi, Shizuoka 425, Japan Received 7 February 1992/Accepted 15 April 1992 To confirm the role of fermentation conditions on flavor stability of beer and to brew a more stable beer, the effect of several fermentation conditions on the sulfite content in the finished beer and the flavor stability was studied using a pilot scale brewing process. Yeast strain significantly affected the suifite content, chemiluminenscence (CL) production which serves as an index of the susceptibility of beer to staling, and flavor stability of the resulting beer. Lower dissolved oxygen in pitching wort, higher pitching rate of yeast and clearer pitching wort led to the higher level of sulfite content, the inhibition of CL production, and better flavor stability of the resulting beers. These fermentation conditions did not significantly injure the fermentation process and change the flavor character of the fresh beer with the exception of the yeast strain. It was, therefore, expected that the flavor stability of beer could be controlled by the fermentation conditions without harming the fermentation process and the flavor character of the fresh beer.
Recently, the importance of shelf life of foods and beverages has greatly increased and that of beer is not exceptional. During the storage of beer, its quality gradually decreases; production of a stale flavor, formation of haze, and browning occurs. During these decreases in beer quality, flavor staling proceeds most rapidly and it is the most important problem for brewers. It has been thought that trans-2-nonenal produced during the process of beer oxidation is mainly responsible for the stale flavor of beer (1-3). So, brewers have tried to reduce the oxygen level in packaged beer and add antioxidants in order to inhibit the carbonyl producing reactions in beer. Sulfite is usually considered to prevent the flavor staling of beer and some breweries have used it for the stabilization of beer quality (4, 5). In terms of the contribution of sulfite to beer stabilization, two main opinions have been proposed but the actual conclusion has not been decided. First, sulfite prevents beer oxidation during storage, acting as an antioxidant. Kaneda et ai. (6) showed that sulflte inhibited the production of active oxygens and the progress of free radical reactions in beer which lead to flavor stability in beer. Recently, a new opinion that stale-flavor carbonyls such as trans-2-nonenal can be masked by the adducts formation with hydrogen sulfite has received particular attention (7-10). But the evidence to confirm this speculation has not been reported yet. It is well known that sulfite is excreted from yeasts during fermentation and survives in the finished beer. Many researchers have studied factors which influence sulfite formation by yeast during fermentation for the control of sulfite level in beer and for the stabilization of beer (11-13). These factors have been reported as being the yeast strain, wort pH, wort aeration, wort trub content, wort composition and nutritional deficiency, and fermentation temperature. However, there are very few studies to comfirm the relationship between wort fermentation and flavor stability of the resulting beer. Therefore, it is still a common question for brewers whether sulfite nat-
urally produced by yeast during wort fermentation can contribute to flavor stability of the finished beer. In a previous report (14), we studied the effect of fermentation temperature on flavor stability of the beer, using new analytical methods to determine the sulfite level in beer and flavor stability. It was shown that sulfite content in beer increased with decreasing fermentation temperatures and that control of the wort fermentation temperature could control the sulfite level and flavor stability of the finished beer. However, there is a danger that the fermentation temperature delays the fermentation rate of yeast and changes the flavor and taste character of the resulting beer, because it can significantly affect the yeast metabolism. In this report, effects of several fermentation conditions on flavor stability of the finished beer were studied in order to brew a more flavor-stable beer without degrading the fermentation process and flavor characteristics of the fresh beer. M A T E R I A L S A N D METHODS Wort production Wort was produced from malt, starch, rice, corn grits, and hop pellet by a decoction method in a pilot plant (400/). Wort clarification was carried out as follows; perlite was added to wort at a dosage of 100mg/l and the wort was then filtered with filter sheets. Beer production Yeasts (I) and (II) were bottom fermenting brewer's yeasts, Saccharomyces cerevisiae, which were stored in our laboratory. The wort was aerated before the start of the fermentation, placed in a fermentation tank (30/), and then fermented at 8°C. Five milliliters of the ethanol solution including 0.3 g of oleic acid and 0.3 g of linoleic acid was added to the wort (30/) after the clarification of pitching wort and just before the start of fermentation. Fermentation conditions used in this study are shown in Table 1. The young beer after fermentation was lagered at 0°C for 40 d. Treatments of the resulting beer and bottling were carried out as previously described
* Corresponding author. 456
FERMENTATION CONDITIONS AND BEER STABILITY
VOL. 73, 1992
457
TABLE 1. Fermentation conditions Brewing
Brewer's yeast
Dissolved oxygen (rag 02//)
Pitching rate ( x l07 cells/ml)
Wort clarification
Additions of fatty acidsa
A B C D E F
yeast (I) yeast (I) yeast (I) yeast (I) yeast (I) yeast (II)
8 4~5 8 8 8 8
1.5 1.5 2.0 1.5 1.5 1.5
No No No Yes Yes No
No No No No Yes No
" Ten mg/I of oleic and 10 mg/l of linoleic acids were added to pitching wort just before fermentation. (14).
Measurement o f wort turbidity Turbidity of pitching wort was measured with a NDH-1001 DP haze meter (Nippon Denshoku Kogyo Co. Ltd., Tokyo) and was shown as a kaolin concentration (mg//). D e t e r m i n a t i o n o f fatty acids and sulfate in wort or beer Fatty acids in wort or beer were extracted with ether-pentane (I : 1), concentrated, and determined using gas chromatography with n-pentadecanoic acid as the internal standard. Sulfate content was determined by a H P L C (ion exchange chromatography)-conductivity detection method. M e a s u r e m e n t o f c h e m i l u m i n e s c e n c e and suiflte in beer Production of chemiluminescence in beer was measured as previously described (14) and the total emission counts in the first 2 h after measurement were shown. Sulfite was determined with a HPLC-electrochemical detection method as previously reported (14). Sensory evaluation The staling degree of each beer stored at 37°C for 8 d was evaluated as previously described (14).
RESULTS A N D DISCUSSION Figure I shows the effect of dissolved oxygen in pitching wort on consumption curves of weft extract, n u m b e r of suspending yeast cells, and sulfite contents in young beers during fermentation. The consumption rate of wort extract and yeast cell growth were inhibited by a decrease in the level of dissolved oxygen. The formation of endogenepus sulfite into wort from yeast started after the maximum n u m b e r of yeast cells was reached, and accelerated in the low dissolved oxygen sample. It has been reported that sulfite production in yeast cells has some close relation120F
oot
10
~S 6
~
60
4
~-~ ~
40
---
2O
ship with respiration systems (12, 15). Ohno and Takahashi studied the effect of wort aeration on sulfite production in the brewing process and concluded that the activation of growth and inhibition of sulfite production by oxygen supply necessarily occur at the same time. A high pitching rate of yeast and wort clarification accelerated the excretion of endogeneous sulfite from the yeast during the fermentation (Fig. 2). The wort clarification inhibited the consumption rate of wort extract and yeast cell growth. In the high pitching rate, the specific growth rate of yeast ceils was lower than that in the low pitching rate, though it led to a higher consumption rate of wort extract and a higher n u m b e r of suspended yeast during the fermentation. It seems that the high pitching rate results in lowering the oxygen consumption by each yeast cell, leading to the inhibition of yeast cell growth and the acceleration of sulfite production. These conditions did not significantly delay nor stop the fermentation process. For these fermentation conditions, an increase in the sulfite content was observed during the lagering periods (5-20/~mol//). Tables 2--4 show the effect of dissolved oxygen in pitching wort, pitching rate, and wort clarification on sulfite content of the finished beer, chemiluminescence (CL) production of the beer before and after storage at 37°C for 8 d, and flavor stability of the beer. Low dissolved oxygen in pitching wort, high pitching rate, and weft clarification led to an increase in the sulfite content of the finished beer and the inhibition of the CL production of the fresh and aged beer, and improved the flavor stability of the beer stored at 37°C for 8 d. The differences in the CL intensities of fresh and aged beers which shows the increase in the CL intensity with beer aging were significantly decreased
120r
I
,,,/
5~
lO
ta 2
I
I
I
I
2 4 6 8 Ferment, period (d)
/0 10
J 0
10 2 4 6 8 Ferment, period (d)
FIG. 1. Effectof dissolved oxygen in pitching wort on consumption curve of wort extract, number of yeast cells, and sulfite content in young beer during fermentation. (A) Open symbols show the consumption curves of wort extract and closed symbols show the number of yeast cells. O and o, brewing condition A in Table 1; zx and A, brewing condition B. (B) O, brewing condition A; zx, brewing condition B.
