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Control of Superoxide Dismutase Activity

during Malting Using Plackett-Burman


and Box-Behnken Experimental Design
and Its Effect on Reducing Power of Wort
De-Jing Meng1,2, Jian Lu1,2,4, Wei Fan3, Jian-Jun Dong3, Yan Lin3, and Lian-Ju Shan3

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

VOL. 113, NO. 4, 2007 365


in steeping water, germination temperature, germination grade and obtained from Sinopharm Chemical Reagent
time, withering temperature, drying temperature and kiln- Co. Ltd (Shanghai, China).
ing temperature, on SOD activity in malt using Plackett-
Laboratory scale malting and mashing
Burman and Box-Behnken experimental design associated
with response surface methodology. Firstly, the Plackett- The barley varieties including Hamelin, Ganpi-3,
Burman experimental design was used to screen the im- Ganpi-4 and Ken-2 were malted in a Phoenix Automatic
portant effects on SOD activity. Subsequently, the factors Micromalting System (Phoenix Biosystems, Adelaide,
that had significant effects were optimized by the Box- Australia) and the moisture of germination was kept at
Behnken experimental design. At the same time, twenty- 95%. The methods of steeping were four hours of steep
nine samples of malts were employed to investigate the and eight hours of air rest. The other different malting
effect of SOD activity on the reducing power of wort. conditions were carried out according to experimental runs
in the Plackett-Burman and Box-Behnken designs. Worts of
twenty-nine samples of malts that had different SOD
MATERIALS AND METHODS activities were prepared in a mashing bath (BGT-8A, Bioer,
Materials Hangzhou, China) according to the EBC congress method.
Ganpi-3, Ganpi-4 and Ken-2 are the representative Optimization procedure
malting barley varieties in China and were obtained from Ganpi-3 was used as the raw material. The optimiza-
northwestern (Gansu province) and northeastern (Heilong- tion of the malting process for improving SOD activity in
jiang province) China, respectively. Hamelin (Australia) the final malt was carried out in two stages.
barley variety was supplied by Tsingtao Brewery Co. Ltd.
Of the twenty-nine malt samples, the four supplied by Plackett-Burman design to identify
Tsingtao Brewery Co. Ltd were Scarlet, Copeland, Stir- important variables
ling and Metcalfe, and the six supplied by Gansu Guo- Plackett-Burman design based on the first order model:
xiang Malt Workshop (Zhangye, Gansu, China) were
Gang-2, Dan-2, Wupi-1, Humai-16, Humai-8 and Nong- Y = β0 + ΣβiXi (2)
mai. These samples, and the others, which were malted was used to screen the important variables that influenced
under common malting processes under laboratory con- SOD activity during malting. Each variable was repre-
ditions were employed to evaluate the effects of SOD sented at two levels, high and low, which were denoted by
activity in malt on the reducing power of wort. All the (+1) and (-1), respectively. The coded level of each
chemicals and solvents were of the highest commercial variable was as given in Table I. Here eight variables were
screened in twelve trials as shown in Table II. Each
column contained an equal number of positive and nega-
Table I. Malting parameters and their levels employed in the Plackett–
Burman design for the screening of the main conditions affecting SOD tive signs. All experiments were performed in duplicate
activity in the final malt. and the mean values are given. The variables (Table III),
whose confidence levels were greater than 95%, were
Coded Coded level
considered to significantly affect SOD activity.
Independent variables symbol –1 1 The standard error (S.E.) of the concentration effect
Steeping temperature (°C) x1 X1 14 18 was the square root of the variance of an effect and the
Steeping time (h) x2 X2 24 48 significant level (P-value) of each concentration effect
Germination temperature (°C) x3 X3 12 20 was determined using the Student’s t-test:
Peroxide hydrogen concentration
in steeping water (g/L) x4 X4 0 0.2
E( X i )
Germination time (d) x5 X5 4 8 t ( xi ) = (3)
Withering temperature (°C) (10h) x6 X6 35 50 S.E.
Drying temperature (°C) (6h) x7 X7 55 65
Kilning temperature (°C) (4h) x8 X8 70 90
where E(Xi) was the effect of variable Xi.

