Preparative Biochemistry & Biotechnology, 42:406425, 2012

Copyright # Taylor & Francis Group, LLC

ISSN: 1082-6068 print/1532-2297 online
DOI: 10.1080/10826068.2011.635739

STATISTICAL OPTIMIZATION OF A NOVEL LOW-COST MEDIUM

BASED ON REGIONAL AGRO-INDUSTRIAL BY-PRODUCTS
FOR THE PRODUCTION OF PROTEOLYTIC ENZYMES BY
Bacillus cereus

C. E. Kotlar,1,2 M. V. Aguero,1,2 and S. I. Roura1,2

1
Research Group on Food Engineering, Department of Chemical & Food Engineering,
Faculty of Engineering, National University of Mar del Plata, Mar del Plata, Argentina
2
National Council of Scientific and Technical Research (CONICET), Argentina

& Bacillus sp. are specific producers of peptidase amongst bacteria and peptidase enzymes and
are of significant ones due to their multifarious applications. Advances in industrial biotechnology
offer potential opportunities for economic utilization of agro-industrial by-products for many biochemical reactions. Due to their rich organic nature, they can serve as an ideal substrate for the
production of different value added products like peptidases. In the present work, an attempt
was made to optimize different variables by Taguchi methodology for the production of peptidase
using agro-industrial by-products hydrolyzed by a Bacillus cereus strain, resulting in brewers
spent grain (BSG) being the optimal organic substrate. Subsequently, operative variables for the
BSG were investigated using Taguchi methodology in order to maximize the enzyme production.
Additionally, the main medium components were optimized using a mixture design. Finally, the
production of peptidase by B. cereus was investigated; also the possible interaction with other proteolytic microbial strains was evaluated. A notorious synergistic effect was observed when B. cereus
was inoculated with Pseudomonas sp. These brought a triple benefit, first, opening the possibility
to produce technical enzymes at low cost, second, giving greater value to a food industry by-product,
and third, reducing the environmental impact caused by the product removal directly into the
environment.
Keywords medium optimization, mixture design, peptidase, statistical designs, taguchi
design

INTRODUCTION
Microbial enzymes are more advantageous than enzymes derived from
plants or animals because of their great variety of catalytic activities,
Address correspondence to Catalina Elena Kotlar, Food Engineer, Grupo de Investigacion en
Ingeniera en Alimentos, Faculty of Engineering, National University of Mar del Plata, Juan B. Justo
4302, Mar del Plata 7600, Argentina. E-mail: ckotlar@fi.mdp.edu.ar

Statistical Optimization of a Novel Low-Cost Medium

407

possible high yields, stability, easy of genetic manipulation, regular supply

due to absence of seasonal fluctuations, rapid microorganisms growth, and
more convenient and safer protection methods.[1] Bacterial strains are generally more used as they offer higher activities compared to yeast.[2] Of the
microbial enzymes, peptidase is of particular interest due to its primary application. Peptidases account for approximately 60% of all enzyme sales because
of their varied applications.[3] Among bacteria, Bacillus sp. is a specific producer of peptidases.[4] Peptidase production from this genus using various
agricultural residues has been widely described in the literature.[5] In Argentina, a livestock agricultural country, a wide range of agro-industrial byproducts is available in large quantities and these by-products have considerable nutritional potential. These wastes, which represent an environmental
problem to the industry, constitute an important protein source.
The selection of an ideal agro-biotech waste for enzyme production
depends upon several factors, mainly related to cost and availability of
the substrate material, and thus may involve screening of several
agro-industrial residues.
The expansion of biotechnology has created an increasing demand for
new and low-cost microbial growth substrate. In most instances, the growth
medium account for approximately 40% of the production cost of industrial enzymes.[6] Searching for cheap substrates that are effective on bacterial growth can reduce operating costs of technical enzymes.[7]
Conventional optimization procedures involve altering of one parameter
at a time keeping all others constant, which enables one to assay the impact
of those particular parameters on the process performance. These procedures are time-consuming, cumbersome, require more experimental data
sets, and cannot provide information about the mutual parameters interactions.[8] As an alternative to conventional optimization procedure, design
of experiments (DOE) methods and statistical tools helps to gain more
information about the optimization conditions in a few trials. DOE methods have been widely employed in bioprocess optimization because these
methods provide a systematic and efficient plan for experimentation
considering the interactive effects among the control factors.
The developing process of an optimum medium for maximum enzyme
production involves a stage of screening the critical medium components
and process parameters that influence the desired products production.
The primary goal in this step is to study the statistical significance of an
effect exerted on a particular factor on the dependent variable of interest.
Once the critical components to the production are screened, the second
stage of media optimization is to find the optimum concentration of
each component for maximum product formation. While developing an
industrial process, it is imperative to carry out the optimization studies that
can be scaled up at larger scale easily.