'°'f / 80
~s
4
0
(B)
ioo ~-
I
I
I
I
2 4 6 8 Fement, period (d)
3~
~ 60
I0 ~
0
2
4
6
8
I0
Ferment. perlod (d)
FIG. 2. Effectof pitching rate of yeast and weft clarification on consumption curve of wort extract, number of yeast cells, and sulfite content in young beer during fermentation. (A) Open symbols show the const~mptioncurves of wort extract and closed symbols show the number of yeast cells. O and e , brewing condition A in Table l; o and s , brewing condition C; A and A, brewing condition D. 03) O, brewing condition A; o, brewing condition C; A, brewing condition D.
458
J. FERMENT. BIOENG.,
KANEDA ET AL.
TABLE 2.
Effect of dissolved oxygen in pitching wort on flavor stability of the finished beer
Sulfite content CL intensity of beer Brewing of ( x 106 counts/2 h) condition ~ finished beer (umol//) fresh aged b A B
80 125
1.05 0.94
1.74 (0.69) ~ 1.12 (0.18)
Staling degree of aged beer 6 fresh stale 1 , 5 3.5 e 2.8
All data are the averages of three replicated brewings. Brewing conditions were the same as in Table 1. b Beer was stored at 37°C for 8 d. Each value shows a difference between CL intensities of fresh and aged beer. d Staling degree is evaluated as cited in the Materials and Methods. TABLE 3.
Effect of pitching rate of yeast on flavor stability of the finished beer
Sulfite content CL intensity of beer Brewing of ( x 106 counts/2 h) condition~ finished beer 0tmol//) fresh aged b A C
57 82
1.07 1.05
1.90 (0.83)c 1.47 (0.42)
Staling degree of aged beer b fresh stale 1 ) 5 3. I a 2.5
All data are the averages of three replicated brewings. a Brewing conditions were the same as in Table 1. b Beer was stored at 37°C for 8 d. c Each value shows a difference between CL intensities of fresh and aged beer. d Staling degree is evaluated as cited in the Materials and Methods.
b y t h e m . A s p r e v i o u s l y r e p o r t e d (16-18), t h e C L p r o d u c t i o n o c c u r s d u r i n g t h e p r o c e s s o f free r a d i c a l r e a c t i o n s i n beer. T h e C L i n t e n s i t y s h o w s t h e o x i d a t i o n degree o f beer a n d h a s a g o o d c o r r e l a t i o n w i t h t h e s t a l i n g degree o f t h e beer flavor. So, t h e difference in t h e C L intensities i n d i c a t e s t h e s t a l i n g degree o f t h e beer d u r i n g t h e s t o r a g e at 3 7 ° C . It was also s h o w n t h a t sulfite i n h i b i t s t h e p r o d u c t i o n o f active o x y g e n s , t h e p r o c e s s o f free r a d i c a l r e a c t i o n s , a n d t h e C L p r o d u c t i o n o f b e e r as a r a d i c a l scavenger, a n d leads to g o o d flavor s t a b i l i t y (6, 14). It was, t h e r e f o r e , t h o u g h t t h a t b e e r f e r m e n t e d u n d e r t h e c o n d i t i o n s o f low d i s s o l v e d o x y g e n in p i t c h i n g w o r t , h i g h p i t c h i n g r a t e , o r w o r t clarification h a s a h i g h t o l e r a n c e t o o x i d a t i o n b e c a u s e o f its h i g h sulfite c o n t e n t , l e a d i n g to g o o d flavor s t a b i l i t y o f t h e b e e r . T a b l e 5 s h o w s a n effect o f t h e s e f e r m e n t a t i o n c o n d i t i o n s o n c o n t e n t s o f flavor c o m p o n e n t s i n t h e fresh beer. N o significant difference i n t h e f l a v o r c h a r a c t e r s o f t h e s e beers was o b s e r v e d . T h i s r e s u l t was also c o n f i r m e d in a sensory e v a l u a t i o n . I n t e r m s o f t h e r e l a t i o n s h i p b e t w e e n w o r t clarification TABLE 4.
Brewing condition" A D
Contents of flavor components (rag//) in the finished beer
Brewing condition" Isobutanol n-Propanol Isoamyl alcohol Furfuryl alcohol Methionol t%Phenylethy[ alcohol Ethyl acetate Isoamyl acetate Ethyl caproate Ethyl caprylate Ethyl caprate /~-Phenylethyl acetate Caproic acid Caprylic acid Capric acid 9-Decenoic acid Acetaldehyde Acetone Diacetyl Acetoin
A
B
C
D
8. I 9.7 51 0.67 1.2 19 22 1.5 0.13 0.14 0.01 0.35 1.8 3.3 0.38 0.09 9.2 0.8 0.03 2.7
8.1 9.6 50 0.69 1.3 19 24 1.7 0.13 0.14 0.01 0.36 1.8 3.3 0.35 0.06 8.1 0.8 0.03 2.6
8.1 9.8 50 0.62 1.2 16 23 1.5 0.13 0.14 0.01 0.33 1.7 3.0 0.37 0.07 7.8 0.8 0.03 2.3
7.8 9.0 50 0.61 1.1 18 20 1.5 0.14 0.13 0.01 0.34 1.9 3.4 0.46 0.05 8.5 0.8 0.04 2.5
All data are the average of three replicated brewings. Brewing conditions were the same as in Table i.