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

366 JOURNAL OF THE INSTITUTE OF BREWING


The Box-Behnken design to further optimize order response surface. Table IV gives the factors and
the malting process their values in connection with the experimental design.
This methodology allowed the modeling of a second order
The Box-Behnken design and response surface meth-
equation that described the process. The activity of SOD
odology (RSM) were used to optimize the process, find- was analyzed by multiple regressions through the least
ing interactions among significant factors obtained from squares method to fit the following equation:
the Plackett-Burman design. The basic strategy for RSM
had four steps: procedures to move into the optimum 3 3 3
region, behavior of the response in the optimum region, Y = β 0 = ∑ βi X i = ∑ βii X i2 + ∑ ∑ βij X ij
estimation of the optimal condition and verification26. The i =1 i =1 i =1 (5)
i< j
Box-Behnken experimental design was employed in this
regard. According to the Box-Behnken experimental where Y was the predicted response variable; βi, βii and βij
design, the total number of experimental combinations were constant regression coefficients of the model, and Xi,
was 2k+2k+n0, where k was the number of independent Xj (i =1, 3; j = 1, 3, i ≠ j) represented the independent
variables and n0 was the number of repetitions of the variables in the form of coded values. The accuracy and
experiments at the centre point. For statistical calculation, general ability of the above polynomial model could be
the experimental variable xi had been coded as Xi accord- evaluated by the coefficient of determination R2.
ing to the following transformation equation:
Common malting process under laboratory
x − x0 conditions
Xi = i (4)
δx Thin barley kernels (those passing through a 1.98 by
19.05 mm slotted sieve) were removed prior to malting.
where Xi was the dimensionless coded value of the Malts from corresponding barleys were micromalted
variable xi, x0 the value of xi at the centre point and δx the employing 36 h of steeping with varying wet and dry
step change. periods (6 h wet, 9 h dry, 7 h wet, 9 h dry, and 5 h wet) at
In this study, the Box-Behnken experimental design 15°C, 5 days of germination at 16°C, and a 22 h kilning
with three factors and three levels, including three repli- procedure (45°C for 8 h, 55°C for 8 h, 65°C for 3 h, and
cates at the centre point, was used for fitting a second 85°C for 3 h).