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C. E. Kotlar et al.

In a previous work, a Bacillus cereus strain producing alkaline peptidase

was isolated from fermented cabbage.[9] The crude extracellular peptidase
extract exhibited the ability to hydrolyze proteins of low and high molecular weight. This fact leads us to look for the formulation of media bases on
cheap substrate, and it was the objective of this study to investigate the production of Bacillus cereus peptidase in a low-cost medium, using
agro-industrial by-products provided from the local industries. To achieve
this objective, we focused first on the application of the Taguchi method
to test the relative importance of medium components (i.e., organic substrate, inorganic nitrogen, metal ions) and environmental factors (i.e., agitation speed and initial pH) in the peptidase production by this strain.
Then we used mixture design experiments to select the best combination
of solid substrate on peptidase production by this microorganism. The
capacity of B. cereus to exploit growth factors and=or vitamins secreted by
other proteolytic bacteria was also explored.

MATERIALS AND METHODS

Microorganism and Culture Maintenance
The microorganisms used in the present study were Bacillus cereus,
Pseudomonas not classified, Pseudomonas putida, and Enterococcus hirae, isolated
from fresh cabbage, and Lactococcus lactis subsp. lactis, which was isolated
from fermented cabbage.[9] These organisms were identified in the
CERELA center (CONICET [Tucuman, Argentina], 2008). The culture
was routinely maintained on soft brain and heart agar (3.5% w=v of
agar-agar) at
18 C. The organism was subcultured every 6 months.

Seed Preparation
The mentioned microorganisms were activated in two steps. The strains
were subcultured in BHI and incubated at 32 C for 24 hr. First, a loop was
inoculated in 8 mL BHI; after that 2 mL of culture was centrifuged at
1,000 rpm for 3 min at 4 C. The precipitate was then added to 25 mL fresh
BHI and statically incubated at 32 C for 24 hr.
The B. cereus culture (0.5% v=v) was allow to grow in minimal broth
(MB), an agarless modified basal medium (MM) containing 0.1% bacteriological glucose (w=v) (Britannia, lot 095, Buenos Aires, Argentina) and
0.25% yeast extract (w=v) (Acumedia, lot 66-22, Maryland), buffered at
pH 8 and at 32 C, which represents the optimal conditions for peptidase
production.[10] The bacterial cells were grown in MB in a 250-mL
Erlenmeyer flask on an orbital shaker (TS-1000, Zhejiang, China).