a n d flavor stability o f b e e r , several i n v e s t i g a t i o n s h a v e b e e n c a r r i e d o u t . It h a s b e e n r e p o r t e d t h a t p o o r s e p a r a t i o n o f t h e s p e n t g r a i n s a n d t r u b results in c l o u d y w o r t w i t h h i g h e r lipid c o n t e n t a n d u n e q u i v o c a l l y d a m a g e s t h e f l a v o r s t a b i l i t y o f t h e finished beer (9, 19, 20). It h a s b e e n t h o u g h t t h a t the c a r r y - o v e r o f u n s a t u r a t e d f a t t y acids s u c h as oleic, linoleic, a n d l i n o l e n i c acids i n t o p i t c h i n g w o n is a n imp o r t a n t p r o b l e m , b e c a u s e a p a r t o f t h e m is s u r v i v e d in p a c k a g e d b e e r a n d o x i d i z e d to p r o d u c e trans-2-nonenal w h i c h is m a i n l y r e s p o n s i b l e for a n y staling off-flavor. D r o s t et al. (9) d e m o n s t r a t e d t h a t t h e o x i d a t i o n p r o d u c t s o f m a l t lipids p r o d u c e d e n z y m a t i c a l l y or n o n e n z y m a t i c a l l y d u r i n g w o r t m a k i n g s u r v i v e d in t h e finished beer a n d was r e s p o n s i b l e f o r f l a v o r staling. O n t h e o t h e r h a n d , D u f o u r et al. (12) s t u d i e d t h e r e l a t i o n s h i p b e t w e e n w o r t clarificat i o n a n d sulfite p r o d u c t i o n b y yeast d u r i n g f e r m e n t a t i o n a n d c o n c l u d e d t h a t c l o u d y w o r t w i t h h i g h e r lipid c o n t e n t a c c e l e r a t e d t h e yeast cell g r o w t h d u r i n g f e r m e n t a t i o n a n d t h e c o n s u m p t i o n o f e n d o g e n e o u s sulflte f o r t h e p r o d u c t i o n o f s u l f u r - c o n t a i n i n g a m i n o acids, l e a d i n g to t h e low level o f sulfite c o n t e n t in y o u n g b e e r . T h e a d d i t i o n o f oleic a n d linoleic acids t o clarified w o r t a c c e l e r a t e d t h e yeast cell g r o w t h a n d the c o n s u m p t i o n r a t e o f w o r t e x t r a c t b y yeast a n d t h o r o u g h l y i n h i b i t e d t h e exc r e t i o n o f sulfite i n t o w o r t d u r i n g f e r m e n t a t i o n (Fig. 3). H o w e v e r , t h e a d d i t i o n o f t h e f a t t y acids a n d w o r t clarifica-
Effect of clarification of pitching wort on flavor stability of the finished beer
Turbidity of pitching wort(mg//) 43 3
TABLE 5.
CL intensity of beer ( x 10~counts/2 h)
Sulfite content of finished beer ~mol//)
fresh
aged b
65 91
1.01 0.93
1.62 (0.61) c 1.23 (0.30)
All data are the averages o f three replicated brewings. * Brewing conditions were the same as in Table I. b Beer was stored at 37eC for 8 d. c Each value shows a difference between CL intensities of fresh and aged beer. d Staling degree is evaluated as cited in the Materials and Methods.
Staling degree of aged beer b fresh 1
, 3.0d 2.4
stale 5
Vot. 73, 1992
FERMENTATION CONDITIONS AND BEER STABILITY 120
10
15 ~
"
,--t
TABLE 7.
(B)
100
8
4~ ==
6
3~
~ 6o
4
2o.~
~_
Effectof addition of oleic and linoleic acids on fermenta tion on flavor stability of the finished beer
Sulfite content CL intensity of beer Brewing of ( x I0~counts/2 h) condition * finished beer (pmol//) fresh agedb
A
8o
D E
40'
45t.
120 39
1.00 1.18
Staling degree of aged beerb fresh stale I , 5
1.21 (0.21)c 2.01 (0.83)
2.6d 4.0
I
i-
2 I
I
I
I
2 4 6 8 Ferment. period (d)
i0
Brewing conditions were the same as in Table 1. b Beer was stored at 37°C for 8 d. ¢ Each value shows a difference between CL intensities of fresh am aged beer. e Staling degree is evaluated as cited in the Materials and Methods
2C I
o~ 0 2 4 6 8 FeFment, PerloO (0}
-
I0
FIG. 3. Effectof oleic and linoleic acids on consumption curve of wort extract, number of yeast cells, and sulfite content in young beer during fermentation. (A) Open symbols show the consumption curves of wort extract and closed symbols show the number of yeast cells. (3 and o , brewing condition D in Table l; ,x and A, brewing condition E (B) O, brewing condition D; zx, brewing condition E.
before and after storage at 37°C for 8 d was acceleratet and flavor stability was decreased by the addition o f thesq fatty acids, indicating that oleic and linoleic acids in wor affected the flavor stability o f the resulting beer. In Table 6, the contents o f fatty acids in wort, younl beer, and finished beer with different brewing co n di t i on are shown. A b o u t 50-60% o f unsaturated fatty acids sucl as oleic, linoleic, and linoleic acids was excluded by thq wort clarification. The turbidity o f pitching wort was sig nificantly decreased by this treatment (Table 4). It seem that the several hydrophobic materials, which contain fat ty acids, may be excluded by this treatment. The content o f myristic acid (C 14 : 0), palmitic acid (C 16 : 0), olei, acid (C 18 : I), linoleic acid (C 18 : 2), and linolenic acid (( 18 : 3) decreased during the fermentation, while cupric acit (C 10 : 0), 9-decenoic acid (C 10 : 1), and laurie acid (( 12 : 0) increased. When oleic and linoleic acids were adde( to wort just before the fermentation, the contents o f thes, unsaturated fatty acids significantly decreased during th, fermentation and were finally at trace levels in the finisher beer. The significant differences in the other acids durinj fermentation were not observed. H a s h i m o t o (21) ant Kaneda et al. (22) reported that longer fatty acids that sur vived in beer could not significantly contribute to th, flavor staling. Therefore, it is unlikely that high content o f longer unsaturated fatty acids in pitching wort can sur rive in the finished beer and participate in the deterioratio] o f the beer flavor. It seems that the kinds and the content o f fatty acids in pitching wort are important factors to de
tion did not significantly affect the consumption o f sulfate, which is a precursor o f sulfite in the yeast metabolism, in pitching wort during fermentation (Table 6). D u f o u r et al. (12) reported that SO2 excretion drastically increased when yeast growth slowed down or stopped. They concluded that in the absence o f growth but under fermenting conditions, yeast cells do not further synthesize sulfur-containing amino acids. But the reactions leading to the synthesis of SO2 can still progress with a concomitant intracellular accumulation o f SO2 level which is excreted in the wort. The sulfite level in young beer is thus dependent on yeast growth. However, Ohno and Takahashi (15) speculated that the decrease in sulfite producing ability o f yeast by the addition o f lipids into wort might not depend on cell growth, but on change in the activity o f the corresponding enzyme. The mechanism o f sulfite production in yeast cells needs to be clarified with more detailed studies. Table 7 shows sulfite content, CL intensity, and flavor stability of the beer fermented with the addition o f oleic and linoleic acids. Sulfite content in the finished beer was 39 p m o l / l though the excretion o f sulfite into wort was thoroughly inhibited during the main fermentation (Fig. 3), indicating the excretion o f endogeneous sulfite f r o m yeast during lagering. The CL production o f the beer TABLE 6. Sample Brewing condition"
Changes in contents of fatty acids (mg//) and sulfate (mg//) in brewing
A
Pitching wort D
E
A
Young beer D
E
A
Packaged beer D
E
Fatty acid C 10 : 0 C 10 : 1 C 11 : 0 C 12 : 0 C 14 : 0 C 16 : 0 C 16 : 1 C 18 : 0 C 18 : 1 C 18 : 2 C 18 : 3 C 20 : 0
0.01 0.03 0.02 0.01 0.05 0.14 tr. b 0.02 0.04 0.17 0.17 0.01
0.01 0.02 0.01 0.01 0.04 0.10 tr. 0.02 0.02 0.06 0.06 tr.
0.01 0.02 0.01 0.01 0.04 0.10 tr. 0.02 10.00 10.10 0.06 tr.