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

VOL. 113, NO. 4, 2007 367


Preparation of extracts for determination process of Ganpi-3. Table II represents twelve different
of SOD activity from malt combinations with two levels for each variable and corre-
sponding SOD activity. The variable x1-x8 represented
Malt was finely ground in a laboratory mill from
eight different malting parameters. Table III shows the
Bühler-Miag (Braunschweig, Germany). Five grams of effect, standard error, t-ratio, P- value and confidence
ground sample was extracted (4°C with stirring in 50 mL level of the Plackett-Burman design. It was found that the
of potassium phosphate buffer (50 mM, pH 7.8) for 1 h.
variables x2 (steeping time), x3 (germination temperature)
Samples were then centrifuged at 5000 g for 20 min at and x8 (kilning temperature) had confidence levels above
4°C and the resulting supernatant was used to determine 95% and hence were considered to significantly affect
SOD activity6.
SOD activity.
Analytical method Neglecting the terms which were insignificant on the
basis of regression coefficients and P- value, the linear
The activity of SOD was determined according to the regression equation (6) that had an important influence on
SOD kit purchased from Nanjing Jiancheng Bioengineer- SOD activity from Plackett-Burman design could be
ing Co., Ltd. (Nanjing, China) The SOD activity of the written as:
respective extract was expressed as units/gram dry weight
malt. Y = 2019 + 390.4 × X2 + 263.0 × X3 + 78.40 × X8 (6)
Statistical analysis
where Y was the predicted response (SOD activity) from
Data from the Plackett-Burman design and Box- the Plackett-Burman design and X2, X3, X8 were coded
Behnken design were analyzed using the RSREG proce- values of steeping time, germination temperature and
dure of the SAS statistical package (version 8.1, SAS kilning temperature, respectively.
Institute, Cary, NC, USA)28.
Regression model of response from the
Verification of the optimum malting process Box-Behnken design and RSM strategies
In addition to the experimental barley Ganpi-3, three Based on the results of the Plackett-Burman design,
barley varieties including Ganpi-4, Ken-2 and Hamelin steeping time, germination temperature and kilning tem-
were malted under optimal conditions. Verification of the perature were identified to have significant effects on
procedure was performed by analyzing SOD activity be- SOD activity and were further optimized using the Box-
tween malts from the common and the optimum malting Behnken design and these three variables used for the
process on a laboratory scale. factorial analysis were renamed X1, X2 and X3 in Table IV,
respectively. For response surface methodology (RSM)
Analysis of reducing power of wort based on the Box-Behnken design, which was used for the
The reducing power of wort was determined as de- optimization of the malting process for higher SOD
scribed by Oktay et al.25 Briefly, 1 mL of properly diluted activity, fifteen experimental runs with different combina-
wort was mixed with phosphate buffer (2.5 mL, 0.2 M, tions of three factors along with their experimental values
pH 6.6) and K3 Fe(CN)6 (2.5 mL, 1%, w/v). The mixture are shown in Table IV. It can be seen from Table IV, that
was incubated at 50°C for 20 min. A portion (2.5 mL) of there was considerable variation in the SOD activity
TCA (10%, w/v) was added to the mixture, which was depending on these three malting parameters. The first
then centrifuged at 10000 g for 10 min. The upper layer of and thirteenth run showed a minimum SOD activity of
solution (2.5 mL) was mixed with deionized water (2.5 559 U/g and a maximum SOD activity of 2103 U/g,
mL) and FeCl3 (0.5 mL, 0.1%, w/v), and the absorbance respectively. The centre point in the design was repeated
was measured at 700 nm. A vitamin C calibration curve three times for the estimation of the error.
was plotted as a function to assess the reducing power of By applying multiple regression analysis on the exper-
the wort. The final results were expressed as mmol vita- imental data, the following second order polynomial
min C/mL. equation (7) was found to explain SOD activity by only
considering the significant terms and is shown as below:
Analysis of physical and chemical
properties of malt Y = 2076 + 484.4 × X1 + 183.2 × X2 + 80.74
× X3 – 467.9 × X12 – 351.3 × X22 – 208.1 (7)
β-Glucan in wort was measured by the Congo red
× X32 + 83.60 × X2X3
method27. Other physical and chemical properties of the
malt were analyzed according to the Methods of Analysis where Y was the predicted response (SOD activity) from
of the American Society of Brewing Chemists21. the Box-Behnken design, X1, X2, X3 were coded values of
steeping time, germination temperature and kilning tem-
RESULTS AND DISCUSSION perature, respectively.
Statistical testing of the model was done by the
Identification of important variables Fisher’s statistical test for analysis of variance (ANOVA)
Table I shows the maximum and minimum levels of and the results are shown in Table V. The goodness of
variables chosen for trials in the Plackett-Burman exper- model could be checked by the determination coefficient
imental design for screening important factors that re- (R2), correlation coefficient (R), coefficient of variation
markably developed SOD activity during the malting (CV) or F-test15. Analysis of variance (F-test) showed that

368 JOURNAL OF THE INSTITUTE OF BREWING


Table V. Analysis of variance (ANOVA) for the quadratic polynomial model of SOD activity in final malt a
Degree Sum Mean
Source of freedom of squares square F–value P>F
Linear 3 2197550 » 200.27 <0.0001
Quadratic 3 1347561 » 122.81 <0.0001
Crossproduct 3 46122 » 4.20 0.0781
Total model 9 3591233 » 109.09 <0.0001
Total error 5 18288 3657.66 » »
a Coefficient 2
of variation (CV) = 4.01; coefficient determination (R ) = 0.9949; correlation coefficient (R) =
0.9974