Statistical Optimization of a Novel Low-Cost Medium

409

In order to assess the ability of B. cereus to use any growth factors

excreted by other proteolytic strains, the seed was prepared by mixing
equal volumes of fresh culture from B. cereus in MB and other proteolytic
strains. To achieve an equal inoculums proportion, the optical density
at 600 nm of these cultures was measured and was been adjusted to 1.2
by adding sterile water to each culture under sterile conditions.[11]
Preparation of Fermentation Medium
Peptidase production by Bacillus cereus was carried out in a medium
consisting of the following composition of constant constituents:
Na3C6H5O7  2H2O (Alun Metroqumica, Argentina), 4 g=L; K2HPO4
(Biopack, Argentina), 4 g=L; CaCl2 (Anedra, Argentina), 0.002 g=L; and
MgSO4  7H2O (Timpar, Argentina), 0.5 g=L. A 10-g=L Na2CO3(Lennox,
England) solution was sterilized separately and added to the rest of the
medium after cooling.[12] This was the base level ingredient concentration
used for the experiments, called mineral based medium (MBM).
Brewers spent grains (BSG), sunflower cake (SFC), soybean cake
(SBC), and wheat bran (WB) were probed as organic substrate in the fermentation medium. In a previous work,[10] the peptidase production
medium, referred as minimal broth (MB), contained 2.5 g=L of yeast
extract and 1 g=L of bacteriological glucose as sole sources of both protein
and carbohydrate. The yeast extract was the unique source of vitamins and
nucleic acids. Considering the protein content in MM and in these four
agro-industrial by-products, 12.0, 4.1, 3.9, and 6.5 g=L of BSG, SC, SBC,
and WB, respectively, was added to the fermentation medium, following
the experimental matrix presented in Table 1.
The importance of the inorganic nitrogen source for the peptidase
production by Bacillus cereus strain was evaluated adding 0.5% w=v of different inorganic nitrogen sources: ammonium sulfate (Baker Chemical, USA),
potassium nitrate (Timpar, Argentina), sodium nitrate (Baker Chemical,
TABLE 1 Factors and Their Levels Employed in the Taguchi Experimental Design for Peptidase
Production by Bacillus cereus
Organic nitrogen source

Variables
Inorganic nitrogen
source (0.5%)
Metal ions
Agitation speed (rpm)
Initial pH

Brewers spent
grains (1)

Sunflower
cake (2)

Soybean
cake (3)

Wheat
bran (4)

(NH4)2SO4 (1)

NO3K (2)

NO3Na (3)

NH4Cl (4)

Mn (1)
0 (1)
5 (1)

Fe (2)
40 (2)
7 (2)

Cu (3)
80 (3)
9 (3)

Zn (4)
120 (4)
11 (4)

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C. E. Kotlar et al.

USA), and ammonium chloride (Merck, Germany), according to the

experimental matrix presented in Table 1. In the same way, the effect of
metallic ions was assayed by supplementing the fermentation medium with
the following salts: CuSO4  5H2O (Alun Metroqumica, Argentina), 0.2 g=
L; ZnSO4(Anedra, Argentina), 0.2 g=L; FeSO4 (Baker chemical Co.,
USA), 0.2 g=L; and MnSO4  H2O (Anedra, Argentina), 0.5 g=L.
The experiments were performed with and without 1.2 g=L starch as
exogenous carbon source.
Substrate Preparation
After an initial screening, the more suitable organic substrate was
chosen for further study in terms of the effect of their size reduction, polyphenols extraction steps, and BSG varieties on the peptidase production.
For size reduction assay, 10 g of dry sample was agitated in the mill at a
rate of 24,000 revolutions per minute (rpm) for 0, 15, and 30 s.
For polyphenols extraction steps, the sample was extracted with an
aqueous 1:4 alcohol solution (ethanol concentration 30% v=v) in a shaker
at 50 rpm at room temperature for 60 min. The mixture was filtrated
and the retained was collected. This procedure was followed none, one, or
two times.
Three batches of brewers spent grain from different raw material varieties comprised of (1) 77.8% Pilsner malt, 17% caramel malt, 4.5% chocolate malt, and 0.7% black malt; (2) 93% Pilsner malt and the remaining
part caramel malt; and (3) 100% Pilsner malt were kindly supplied by
Antares S.A. (Mar del Plata, Argentina). These treatments were combined
according to Table 2.
Submerged Fermentation
Organic substrate and inorganic nitrogen source and metallic ions were
added into 100 mL of the fermentation medium in a 15-mL Erlenmeyer
TABLE 2 Factors and Their Levels Employed in the Taguchi Experimental Design for Peptidase Production by Bacillus cereus with BSG as Organic Substrate
BSG variety

Variable
Grinding time (s)
Polyphenols
extraction runs

77.8% Pilsner malt. 17% caramel

malt. 4.5% chocolate malt and
93% of Pilsner malt and
0.7% black malt (1)
7% caramel malt (2)
0 (1)
0 (1)

15 (2)
1 (2)

100% of Pilsner
malt (3)
30 (3)
2 (3)