0.67 0.11 0.01 0.25 0.03 0.04 tr. 0.02 0.02 tr. n.d. c n.d.
0.71 0.11 0.01 0.28 0.03 0.04 tr. 0.01 0.01 tr. tr. n.fl.
0.69 0.11 0.01 0.20 0.02 0.08 tr. 0.04 0.37 0.17 n.d. n.d.
0.79 0.13 0.01 0.08 0.01 0.06 0.01 0.01 tr. tr. 0.01 n.d.
0.80 0.09 0.01 0.08 0.01 0.06 0.01 0.01 tr. tr. 0.01 n.d.
0.90 0.17 0.01 0.08 0.01 0.06 0.01 0.01 tr. tr. 0.01 n.d.
Sulfate
70
72
74
51
57
59
55
54
60
" Brewing conditions were the same as in Table 1. b tr., Trace. c n.d., No detect.
460
J. FERMEI~T. BIOENG.,
KANEDA ET AL.
TABLE 8.
Effect of brewer's yeast strain on flavor stability of the finished beer
Sulfite content CL intensity of beer Brewing of ( × 106 counts/2 h) conditiona finished beer ~mol//) fresh aged b A F
73 35
1.00 1.28
Staling degree of aged beerb fresh stale I ----, 5
1.58 (0.58) ~ 2.16 (0.88)
3.0'1 4.0
• Brewing conditions were the same as in Table I. b Beer was stored at 37°C for 8d. c EachvalueshowsadifferencebetweenCLintensitiesoffreshand
aged beer. e Staling degree is evaluated as cited in the Materials and Methods. t e r m i n e t h e flavor s t a b i l i t y o f t h e r e s u l t i n g beer, b e c a u s e they affect yeast m e t a b o l i s m d u r i n g f e r m e n t a t i o n a n d t h e sulflte c o n t e n t o f t h e finished beer. In this s t u d y , it c o u l d n o t b e clarified w h e t h e r t h e o x i d a t i o n p r o d u c t s o f lipids, w h i c h o c c u r r e d d u r i n g t h e w o r t m a k i n g process, h a d a r e l a t i o n s h i p w i t h flavor staling o f t h e r e s u l t i n g beer. T h e s t u d y t o clarify t h e rote o f t h e w o r t m a k i n g p r o c e s s e s o n t h e flavor s t a b i l i t y o f beer is i n p r o g r e s s . T a b l e 8 s h o w s the effect o f yeast s t r a i n o n t h e sulfite c o n t e n t i n t h e finished b e e r , t h e C L p r o d u c t i o n o f t h e beer b e f o r e a n d a f t e r its s t o r a g e a t 370C f o r 8 d, a n d t h e degree o f f l a v o r staling o f t h e b e e r s t o r e d at 3 7 ° C f o r 8 d. T h e sulrite c o n t e n t in t h e beer b r e w e d w i t h yeast (II) was lower t h a n t h a t w i t h yeast (I). T h e b e e r o f yeast (II) h a d a h i g h e r C L p r o d u c i n g activity a n d less f l a v o r s t a b i l i t y t h a n t h a t o f y e a s t (I). T h e flavor c h a r a c t e r o f t h e r e s u l t i n g beers were different i n a s e n s o r y test; t h e b e e r b r e w e d w i t h yeast (I) s h o w e d a m o r e esteric c h a r a c t e r t h a n w i t h yeast (II). It was t h o u g h t t h a t yeast s t r a i n s c o u l d significantly affect n o t o n l y flavor c h a r a c t e r b u t also f l a v o r s t a b i l i t y o f beer. B a s e d o n t h e results p r e s e n t e d so far, it was c o n c l u d e d t h a t f e r m e n t a t i o n c o n d i t i o n s p l a y a n i m p o r t a n t role in flavor s t a b i l i t y o f beer. It was e x p e c t e d t h a t flavor stability o f b e e r c o u l d b e c o n t r o l l e d b y t h e f e r m e n t a t i o n c o n d i tions without harming the fermentation process and the flavor c h a r a c t e r s o f t h e f r e s h b e e r . It w a s also r e p o r t e d t h a t s t a r v i n g t i m e o f yeast a n d " d a r a u f l a s s e n " , fresh w o r t being a d d e d to t h e f e r m e n t i n g w o r t a t a n i n t e r v a l o f several h o u r s , significantly affected t h e c o n t e n t o f s u l f u r d i o x i d e in y o u n g b e e r (13). T h e s t u d y o f t h e r e l a t i o n s h i p b e t w e e n ferm e n t a t i o n c o n d i t i o n s a n d f l a v o r s t a b i l i t y o f beer will b e s t e p p e d u p f r o m t h e p i l o t line t o f a c t o r y scale, i n o r d e r t o b r e w a m o r e s t a b l e a n d f a v o r a b l e beer. ACKNOWLEDGMENTS The authors would like to thank Mr. T. Kirnura and Mr. M. Kobayashi, Brewing Research Laboratories, Sapporo Breweries Ltd., for analyzing the beer and won components and Mr. J. Murakami for his helpful advice.
REFERENCES 1. Jamieson, A. M. and van Gheluwe, J. E. A.: Identification of a compound responsible for cardboard flavor in beer. Pro¢. Am. So¢. Brew. Chem., 28, 192-197 (1970). 2. Meilgaard, M.: Stale flavor carbonyls in brewing. The Brewers Digest, 47 (4), 48-57 (1972). 3. Wang, P. S. and Siebert, K. J.: Determination of t r a n s - 2 - n o n e n a l in beer. Tech. Quart. Master Brew. Ass. Am., 11, 110-117 (1974). 4. Ryder, D. S., Power, J., and Siebel, R. E.: A question of flavor stability or instability. Proc. Inst. Brew., Central & Southern Africa Section, 366-391 (1989). 5. Raymond, ,I., Klimovitz, R. J., and Kindnaka, J. A.: The impact of various antioxidants on flavor stability. Tech. Quart. Master Brew. Ass. Am., 29, 70-74 (1989). 6. Kaneda, H., Kano, Y., Osawa, T., Kawakishi, S., and Koshino, S.: Role of active oxygens on deterioration of beer flavor. Eur. Brew. Cony. Proc. 23rd Congr., 433-440 (1991). 7. Barker, R. L,, Gracey, D. E. F., Irwin, A.J., Pipasts, P., and Leiska, E.: Liberation of staling aldehydes during storage of beer. J. Inst. Brew., 89,411--415 (1983). 8. Graeey, D. E. F., Barker1 R.L., Irwin, A.J., Pipasts, P., and Leiska, E.: Chemical equilibria of beer flavor stability. Proc. 18th Inst. Brew., Australia & New Zealand Section, 50-58 (1984). 9. Drost, B. W., van den Berg, R., Freijee, F. J. M., van der Velde, E. G., and Hollemans, M.: Flavor stability. J. Am. Soc. Brew. Chem., 48, 124-131 (1990). I0. Nordi6v, H. and Winell, B.: Beer flavour stabilization by interaction between bisulfite and trans-2-nonenal. Eur. Brew. Cony., Proc. 20th Congr., 291-298 (1985). 11. Brewer, J. D. and Fenton, M. S.: The formation of sulphur dioxide during fermentation. Proe. Cony. Inst. Brew., Australia & New Zealand Section, 16, 155-164 (1980). 12. Dufour, J.-P., Carpentier, B., Kulalumba, M., Haeeht, J. L., and Devreux, A.: Alteraction of SO2 production during fermentation. Eur. Brew. Cony., Proe. 22nd Congr., 331-338 (1989). 13. Dufour, J.-P.: Influence of industrial brewing and fermentation working conditions on beer SO2 level and flavor stability. Eur. Brew. Cony., Proc. 23rd Congr., 209-216 (1991). 14. Kaneda, H., Kimura, T., Kano, Y., Koshino, S., Osawa, T., and Kawakishi, S.: Role of fermentation conditions on flavor stability of beer. J. Ferment. Bioeng., 72, 26-30 (1991). 15. Ohno, T. and Takahashi, R.: Production of sulfite in the brewing process. Rept. Res. Lab. Kirin Brew. Co. Ltd., 30, 15-19 (1987). 16. Kaneda, H., Kano, Y., Kamimura, M., Osawa, T., and Knwakishi, S.: Role of free radicals in chemiluminescence produced during beer oxidation. Agric. Biol. Chem., 54, 21652166 (1990). 17. Kaneda, H., Kano, Y., Kamimura, M., Osawa, T., and Kawakishi, S.: Evaluation of beer deterioration by chemiluminescence. J. Food Sci., 55, 1361-1364 (1990). 18. Kaneda, H., Kano, Y., Osawa, T., Kawakishi, S., and Kamimura, M.: The study of beer staling by chemiluminescence analysis..i.. Inst. Brew., 97, 105-109 (1991). I9. Olsen, A.: The role of wort turbidity in flavour and flavour stability. Eur. Brew. Cony. Monogr., 7, 223-236 (1981). 20. Narziss, L.: Centenary review: technological factors of flavour stability. J. Inst. Brew., 92, 346-353 (1986). 21. Hashimoto, N.: Melanoidin-mediated oxidation; a greater involvement in flavor staling. Techn. Rep. Kirin, 31, 19-32 (1988). 22. Kaneda, H., Kano, Y., Kamimura, M., Osawa, T., and Kawakishi, S.: Analysis of long-chain fatty acids in beer by HPLC-fluorescence detection method. J. Agric. Food Chem., 38, 1363-1367 (1990).