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)

the second model was well adjusted to the experimental


data ((Pmodel>F) < 0.0001). Based on the report by
Joglekar and May17, if a model had a high goodness, its R2
should not be lower than 0.8 and CV not be higher than
10. Generally, the higher the value of CV, the lower was
the reliability of the experiment. Here, a lower value of
CV (4.01) indicated a greater reliability of the experiments
performed. The determination coefficient (R2) implied that
the sample variation of 99.49% for SOD activity was
attributed to the independent variables, and only about
0.51% of the total variation could not be explained by the
model. The closer value of the R (correlation coefficient)
to 1, the better was the correlation between the exper-
imental and predicted values. Here the value of R (0.9974)
for equation (7) being close to 1 indicated a close agree-
ment between the experimental results and the theoretical
values predicted by the model equation. Linear and
Fig. 1. Observed SOD activity versus predicted SOD activity
quadratic terms were significant at the 1% level.
from the empirical model (y = 0.9899x+15.231, R2 = 0.9899).
The Student t-distribution and the corresponding P-
value, along with the parameter estimate, are presented in
Table VI. The P-values were used as a tool to check the
significance of each of the coefficients, which in turn Comparison of observed and predicted
were necessary to understand the pattern of the mutual SOD activity
interactions between the best variables. The parameter A regression model could be used to predict future
estimates and the corresponding P-values (Table VI) sug- observations on the response Y (SOD activity) corre-
gested that among the independent variables, X1 (steeping sponding to particular values of the regressor variables. In
time), X2 (germination temperature), and X3 (kilning tem- predicting new observations and in estimating the mean
perature) had a significant effect on SOD activity. Positive response at a given point, one must be careful about
coefficients for X1, X2, and X3 indicated a linear effect to extrapolating beyond the region containing the original
increase SOD activity. The quadratic term of these three observations. It is very possible that a model that fits well
variables also had a significant effect. Evident interactions in the region of the original data would no longer fit well
between X2 and X3 were also found to contribute to the outside the region. Fig. 1 showed observed SOD activities
response at a significant level. When P-values were less (the response) versus those from the empirical model
than 0.05, model terms were significant. In this case, X1, equation (7). The figure proved the predicted data of the
X2, X3, X12, X22, X32, X2X3 were significant model terms, response from the empirical model was in agreement with
respectively. the observed ones in the range of the operating variables.

VOL. 113, NO. 4, 2007 369


Localization of optimum condition between steeping time and germination temperature were
The fitted response surface plots and their correspond- found to contribute to the response at a significant level.
ing contour plots for the SOD activity by the above model This fact also could be accounted for by the P-value
are shown in Figs. 2-4, respectively. The contour plots (0.1343>0.05) in Table VI. It can be seen from Fig. 4,
affirmed that the objective function was unimodal in when steeping time was at a low level, the effect of germi-
nature, which showed an optimum in the boundaries. The nation temperature on the response was insignificant. It
boundary optimum point was evaluated using the gradient was evident that SOD activity was higher than 2200 U/g
method in the direction of steepest ascent. The graphical when steeping time was in the range of 0.3 to 0.7 (coded
representation provided a method to visualize the relation- value) and germination temperature was in the range of 0
ship between the response and experimental levels of each to 0.5 (coded value). Eneje et al.9 investigated the effect of
variable, and the type of interactions between test varia- steeping time on germination of maize, suggesting that
bles in order to deduce the optimum conditions. proper steeping time was not only beneficial to extract of
Fig. 2 depicts the three-dimensional plot and its respec- maize, but also could improve development of enzymes
tive contour plot showing the effects of steeping time and during germination. Iwuoha and Aina16 also reported that
germination temperature on SOD activity in the final different steeping conditions could remarkably affect the
malt, while kilning temperature was fixed at the middle formation of α-amylase during germination of sorghum.
level. The interaction relationships between the two These facts adequately indicated that control of appro-
chosen variables and the response variable can be easily priate steeping time seemed very important for enzymes
understood by examining the contour plot. The circinal in germinated grains, where one wanted to keep higher
nature of the contour plot indicated that no interactions activities..