Statistical Optimization of a Novel Low-Cost Medium

411

flask separately, sterilized at 121.5 C for 15 min, and cooled. The initial pH
(5, 7, 9, and 11) was adjusted under sterile conditions using the previously
sterilized sodium carbonate solution. Then the fermentation medium was
inoculated with 5% v=v microorganism culture and incubated at 32 C for
36 hr in an orbital shaker at 0, 40, 80, and 120 rpm, depending on the
experimental matrix.
After incubation the crude enzyme was obtained by centrifuging the
culture broth at 10,000 rpm for 10 min at 4 C. The cell-free supernatant,
which contains the enzyme, was assayed for peptidase activity.
Enzyme
Proteolytic activity of the cell-free culture supernatants was assessed by
using azocasein as substrate. Briefly, 120 mL of supernatant was incubated
with 480 mL of 10 g=L azocasein in buffer, 100 mM Tris, pH 7, for 30 min
at 32 C. The reaction was stopped by the addition of 480 mL trichloroacetic
acid (TCA) to a final concentration of 100 g=L and incubated for 30 min at
4 C before being centrifuged at 10,000 rpm for 10 min; 800 mL of the supernatant from the centrifuged reaction was added to 200 mL of 1.8 N sodium
hydroxide and the absorbances at 420 nm were measured in a Spectrum
SP-2000 ultraviolet (UV) spectrophotometer (Zhejiang, China). For the
control, the reaction was stopped with TCA immediately after the supernatant was added. One enzyme activity unit (U) was expressed as the
amount of enzyme that caused a change of absorbance of 0.01 at 420 nm
under the assay conditions (120 mL of enzyme source, 30 min at 32 C).
Experimental Design
Taguchi Method
The Taguchi experimental design allowed us to determine the most
suitable organic substrate, the metal ion that promotes the hydrolysis and
the initial pH optimum to start the fermentation process (Tables 1 and 3).
Having established the main effects, it was determined also through the
Taguchi method the effects of organic substrate variety, the milling degree,
and the extraction polyphenols level over the hydrolysis ability of Bacillus
cereus (Tables 2 and 4).
These experimental designs allowed examining five factors in four
levels and three factors in three levels, respectively. As already mentioned,
the levels of the factors studied and the layout of the Taguchis array are
shown in Tables 1 and 2.
The results were analyzed to extract independently the main factors
effects; the variance technique analysis was then applied to determine
which factors were statistically significant. The controlling factors were

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C. E. Kotlar et al.

TABLE 3 L16 Orthogonal Array of Taguchi Experimental Design and Corresponding Peptidase
Production by Bacillus cereus in a Submerged Culture
Proteolytic activity in a submerged
medium (U)

Run
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

Organic
substrate

Inorganic
nitrogen

Metal
ions

1
1
1
1
2
2
2
2
3
3
3
3
4
4
4
4

1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4

1
2
3
4
2
1
4
3
3
4
1
2
4
3
2
1

Agitation Initial Without exogenous

speed
pH carbon source
1
2
3
4
3
4
1
2
4
3
2
1
2
1
4
3

1
2
3
4
4
3
2
1
2
1
4
3
3
4
1
2

0.113
0.351
0.075
0.097
0.062
0
0.028
0
0.093
0
0.05
0.176
0
0
0.004
0

With exogenous
carbon source
0.188
0.359
0.211
0.346
0.196
0.050
0.018
0.062
0.059
0.031
0.032
0.035
0.019
0.037
0.116
0

Note. Each result is the mean of three determinations; standard errors were less than 10% of the
means.

identified, through the magnitude of the quantified effects, and the statistically significant effects were determined. Accordingly, the optimal conditions were determined by combining the factors levels that had the
highest main effect value. All calculations were performed using SAS software (version 8.0, Cary, NC).
TABLE 4 L9 Orthogonal Arrays of Taguchi Experimental Design and Corresponding Peptidase
Production by Bacillus cereus in a Submerged Culture With BSG as Organic Substrate

Run
1
2
3
4
5
6
7
8
9

Variety

Grinding time

Polyphenols
extraction runs

Proteolytic activity in a
submerged medium (U)

1
1
1
2
2
2
3
3
3

1
2
3
1
2
3
1
2
3

1
2
3
2
3
1
3
1
2

0.489
0.693
0.641
0.615
0.661
0.330
0.207
0.647
0.536

Note. Each result is the mean of three determinations; standard errors were less than 10% of the
means.