Effect of Pitching Yeast and Wort Preparation on Flavor Stability of Beer HIROTAKA KANEDA,* YUKINOBU KANO, TAKATOSHI SEKINE, SHUICHI ISHII, KATSUHISA TAKAHASHI, ArrD SHOUHEI KOSHINO
Brewing Research Laboratories, Sapporo Breweries Ltd., 10, Okatohme, Yaizu-shi, Shizuoka 425, Japan Received 7 February 1992/Accepted 15 April 1992 To confirm the role of fermentation conditions on flavor stability of beer and to brew a more stable beer, the effect of several fermentation conditions on the sulfite content in the finished beer and the flavor stability was studied using a pilot scale brewing process. Yeast strain significantly affected the suifite content, chemiluminenscence (CL) production which serves as an index of the susceptibility of beer to staling, and flavor stability of the resulting beer. Lower dissolved oxygen in pitching wort, higher pitching rate of yeast and clearer pitching wort led to the higher level of sulfite content, the inhibition of CL production, and better flavor stability of the resulting beers. These fermentation conditions did not significantly injure the fermentation process and change the flavor character of the fresh beer with the exception of the yeast strain. It was, therefore, expected that the flavor stability of beer could be controlled by the fermentation conditions without harming the fermentation process and the flavor character of the fresh beer.
Recently, the importance of shelf life of foods and beverages has greatly increased and that of beer is not exceptional. During the storage of beer, its quality gradually decreases; production of a stale flavor, formation of haze, and browning occurs. During these decreases in beer quality, flavor staling proceeds most rapidly and it is the most important problem for brewers. It has been thought that trans-2-nonenal produced during the process of beer oxidation is mainly responsible for the stale flavor of beer (1-3). So, brewers have tried to reduce the oxygen level in packaged beer and add antioxidants in order to inhibit the carbonyl producing reactions in beer. Sulfite is usually considered to prevent the flavor staling of beer and some breweries have used it for the stabilization of beer quality (4, 5). In terms of the contribution of sulfite to beer stabilization, two main opinions have been proposed but the actual conclusion has not been decided. First, sulfite prevents beer oxidation during storage, acting as an antioxidant. Kaneda et ai. (6) showed that sulflte inhibited the production of active oxygens and the progress of free radical reactions in beer which lead to flavor stability in beer. Recently, a new opinion that stale-flavor carbonyls such as trans-2-nonenal can be masked by the adducts formation with hydrogen sulfite has received particular attention (7-10). But the evidence to confirm this speculation has not been reported yet. It is well known that sulfite is excreted from yeasts during fermentation and survives in the finished beer. Many researchers have studied factors which influence sulfite formation by yeast during fermentation for the control of sulfite level in beer and for the stabilization of beer (11-13). These factors have been reported as being the yeast strain, wort pH, wort aeration, wort trub content, wort composition and nutritional deficiency, and fermentation temperature. However, there are very few studies to comfirm the relationship between wort fermentation and flavor stability of the resulting beer. Therefore, it is still a common question for brewers whether sulfite nat-
urally produced by yeast during wort fermentation can contribute to flavor stability of the finished beer. In a previous report (14), we studied the effect of fermentation temperature on flavor stability of the beer, using new analytical methods to determine the sulfite level in beer and flavor stability. It was shown that sulfite content in beer increased with decreasing fermentation temperatures and that control of the wort fermentation temperature could control the sulfite level and flavor stability of the finished beer. However, there is a danger that the fermentation temperature delays the fermentation rate of yeast and changes the flavor and taste character of the resulting beer, because it can significantly affect the yeast metabolism. In this report, effects of several fermentation conditions on flavor stability of the finished beer were studied in order to brew a more flavor-stable beer without degrading the fermentation process and flavor characteristics of the fresh beer. M A T E R I A L S A N D METHODS Wort production Wort was produced from malt, starch, rice, corn grits, and hop pellet by a decoction method in a pilot plant (400/). Wort clarification was carried out as follows; perlite was added to wort at a dosage of 100mg/l and the wort was then filtered with filter sheets. Beer production Yeasts (I) and (II) were bottom fermenting brewer's yeasts, Saccharomyces cerevisiae, which were stored in our laboratory. The wort was aerated before the start of the fermentation, placed in a fermentation tank (30/), and then fermented at 8°C. Five milliliters of the ethanol solution including 0.3 g of oleic acid and 0.3 g of linoleic acid was added to the wort (30/) after the clarification of pitching wort and just before the start of fermentation. Fermentation conditions used in this study are shown in Table 1. The young beer after fermentation was lagered at 0°C for 40 d. Treatments of the resulting beer and bottling were carried out as previously described
* Corresponding author. 456
FERMENTATION CONDITIONS AND BEER STABILITY
VOL. 73, 1992
457
TABLE 1. Fermentation conditions Brewing
Brewer's yeast
Dissolved oxygen (rag 02//)
Pitching rate ( x l07 cells/ml)
Wort clarification
Additions of fatty acidsa
A B C D E F
yeast (I) yeast (I) yeast (I) yeast (I) yeast (I) yeast (II)
8 4~5 8 8 8 8
1.5 1.5 2.0 1.5 1.5 1.5
No No No Yes Yes No
No No No No Yes No
" Ten mg/I of oleic and 10 mg/l of linoleic acids were added to pitching wort just before fermentation. (14).