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.

370 JOURNAL OF THE INSTITUTE OF BREWING


Fig. 4. Response surface plot and contour plot of the combined effects of germination temperature and kilning
temperature on SOD activity in the final malt.

Fig. 3 shows the effects occurring between steeping


time and kilning temperature, while germination temper-
ature was fixed at its middle level. In the same way, there
were no evident interaction relationships existing between
the two independent variables and the response variable.
It was noticed that when kilning temperature was more
than 0.4 (coded value), SOD activity gradually decreased
with increasing steeping time. It was possible that the
higher kilning temperature caused inactivation of SOD.
Bamforth5 also found that SOD in green malt was 5-fold
higher than in barley, but was largely destroyed after
kilning. Clarkson and Large6 reported that the level of
SOD in lager malt was about 2-fold higher than that in ale
malt after kilning. So kilning temperature occupies an
important part in the SOD activity of the final malt.
Fig. 4 pictures the intersectant effects of germination
temperature and kilning temperature on SOD activity in
final malt, while steeping time was fixed at its middle Fig. 5. Plot of internally studentized residuals versus predicted
level. The interaction relationships between the two inde- response.
pendent variables and the response variable can easily be
understood by examining the contour plot. It is evident
from Fig. 4 that the elliptical nature of the contour plot sents a plot of residuals versus the predicted response.
indicates that the interactions between the two variables The general impression was that the residuals scatter ran-
were significant. This can also could be seen from the P- domly on the display, suggesting that the variance of the
value (0.0396<0.05) in Table VI. Clearly, when the coded original observation was constant for all values of Y. As
value of germination temperature was from 0.15 to 0.45, from Fig. 5, it was satisfactory based on the judging of
and the coded value of kilning temperature from 0.1 to model adequacy. Thus it was concluded that the empirical
0.5, SOD activity was easily more than 2100 U/g. Thus model was adequate to describe the SOD activity by re-
the control of appropriate germination temperature and sponse surface.
kilning temperature in industrial production, namely the
proper supply of energy, appears to be an important factor. Verification of optimum malting process
The process of verification was carried out by com-
Model adequacy checking paring SOD activities in malts from a common malting
Usually, it is necessary to check the fitted model to process with those from the optimum malting conditions
ensure that it has provided an adequate approximation to (Fig. 6). Besides the experimental barley Ganpi-3, three
the real system. Unless the model shows an adequate fit, other barley varieties including Ganpi-4, Ken-2 and
proceeding with the investigation and optimization of the Hamelin were also malted under optimal conditions to
fitted response surface will likely give poor or misleading evaluate the reliability of the regression model. Under
results. The residuals from the least squares fit played an optimal conditions, the model predicted a SOD activity of
important role in judging model adequacy23. Fig. 5 pre- 2234 U/g and a verification value of 2220 U/g was ob-