Statistical Optimization of a Novel Low-Cost Medium

413

Mixture Design
The mixture design method was used to obtain the possible proportions of the selected solid substrates. In the mixture experiment, the independent factors were proportions of different components in a blend and
the total proportions of the different factors had to be 100%.
Thus, the component concentrations cannot be independently changed.[13] However, one cannot design experiments where the concentration
of each media component equals one. To overcome this, the concentration
of each medium component in a given experiment was expressed in terms
of a fraction of the maximum value. These calculated proportions are
named as coded values.
An augmented simplex centric design from Scheffes special cubic
model (3, 2), consisting of 7 (23
1) runs in addition to three replicated
vertices, was used (see Table 8, shown later).[14]
As the presence of three components always generates higher responses
than the pure components, it was necessary to use constraints. In a preliminary experiment that was carried out without establish constraints conditions, a maximum response was not found in the boundary region
(data not shown).
Algebraically, the experimental design constraints are as follows. Lets Xi
denote the proportion of i in the mixture and n the number of components:
Mixture design constraints were:
n
X

Xi 1

i1

0:5  X1  0:7
0:25  X2  0:45
0:05  X3  0:25
where X1, X2, and X3 are the BSG, starch, and FeSO4 proportions, respectively. For each component the equations established low and high
constraints.
The model that represents the response as a function of the mixture
variables consists of a first-order function (linear), described as follows:
Y

X3
i1

bi xi

where Y is the response and bi is a linear coefficient.

All calculations were performed using SAS software (version 8.0,
Cary, NC).

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C. E. Kotlar et al.

Validation
To validate the proposed experimental methodology, fermentation
experiments were performed in triplicate for peptidase production by
employing the obtained optimized culture conditions and composition.
RESULTS AND DISCUSSIONS
Effect of Medium Components and Environment Conditions on
Peptidase Production
Fermentation Medium without Starch
Taguchi experimental results designed in 16 runs, for the five factors,
that is, nitrogen organic source, nitrogen inorganic source, metal ions,
agitation speed, and initial pH, were chosen for the peptidase production
optimization by Bacillus cereus spp. strain.
Table 3 shows the proteolytic activity ranging from 0 to 0.359 U
corresponding to the combined effect of the five factors in their specific
ranges. The experimental results suggested that these factors at optimum
level strongly support the peptidase production. In run 16, with wheat bran
as organic substrate, NH4Cl (0.5% w=v) as inorganic nitrogen source, Mn
(0.5 g=L) as metal ions, 80 rpm, and neutral initial pH, peptidase production was not observed in fermentation medium with and without starch
as an exogenous carbon source. The higher enzyme production (0.351 U)
was observed in run 2 with a combination of brewers spent grain (BSG),
NO3K (0.5% w=v), Fe (0.2 g=L), 40 rpm, and neutral initial pH. Figure 1

FIGURE 1 Contribution of five factors on protease production and microorganism growth by Bacillus
cereus in a submerged culture: & without exogenous carbon source, and with starch as exogenous
carbon source, using Taguchi experimental design. A: organic substrate, B: inorganic nitrogen source;
C: metal ions; D: agitation speed; E: initial pH.

Statistical Optimization of a Novel Low-Cost Medium

415

presents the contribution of selected factors on the peptidase production

in fermentation medium with and without starch. It can be observed that
organic nitrogen source, metal ions, and initial pH showed the highest
positive impact on peptidase production, with contributions of 45.89%,
28.50%, and 13.37%, respectively. Figure 2 shows the effect of these major
contribution factors on the peptidase activity. The peptidase activity
enhancement in media containing BSG may be due to the presence of
bioactive substance that could act as peptidase inducers. Although BSG
contains iron, supplementing it with exogenous Fe2 led to a peptidase
activity increase.[16] Probably this metallic ion acts as an inducer in the
enzymatic hydrolysis by B. cereus.