Measurement o f wort turbidity Turbidity of pitching wort was measured with a NDH-1001 DP haze meter (Nippon Denshoku Kogyo Co. Ltd., Tokyo) and was shown as a kaolin concentration (mg//). D e t e r m i n a t i o n o f fatty acids and sulfate in wort or beer Fatty acids in wort or beer were extracted with ether-pentane (I : 1), concentrated, and determined using gas chromatography with n-pentadecanoic acid as the internal standard. Sulfate content was determined by a H P L C (ion exchange chromatography)-conductivity detection method. M e a s u r e m e n t o f c h e m i l u m i n e s c e n c e and suiflte in beer Production of chemiluminescence in beer was measured as previously described (14) and the total emission counts in the first 2 h after measurement were shown. Sulfite was determined with a HPLC-electrochemical detection method as previously reported (14). Sensory evaluation The staling degree of each beer stored at 37°C for 8 d was evaluated as previously described (14).
RESULTS A N D DISCUSSION Figure I shows the effect of dissolved oxygen in pitching wort on consumption curves of weft extract, n u m b e r of suspending yeast cells, and sulfite contents in young beers during fermentation. The consumption rate of wort extract and yeast cell growth were inhibited by a decrease in the level of dissolved oxygen. The formation of endogenepus sulfite into wort from yeast started after the maximum n u m b e r of yeast cells was reached, and accelerated in the low dissolved oxygen sample. It has been reported that sulfite production in yeast cells has some close relation120F
oot
10
~S 6
~
60
4
~-~ ~
40
---
2O
ship with respiration systems (12, 15). Ohno and Takahashi studied the effect of wort aeration on sulfite production in the brewing process and concluded that the activation of growth and inhibition of sulfite production by oxygen supply necessarily occur at the same time. A high pitching rate of yeast and wort clarification accelerated the excretion of endogeneous sulfite from the yeast during the fermentation (Fig. 2). The wort clarification inhibited the consumption rate of wort extract and yeast cell growth. In the high pitching rate, the specific growth rate of yeast ceils was lower than that in the low pitching rate, though it led to a higher consumption rate of wort extract and a higher n u m b e r of suspended yeast during the fermentation. It seems that the high pitching rate results in lowering the oxygen consumption by each yeast cell, leading to the inhibition of yeast cell growth and the acceleration of sulfite production. These conditions did not significantly delay nor stop the fermentation process. For these fermentation conditions, an increase in the sulfite content was observed during the lagering periods (5-20/~mol//). Tables 2--4 show the effect of dissolved oxygen in pitching wort, pitching rate, and wort clarification on sulfite content of the finished beer, chemiluminescence (CL) production of the beer before and after storage at 37°C for 8 d, and flavor stability of the beer. Low dissolved oxygen in pitching wort, high pitching rate, and weft clarification led to an increase in the sulfite content of the finished beer and the inhibition of the CL production of the fresh and aged beer, and improved the flavor stability of the beer stored at 37°C for 8 d. The differences in the CL intensities of fresh and aged beers which shows the increase in the CL intensity with beer aging were significantly decreased
120r
I
,,,/
5~
lO
ta 2
I
I
I
I
2 4 6 8 Ferment, period (d)
/0 10
J 0
10 2 4 6 8 Ferment, period (d)
FIG. 1. Effectof dissolved oxygen in pitching wort on consumption curve of wort extract, number of yeast cells, and sulfite content in young beer during fermentation. (A) Open symbols show the consumption curves of wort extract and closed symbols show the number of yeast cells. O and o, brewing condition A in Table 1; zx and A, brewing condition B. (B) O, brewing condition A; zx, brewing condition B.
'°'f / 80
~s
4
0
(B)
ioo ~-
I
I
I
I
2 4 6 8 Fement, period (d)
3~
~ 60
I0 ~
0
2
4
6
8
I0
Ferment. perlod (d)
FIG. 2. Effectof pitching rate of yeast and weft clarification on consumption curve of wort extract, number of yeast cells, and sulfite content in young beer during fermentation. (A) Open symbols show the const~mptioncurves of wort extract and closed symbols show the number of yeast cells. O and e , brewing condition A in Table l; o and s , brewing condition C; A and A, brewing condition D. 03) O, brewing condition A; o, brewing condition C; A, brewing condition D.
458
J. FERMENT. BIOENG.,
KANEDA ET AL.
TABLE 2.
Effect of dissolved oxygen in pitching wort on flavor stability of the finished beer
Sulfite content CL intensity of beer Brewing of ( x 106 counts/2 h) condition ~ finished beer (umol//) fresh aged b A B
80 125
1.05 0.94
1.74 (0.69) ~ 1.12 (0.18)
Staling degree of aged beer 6 fresh stale 1 , 5 3.5 e 2.8
All data are the averages of three replicated brewings. Brewing conditions were the same as in Table 1. b Beer was stored at 37°C for 8 d. Each value shows a difference between CL intensities of fresh and aged beer. d Staling degree is evaluated as cited in the Materials and Methods. TABLE 3.
Effect of pitching rate of yeast on flavor stability of the finished beer
Sulfite content CL intensity of beer Brewing of ( x 106 counts/2 h) condition~ finished beer 0tmol//) fresh aged b A C
57 82
1.07 1.05
1.90 (0.83)c 1.47 (0.42)
Staling degree of aged beer b fresh stale 1 ) 5 3. I a 2.5
All data are the averages of three replicated brewings. a Brewing conditions were the same as in Table 1. b Beer was stored at 37°C for 8 d. c Each value shows a difference between CL intensities of fresh and aged beer. d Staling degree is evaluated as cited in the Materials and Methods.
b y t h e m . A s p r e v i o u s l y r e p o r t e d (16-18), t h e C L p r o d u c t i o n o c c u r s d u r i n g t h e p r o c e s s o f free r a d i c a l r e a c t i o n s i n beer. T h e C L i n t e n s i t y s h o w s t h e o x i d a t i o n degree o f beer a n d h a s a g o o d c o r r e l a t i o n w i t h t h e s t a l i n g degree o f t h e beer flavor. So, t h e difference in t h e C L intensities i n d i c a t e s t h e s t a l i n g degree o f t h e beer d u r i n g t h e s t o r a g e at 3 7 ° C . It was also s h o w n t h a t sulfite i n h i b i t s t h e p r o d u c t i o n o f active o x y g e n s , t h e p r o c e s s o f free r a d i c a l r e a c t i o n s , a n d t h e C L p r o d u c t i o n o f b e e r as a r a d i c a l scavenger, a n d leads to g o o d flavor s t a b i l i t y (6, 14). It was, t h e r e f o r e , t h o u g h t t h a t b e e r f e r m e n t e d u n d e r t h e c o n d i t i o n s o f low d i s s o l v e d o x y g e n in p i t c h i n g w o r t , h i g h p i t c h i n g r a t e , o r w o r t clarification h a s a h i g h t o l e r a n c e t o o x i d a t i o n b e c a u s e o f its h i g h sulfite c o n t e n t , l e a d i n g to g o o d flavor s t a b i l i t y o f t h e b e e r . T a b l e 5 s h o w s a n effect o f t h e s e f e r m e n t a t i o n c o n d i t i o n s o n c o n t e n t s o f flavor c o m p o n e n t s i n t h e fresh beer. N o significant difference i n t h e f l a v o r c h a r a c t e r s o f t h e s e beers was o b s e r v e d . T h i s r e s u l t was also c o n f i r m e d in a sensory e v a l u a t i o n . I n t e r m s o f t h e r e l a t i o n s h i p b e t w e e n w o r t clarification TABLE 4.