VOL. 113, NO. 4, 2007 371


served for Ganpi-3. Moreover, to some extent, SOD activ- physical and chemical indices, which indicated that the
ities were higher in malts from the optimal malting optimal malting process was applicable to produce malt in
process than those from the common malting process. In the malting industry.
particular, Ganpi-3 and Hamelin showed a more remark-
able trend, with an increasing SOD activity of 351 U/g Relationship between SOD activity
and 295 U/g, respectively. These observations showed that and reducing power of wort
the experimental data could be reliably predicted by the SOD activities in twenty-nine samples of malts, includ-
polynomial model. ing eleven imported malts and eighteen domestic malts,
and the reducing power of the respective congress worts,
Effects of the optimum malting process
were analyzed. Correlation analysis between the samples
on the physical and chemical property was carried out and are shown as Fig. 7. As can be seen,
of the malt the reducing power of the congress wort was much higher
Laboratory scale mashing experiments were carried if the SOD activity of the corresponding malt was rela-
out in a mashing bath for Ganpi-3 malts from the optimal tively high. Reasonable explanations were possible, such
and common malting process. Physical and chemical tests as differences of SOD activity in different malts were
of wort are shown in Table VII. From KI, FAN, diastatic caused by the degree of protection reducing compounds,
power and β-glucan of the two worts, there were no evi- such as flavanoids, phenolic acids and so on that exhibited
dent differences among the samples, suggesting that the antioxidant activity through their reductive capacity in
optimal malting process had no negative effect on pro- Fe3+-Fe2+ and from oxidation by superoxide radical dis-
teinase, amylase and β-glucanase. Compared with malt tinct during the onset of mashing8,13. Also possible was
obtained from a high quality malting barley variety (Lofty that, polyphenols, melanioidins and reduced thiol groups
Nijo)24, the β-glucan content (27.2 mg/L and 18.5 mg/L) of proteins were responsible for notable variations in the
of the two Ganpi-3 malts was much lower than the Lofty reducing power in mashing22. The higher reducing power
Nijo malt (77 mg/L). However, extract of Lofty Nijo malt of wort could better restrain oxidation of unsaturated fatty
(81.1%) was higher than Ganpi-3 malts (78.1% and acids, producing less trans-2-nonenol during wort boiling
77.6%). Differences in barley varieties and in the malting and ultimately bringing about a better flavor stability in
process might account for discrimination among the sam- the beer2,20.
ples. There were no other significant differences in these
CONCLUSIONS
To the best of our knowledge, there are currently no
reports on the optimization of the malting process for
higher SOD activity in final malt using statistical exper-
imental design. The Plackett-Burman design and the Box-
Behnken design appeared to be a powerful tool for the
prediction of SOD activity. The Plackett-Burman design
made it possible to consider a large number of variables
and avoid the loss of information that might be essential
in the optimization of the process. Steeping time, germi-
nation temperature and kilning temperature were identi-
fied to have significant effects on SOD activity using the

Fig. 6. Comparison of SOD activity in the malt from optimal


and common malting processes.

Table VII. Comparison of physical and chemical properties of malt


under different malting processes.
Malt Malt
from common from optimal
Item malting process malting process
Moisture (%) 5.8 6.4
Extract (%) 78.1 77.6
Protein (%) 11.5 11.4
Soluble nitrogen (%) 4.88 4.96
KI (%) 42.7 43.5
FAN (mg/L) 123 143
Total acidity (mL 1N NaOH) 1.60 1.64
pH 5.77 5.86
Color (EBC) 1.78 1.38
Turbidity (EBC) 4.93 4.80
Diastatic power (WK) 249 254 Fig. 7. Relationship between SOD activity and reducing power
β-glucan (mg/L) 27.2 18.5 of wort (y = 0.0027x - 2.9719, R2 = 0.8069).

372 JOURNAL OF THE INSTITUTE OF BREWING


Plackett-Burman experimental design. Their levels were varieties. Process Biochem., 2004, 39, 1013-1016.
further optimized by the Box-Behnken design. The pre- 10. Eriksson, C. E., Lipid oxidation catalysts and inhibitors in raw
dicted and the actual SOD activities, verified by exper- materials and processed foods. Food Chem., 1982, 9, 3-19.
11. Fridovich, I., Superoxide dismutase. Annu. Rev. Biochem., 1975,
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(Hordeum vulgare L.). J. Agric. Food Chem., 2006, 54, 7277-
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16. Iwuoha C. I. and Aina J. O., Effects of steeping condition and
The authors gratefully acknowledge the program for Chang- germination time on the alpha-amylase activity, phenolics con-
jiang Scholars and Innovative Research Team in University, tent and malting loss of Nigerian local red and hybrid short
IRT0532 and the Qinglan Project of Jiangsu province for finan- kaura sorghum malts. Food Chem., 1997, 58, 289-295.
cial support. 17. Joglekar A. M. and May A. T., Product excellence through
design of experiments. Cereal Foods World, 1987, 32, 857-868.
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