FIGURE 2 Effect of organic substrate (a and b), metal ion (c and d) and initial pH (e) in a submerged
culture without exogenous carbon source (a, c and e) and with starch (b and d) on protease
production, measured as the absorbance at 420 nm.

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C. E. Kotlar et al.

Furthermore, peptidase production by microbial strains depends on

the extracellular pH because culture pH strongly influences many enzymatic processes and various components transport across the cell membranes, which in turn support the cell growth and product
production.[17] Bacillis cereus strain exhibited its maximum peptidase production at pH 7 (Figure 2) regardless of the addition of exogenous starch
as carbon source. This is in complete accordance with the findings of many
workers. The optimum pH values for the maximum peptidase production
were 77.5 for Bacillus subtilis and 7 for Bacillus sp. TKU0004.[18,19]
Inorganic nitrogen source and agitation speed showed least impact
among the factors studied with the assigned variance of values. Results of
Table 3 exhibited the fact that the ammonium phosphate in the production
media has a slight inhibitory effect (runs 1, 5, 9, and 13).
Based on L16 orthogonal array design, 16 experiments were carried out
in triplicate. In full-factorial experimental designs and working with the
same factor and level numbers, to reach the same results as those of the
orthogonal array method, at least 625 experiments are necessary. Therefore, the Taguchi experimental design is a better option for the optimization of biotechnological processes for microbial enzymes production.
The fermentation medium was formulated limiting the energy source
from sugars and relatively poor in protein content. This poor growing condition would be expected to enhance the microbial extracellular peptidases
production.[9]
Conflicting results regarding the effects of organic substrate on alkaline
peptidase production by Bacillus sp. have been reported in the literature.
Do Nascimento and Martins reported maximum enzyme activity by thermophilic Bacillus sp. strain SMIA-2 with wheat and fish powders enhancing significantly the peptidase production with comparison with commercial
substrate.[15] The main chemical compositions of the organic substrates
used by other authors are given in Table 5.
Of interest was the fact that B. cereus strain was successful in producing
peptidase in the absence of any of the inorganic nitrogen source supplied
to the medium (Table 6). This was in complete accordance with the results
TABLE 5

Proximate Composition of Used Organic Substrates

Organic
substrate

CarboIron
Ca
P
Water Protein Fat hydrate Fiber (mg=100 g) (mg=100 g) (mg=100 g)

BSG
SFC
SBC
WB

78.00
6.72
6.11
2.17

6.38 1.82 9.21

25.54 6.28 55.34
36.00 14.03 40.67
15.00 4.08 28.1

Note. NA: not analyzed.

4.31
NA
0.21
45.6

NA
NA
2.63
15.1

160
400
13.33
78.3

650
1000
300
560

Reference
[23]
[24]
[25]
[26]

Statistical Optimization of a Novel Low-Cost Medium

417

TABLE 6 ANOVA for Peptidase Production by Bacillus cereus in Medium Without Exogenous Carbon
Source
Source

DF

Sum of squares

Mean square

F value

Pr > F

Model
A
C
E
Error
Total

9
3
3
3
6
15

0.11726656
0.06132519
0.03807669
0.01786469
0.01635837
0.13362494

0.01302962
0.02044173
0.01269223
0.00595490
0.00272640
0.00595490

4.78
7.50
4.66
2.18

0.0352
0.0187
0.0522
0.1909

Note. R-squared: 0.878; coefficient of variation: 79.039; root MSE: 0.052; mean: 0.066. A: Organic substrate; C: metal ions; E: initial pH; asterisk indicates significant terms.