Brewing condition" A D
Contents of flavor components (rag//) in the finished beer
Brewing condition" Isobutanol n-Propanol Isoamyl alcohol Furfuryl alcohol Methionol t%Phenylethy[ alcohol Ethyl acetate Isoamyl acetate Ethyl caproate Ethyl caprylate Ethyl caprate /~-Phenylethyl acetate Caproic acid Caprylic acid Capric acid 9-Decenoic acid Acetaldehyde Acetone Diacetyl Acetoin
A
B
C
D
8. I 9.7 51 0.67 1.2 19 22 1.5 0.13 0.14 0.01 0.35 1.8 3.3 0.38 0.09 9.2 0.8 0.03 2.7
8.1 9.6 50 0.69 1.3 19 24 1.7 0.13 0.14 0.01 0.36 1.8 3.3 0.35 0.06 8.1 0.8 0.03 2.6
8.1 9.8 50 0.62 1.2 16 23 1.5 0.13 0.14 0.01 0.33 1.7 3.0 0.37 0.07 7.8 0.8 0.03 2.3
7.8 9.0 50 0.61 1.1 18 20 1.5 0.14 0.13 0.01 0.34 1.9 3.4 0.46 0.05 8.5 0.8 0.04 2.5
All data are the average of three replicated brewings. Brewing conditions were the same as in Table i.
a n d flavor stability o f b e e r , several i n v e s t i g a t i o n s h a v e b e e n c a r r i e d o u t . It h a s b e e n r e p o r t e d t h a t p o o r s e p a r a t i o n o f t h e s p e n t g r a i n s a n d t r u b results in c l o u d y w o r t w i t h h i g h e r lipid c o n t e n t a n d u n e q u i v o c a l l y d a m a g e s t h e f l a v o r s t a b i l i t y o f t h e finished beer (9, 19, 20). It h a s b e e n t h o u g h t t h a t the c a r r y - o v e r o f u n s a t u r a t e d f a t t y acids s u c h as oleic, linoleic, a n d l i n o l e n i c acids i n t o p i t c h i n g w o n is a n imp o r t a n t p r o b l e m , b e c a u s e a p a r t o f t h e m is s u r v i v e d in p a c k a g e d b e e r a n d o x i d i z e d to p r o d u c e trans-2-nonenal w h i c h is m a i n l y r e s p o n s i b l e for a n y staling off-flavor. D r o s t et al. (9) d e m o n s t r a t e d t h a t t h e o x i d a t i o n p r o d u c t s o f m a l t lipids p r o d u c e d e n z y m a t i c a l l y or n o n e n z y m a t i c a l l y d u r i n g w o r t m a k i n g s u r v i v e d in t h e finished beer a n d was r e s p o n s i b l e f o r f l a v o r staling. O n t h e o t h e r h a n d , D u f o u r et al. (12) s t u d i e d t h e r e l a t i o n s h i p b e t w e e n w o r t clarificat i o n a n d sulfite p r o d u c t i o n b y yeast d u r i n g f e r m e n t a t i o n a n d c o n c l u d e d t h a t c l o u d y w o r t w i t h h i g h e r lipid c o n t e n t a c c e l e r a t e d t h e yeast cell g r o w t h d u r i n g f e r m e n t a t i o n a n d t h e c o n s u m p t i o n o f e n d o g e n e o u s sulflte f o r t h e p r o d u c t i o n o f s u l f u r - c o n t a i n i n g a m i n o acids, l e a d i n g to t h e low level o f sulfite c o n t e n t in y o u n g b e e r . T h e a d d i t i o n o f oleic a n d linoleic acids t o clarified w o r t a c c e l e r a t e d t h e yeast cell g r o w t h a n d the c o n s u m p t i o n r a t e o f w o r t e x t r a c t b y yeast a n d t h o r o u g h l y i n h i b i t e d t h e exc r e t i o n o f sulfite i n t o w o r t d u r i n g f e r m e n t a t i o n (Fig. 3). H o w e v e r , t h e a d d i t i o n o f t h e f a t t y acids a n d w o r t clarifica-
Effect of clarification of pitching wort on flavor stability of the finished beer
Turbidity of pitching wort(mg//) 43 3
TABLE 5.
CL intensity of beer ( x 10~counts/2 h)
Sulfite content of finished beer ~mol//)
fresh
aged b
65 91
1.01 0.93
1.62 (0.61) c 1.23 (0.30)
All data are the averages o f three replicated brewings. * Brewing conditions were the same as in Table I. b Beer was stored at 37eC for 8 d. c Each value shows a difference between CL intensities of fresh and aged beer. d Staling degree is evaluated as cited in the Materials and Methods.
Staling degree of aged beer b fresh 1
, 3.0d 2.4
stale 5
Vot. 73, 1992
FERMENTATION CONDITIONS AND BEER STABILITY 120
10
15 ~
"
,--t
TABLE 7.
(B)
100
8
4~ ==
6
3~
~ 6o
4
2o.~
~_
Effectof addition of oleic and linoleic acids on fermenta tion on flavor stability of the finished beer
Sulfite content CL intensity of beer Brewing of ( x I0~counts/2 h) condition * finished beer (pmol//) fresh agedb
A
8o
D E
40'
45t.
120 39
1.00 1.18
Staling degree of aged beerb fresh stale I , 5
1.21 (0.21)c 2.01 (0.83)
2.6d 4.0
I
i-
2 I
I
I
I
2 4 6 8 Ferment. period (d)
i0
Brewing conditions were the same as in Table 1. b Beer was stored at 37°C for 8 d. ¢ Each value shows a difference between CL intensities of fresh am aged beer. e Staling degree is evaluated as cited in the Materials and Methods
2C I
o~ 0 2 4 6 8 FeFment, PerloO (0}
-
I0
FIG. 3. Effectof oleic and linoleic acids on consumption curve of wort extract, number of yeast cells, and sulfite content in young beer during fermentation. (A) Open symbols show the consumption curves of wort extract and closed symbols show the number of yeast cells. (3 and o , brewing condition D in Table l; ,x and A, brewing condition E (B) O, brewing condition D; zx, brewing condition E.
