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ABSTRACT INTRODUCTION
J. Inst. Brew. 113(4), 365–373, 2007
The superoxide radical, O2●- is generated from oxygen
The Plackett-Burman multifactorial design was employed to in numerous autoxidation and metal-catalysed reactions19.
screen the important malting parameters for superoxide dis- During brewing, very small quantities of superoxide,
mutase (SOD) in final malt of Ganpi-3. The eight factors which lead to the formation of a hydroxyl radical (OH·)
screened for SOD were steeping temperature, steeping time, per-
that is intensely reactive, can have a number of damaging
oxide hydrogen concentration in steeping water, germination
temperature, germination time, withering temperature, drying effects on biochemical systems4. Effects that would seem
temperature and kilning temperature. Variance analysis showed to be of importance in a brewing context, include the per-
that steeping time, germination temperature and kilning tem- oxidation of lipids, the degradation of polysaccharides,
perature were significant for SOD activity. Box-Behnken exper- inactivation of enzymes, the loss in viability of yeast, the
imental design was further used to optimize the levels of the decline of colloidal stability and the increase in color dur-
above three factors. By response surface methodology and ing beer storage7,10,12,14,18.
canonical analysis, the optimal malting factors for higher SOD Superoxide dismutase (SOD) catalyzes the dismutation
activity in final malt were: steeping time 42.2 h, germination reaction shown in equation (1), being capable of scaveng-
temperature 16.9°C and kilning temperature 82.9°C. Under ing superoxide anion radicals.
these conditions, the model predicted a SOD activity of 2234
S
O
D
U/g of dry weight malt. Verification of the optimization showed •−
that a SOD activity of 2220 U/g was observed under optimal 2O 2 + 2H + ⎯⎯
⎯→
H2O2 + O2 (1)
conditions. It showed that the experimental data could be
reliably predicted by the polynomial model. Besides Ganpi-3, Barley contains at least three SOD isozymes, of which
three other barley varieties including Ganpi-4, Ken-2 and two forms, presumed to be copper-and-zinc containing
Hamelin were malted under optimal and common conditions species, predominate5,11. The enzyme activity heavily in-
under laboratory conditions. To some extent, SOD activities
creases during germination and substantially survives in
were higher in malts from the optimal malting process than
those from the common malting process. Especially, SOD activ- final malts3. It has been demonstrated that SOD in barley
ities in Ganpi-3 and Hamelin increased by 18.8% and 15.3%, and malt will prevent the development of rancid notes
respectively. Furthermore, twenty-nine samples of malts, includ- during grain storage and off-flavours in beer by reducing
ing eleven imported malts and eighteen domestic malts, were the oxidation of lipids caused by superoxide1. Moreover,
used. Relationships between SOD activity in malt and the reduc- the activity of SOD in malt, which may all be active at the
ing power of wort were examined. There was significant cor- onset of mashing, not only could provide some protection
relation between SOD activity and the reducing power of wort against oxidative damage brought about by oxygen radi-
(R2 = 0.8069). cals, but could also eliminate the origin of hydroxyl radi-
Key words: Box-Behnken experimental design, malting, opti- cal (OH)6, retain the reducing power of wort to a much
misation, Plackett-Burman experimental design, reducing power greater extent and improve the antioxidant ability of wort
of wort, superoxide dismutase. and the flavor stability of beer4. How to optimize the
malting process to obtain a higher activity of SOD in final
malt is important. Bamforth5 and Clarkson and Large6
1 State
only investigated the changes of SOD during malting and
Key Laboratory of Food Science and Technology,Jiangnan
did not explain corporate effects of all the factors in
University, Wuxi, 214122, P.R. China
2 School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. malting on the SOD activity of malt.
China Therefore, it is important for maltsters and brewers to
3 Tsingtao Brewery Co., Ltd., Qingdao, 266061, P.R. China know which parameters affect SOD activity in malting
4 Corresponding Author. E-mail: jlu@jiangnan.edu.cn and how to make the SOD activity of the final malt much
higher. The present work was aimed at examining inter-
Publication no. G-2007-0128-529 actional effects of malting factors, namely steeping tem-
© 2007 The Institute of Brewing & Distilling perature, steeping time, peroxide hydrogen concentration
Table II. The Plackett–Burman experimental design matrix for the screening of the main factors affecting SOD
activity in the final malt.