before and after storage at 37°C for 8 d was acceleratet and flavor stability was decreased by the addition o f thesq fatty acids, indicating that oleic and linoleic acids in wor affected the flavor stability o f the resulting beer. In Table 6, the contents o f fatty acids in wort, younl beer, and finished beer with different brewing co n di t i on are shown. A b o u t 50-60% o f unsaturated fatty acids sucl as oleic, linoleic, and linoleic acids was excluded by thq wort clarification. The turbidity o f pitching wort was sig nificantly decreased by this treatment (Table 4). It seem that the several hydrophobic materials, which contain fat ty acids, may be excluded by this treatment. The content o f myristic acid (C 14 : 0), palmitic acid (C 16 : 0), olei, acid (C 18 : I), linoleic acid (C 18 : 2), and linolenic acid (( 18 : 3) decreased during the fermentation, while cupric acit (C 10 : 0), 9-decenoic acid (C 10 : 1), and laurie acid (( 12 : 0) increased. When oleic and linoleic acids were adde( to wort just before the fermentation, the contents o f thes, unsaturated fatty acids significantly decreased during th, fermentation and were finally at trace levels in the finisher beer. The significant differences in the other acids durinj fermentation were not observed. H a s h i m o t o (21) ant Kaneda et al. (22) reported that longer fatty acids that sur vived in beer could not significantly contribute to th, flavor staling. Therefore, it is unlikely that high content o f longer unsaturated fatty acids in pitching wort can sur rive in the finished beer and participate in the deterioratio] o f the beer flavor. It seems that the kinds and the content o f fatty acids in pitching wort are important factors to de
tion did not significantly affect the consumption o f sulfate, which is a precursor o f sulfite in the yeast metabolism, in pitching wort during fermentation (Table 6). D u f o u r et al. (12) reported that SO2 excretion drastically increased when yeast growth slowed down or stopped. They concluded that in the absence o f growth but under fermenting conditions, yeast cells do not further synthesize sulfur-containing amino acids. But the reactions leading to the synthesis of SO2 can still progress with a concomitant intracellular accumulation o f SO2 level which is excreted in the wort. The sulfite level in young beer is thus dependent on yeast growth. However, Ohno and Takahashi (15) speculated that the decrease in sulfite producing ability o f yeast by the addition o f lipids into wort might not depend on cell growth, but on change in the activity o f the corresponding enzyme. The mechanism o f sulfite production in yeast cells needs to be clarified with more detailed studies. Table 7 shows sulfite content, CL intensity, and flavor stability of the beer fermented with the addition o f oleic and linoleic acids. Sulfite content in the finished beer was 39 p m o l / l though the excretion o f sulfite into wort was thoroughly inhibited during the main fermentation (Fig. 3), indicating the excretion o f endogeneous sulfite f r o m yeast during lagering. The CL production o f the beer TABLE 6. Sample Brewing condition"
Changes in contents of fatty acids (mg//) and sulfate (mg//) in brewing
A
Pitching wort D
E
A
Young beer D
E
A
Packaged beer D
E
Fatty acid C 10 : 0 C 10 : 1 C 11 : 0 C 12 : 0 C 14 : 0 C 16 : 0 C 16 : 1 C 18 : 0 C 18 : 1 C 18 : 2 C 18 : 3 C 20 : 0
0.01 0.03 0.02 0.01 0.05 0.14 tr. b 0.02 0.04 0.17 0.17 0.01
0.01 0.02 0.01 0.01 0.04 0.10 tr. 0.02 0.02 0.06 0.06 tr.
0.01 0.02 0.01 0.01 0.04 0.10 tr. 0.02 10.00 10.10 0.06 tr.
0.67 0.11 0.01 0.25 0.03 0.04 tr. 0.02 0.02 tr. n.d. c n.d.
0.71 0.11 0.01 0.28 0.03 0.04 tr. 0.01 0.01 tr. tr. n.fl.
0.69 0.11 0.01 0.20 0.02 0.08 tr. 0.04 0.37 0.17 n.d. n.d.
0.79 0.13 0.01 0.08 0.01 0.06 0.01 0.01 tr. tr. 0.01 n.d.
0.80 0.09 0.01 0.08 0.01 0.06 0.01 0.01 tr. tr. 0.01 n.d.
0.90 0.17 0.01 0.08 0.01 0.06 0.01 0.01 tr. tr. 0.01 n.d.
Sulfate
70
72
74
51
57
59
55
54
60
" Brewing conditions were the same as in Table 1. b tr., Trace. c n.d., No detect.
460
J. FERMEI~T. BIOENG.,
KANEDA ET AL.
TABLE 8.
Effect of brewer's yeast strain on flavor stability of the finished beer
Sulfite content CL intensity of beer Brewing of ( × 106 counts/2 h) conditiona finished beer ~mol//) fresh aged b A F
73 35
1.00 1.28
Staling degree of aged beerb fresh stale I ----, 5
1.58 (0.58) ~ 2.16 (0.88)
3.0'1 4.0
• Brewing conditions were the same as in Table I. b Beer was stored at 37°C for 8d. c EachvalueshowsadifferencebetweenCLintensitiesoffreshand
aged beer. e Staling degree is evaluated as cited in the Materials and Methods. t e r m i n e t h e flavor s t a b i l i t y o f t h e r e s u l t i n g beer, b e c a u s e they affect yeast m e t a b o l i s m d u r i n g f e r m e n t a t i o n a n d t h e sulflte c o n t e n t o f t h e finished beer. In this s t u d y , it c o u l d n o t b e clarified w h e t h e r t h e o x i d a t i o n p r o d u c t s o f lipids, w h i c h o c c u r r e d d u r i n g t h e w o r t m a k i n g process, h a d a r e l a t i o n s h i p w i t h flavor staling o f t h e r e s u l t i n g beer. T h e s t u d y t o clarify t h e rote o f t h e w o r t m a k i n g p r o c e s s e s o n t h e flavor s t a b i l i t y o f beer is i n p r o g r e s s . T a b l e 8 s h o w s the effect o f yeast s t r a i n o n t h e sulfite c o n t e n t i n t h e finished b e e r , t h e C L p r o d u c t i o n o f t h e beer b e f o r e a n d a f t e r its s t o r a g e a t 370C f o r 8 d, a n d t h e degree o f f l a v o r staling o f t h e b e e r s t o r e d at 3 7 ° C f o r 8 d. T h e sulrite c o n t e n t in t h e beer b r e w e d w i t h yeast (II) was lower t h a n t h a t w i t h yeast (I). T h e b e e r o f yeast (II) h a d a h i g h e r C L p r o d u c i n g activity a n d less f l a v o r s t a b i l i t y t h a n t h a t o f y e a s t (I). T h e flavor c h a r a c t e r o f t h e r e s u l t i n g beers were different i n a s e n s o r y test; t h e b e e r b r e w e d w i t h yeast (I) s h o w e d a m o r e esteric c h a r a c t e r t h a n w i t h yeast (II). It was t h o u g h t t h a t yeast s t r a i n s c o u l d significantly affect n o t o n l y flavor c h a r a c t e r b u t also f l a v o r s t a b i l i t y o f beer. B a s e d o n t h e results p r e s e n t e d so far, it was c o n c l u d e d t h a t f e r m e n t a t i o n c o n d i t i o n s p l a y a n i m p o r t a n t role in flavor s t a b i l i t y o f beer. It was e x p e c t e d t h a t flavor stability o f b e e r c o u l d b e c o n t r o l l e d b y t h e f e r m e n t a t i o n c o n d i tions without harming the fermentation process and the flavor c h a r a c t e r s o f t h e f r e s h b e e r . It w a s also r e p o r t e d t h a t s t a r v i n g t i m e o f yeast a n d " d a r a u f l a s s e n " , fresh w o r t being a d d e d to t h e f e r m e n t i n g w o r t a t a n i n t e r v a l o f several h o u r s , significantly affected t h e c o n t e n t o f s u l f u r d i o x i d e in y o u n g b e e r (13). T h e s t u d y o f t h e r e l a t i o n s h i p b e t w e e n ferm e n t a t i o n c o n d i t i o n s a n d f l a v o r s t a b i l i t y o f beer will b e s t e p p e d u p f r o m t h e p i l o t line t o f a c t o r y scale, i n o r d e r t o b r e w a m o r e s t a b l e a n d f a v o r a b l e beer. ACKNOWLEDGMENTS The authors would like to thank Mr. T. Kirnura and Mr. M. Kobayashi, Brewing Research Laboratories, Sapporo Breweries Ltd., for analyzing the beer and won components and Mr. J. Murakami for his helpful advice.
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