SOD
No. X1 X2 X3 X4 X5 X6 X7 X8 (U/g)
1 1 –1 1 –1 –1 –1 1 1 1058
2 1 1 –1 1 –1 –1 –1 1 1107
3 –1 1 1 –1 1 –1 –1 –1 1254
4 1 –1 1 1 –1 1 –1 –1 911
5 1 1 –1 1 1 –1 1 –1 1122
6 1 1 1 –1 1 1 –1 1 1338
7 –1 1 1 1 –1 1 1 –1 1303
8 –1 –1 1 1 1 –1 1 1 980
9 –1 –1 –1 1 1 1 –1 1 706
10 1 –1 –1 –1 1 1 1 –1 642
11 –1 1 –1 –1 –1 1 1 1 1102
12 –1 –1 –1 –1 –1 –1 –1 –1 588
Table III. Regression coefficients and their significances for SOD from the results of the Plackett-Burman
design.
Standard Confidence
Factors Effect error t–ratio P–value level (%)
X1 40.83 22.1757 1.8414 0.1628 83.72
X2 390.4 22.1757 17.6034 0.0004 99.96
X3 263.0 22.1757 11.8583 0.0013 99.87
X4 24.50 22.1757 1.1048 0.3499 65.01
X5 –4.900 22.1757 –0.2210 0.8393 16.07
X6 –17.97 22.1757 –0.8102 0.4771 52.29
X7 50.63 22.1757 2.2833 0.1066 89.34
X8 78.40 22.1757 3.5354 0.0385 96.15
Table IV. Maximum and minimum levels of variables used in the Box-Behnken design and experimental values of SOD activity in final malt.
Coded values Real values
X2 X3 x2 x3
X1 Germination Kilning x1 Germination Kilning SOD (U/g)
Trial Steeping time temperature temperature Steeping time temperature temperature
number (h) (°C) (°C) (4 h) (h) (°C) (°C) (4 h) Experimental
1 –1 –1 0 24 12 80 559
2 –1 1 0 24 20 80 949
3 1 –1 0 48 12 80 1600
4 1 1 0 48 20 80 1774
5 0 –1 –1 36 12 70 1264
6 0 –1 1 36 12 90 1246
7 0 1 –1 36 20 70 1548
8 0 1 1 36 20 90 1864
9 –1 0 –1 24 16 70 851
10 1 0 –1 48 16 70 1774
11 –1 0 1 24 16 90 945
12 1 0 1 48 16 90 2029
13 0 0 0 36 16 80 2103
14 0 0 0 36 16 80 2066
15 0 0 0 36 16 80 2059
Table VI. Regression coefficients and their significances for SOD activity in the final malt from the results of
the Box-Behnken design.
Degree Standard
Model term of freedom Estimate error t–value P>∣t∣
Intercept 1 2076 34.92 59.45 <0.0001*
X1 1 484.4 21.38 22.65 <0.0001*
X2 1 183.2 21.38 8.57 0.0004*
X3 1 80.74 21.38 3.78 0.0129*
2
X1 1 –467.9 31.47 –14.87 <0.0001*
X1X2 1 –53.90 30.24 –1.78 0.1343
2
X2 1 –351.3 31.47 –12.31 <0.0001*
X1X3 1 40.30 30.23 1.33 0.2401
X2X3 1 83.60 30.23 2.77 0.0396*
2
X3 1 –208.1 31.47 –6.61 <0.0012*
* Significant at 5% level (P<0.05)
Fig. 2. Response surface plot and contour plot of the combined effects of steeping time and germination temperature
on SOD activity in the final malt.
Fig. 3. Response surface plot and contour plot of the combined effects of steeping time and kilning temperature on
SOD activity in the final malt.