obtained by Massucco et al. and Chantawannakul et al.[20,21] Furthermore,

it should be noted that the fermentation medium included BSG as unique
source of both vitamins and nucleic acids.[22]
Therefore, as suggested by the Taguchi method, the analysis of variance
(ANOVA) for the peptidase production responses was carried out according to the factors with contributions higher than 10%.[23] In the Taguchi
approach, ANOVA is used to analyze the results of the orthogonal array
(OA) experiments and to determine how much variation each factor has
contributed. By studying the main effects of each factor, the general trends
of the factors influence toward the process can be distinguished.
Data analysis for the determination of significant parameters on peptidase
production was performed and the results are shown in Table 6. From the calculated ratios (F), it can be inferred that the organic substrate is statistically
significant at 95% confidence limit. The ANOVA of peptidase production
has a model F value of 4.78, which implies the model is significant. The model
obtained from ANOVA indicated that the multiple correlation coefficient of
R2 is 0.8776that is, the model can explain 87.76% variation in the response.
Fermentation Medium Supplemented with Potato Starch
The experimental procedure mentioned in the preceding subsection was
repeated in order to study the effect of the addition of an exogenous carbon
source (potato starch) in the fermentation medium on the peptidase production. Bacillus cereus has the ability to hydrolyze starch enzymatically.[30]
Figure 1b shows that the organic substrate and metal ions presented the
higher contributions (74.96 and 13.04%, respectively) on the enzyme
production. The other factors contributed less than 10% and were not
considered in the variance analysis.
Noticeable was the low contribution of pH on proteolytic activity,
contrary to what was found without the addition of an exogenous source
of starch. The presence of a quick energy source may alter the cellular
metabolic demands for the release of proteolytic enzymes, making highly

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C. E. Kotlar et al.

TABLE 7 ANOVA for Peptidase Production by Bacillus cereus in Medium With Starch as Exogenous
Carbon Source
Source

DF

Sum of squares

Mean square

F value

Pr > F

Model
A
C
Error
Total

6
3
3
9
15

0.0994
0.0613
0.0381
0.0342
0.1336

0.1650
0.0204
0.0126
0.0036

4.36
5.38
3.34

0.0244
0.0214
0.0698

Note. R-squared: 0.988; coefficient of variation: 41.627; root MSE: 0.046; mean: 0.110. A: Organic substrate; C: metal ions; asterisk indicates significant terms.

significant in this case the organic nitrogen source and metal ions versus
the pH fermentation medium.
Several studies have reported that proteins and peptides are necessary
for effective peptidase production, while carbohydrates repressed peptidase
formation.[2426] However, some works reported better peptidase synthesis
in the presence of a carbon source.[27,28]
The data analysis for the determination of significant parameters on peptidase production has been performed and the results are shown in Table 7.
From the calculated ratio (F), the model F value was 4.36, which implies the
model significance. The minor significance of metallic ions could attribute
to the supplementation with starch that provides an additional iron level.[29]
Although the organic substrates contained endogenous carbohydrates,
in general the starch addition favored the enzyme activity from B. cereus
(Table 5), probably due to the fact that these carbohydrates could not be
use as an immediate energy source. Thus, in the further experiments the
medium was supplemented with this exogenous carbohydrate source.
Effect of Organic Substrate Pretreatment on Peptidase
Production
The organic substrate of the medium was the most significant factor
among all selected optimization parameters at an individual level, with
BSG showing the highest peptidase production (Table 4).
BSG is an interesting raw material; it is rich in nutrients and minerals,
and cheap as well, as it is a readily available by-product from the brewing
industry.[30]
In order to investigate how preliminary pretreatments applied on the
organic substrate affect the enzyme production, an L9 (33) experimental
design was performed with three different factors named BSG varieties,
milling time, and polyphenols extraction runs. The experimental layout
for the peptidase production using the L9 orthogonal array is shown in
Table 2 and 4. The experiments were conducted using three levels

Statistical Optimization of a Novel Low-Cost Medium

419

(Table 2). Table 4 also shows the experimental results for the response
(peptidase production). Pretreatments applied on BSG organic substrate
significantly affected the peptidase activity from B. cereus (Table 4).
The maximal peptidase production (0.693 U) was reached in run 2 with
the following operating conditions: variety 1, 15 s of milling time and one
polyphenols extraction run (2) (Table 4), which gave twice the peptidase
production mentioned in run 3 from Table 3. However, the evaluation of
the effect that each factor had on the peptidase production indicated that
the milling time was the only most significant factor for the peptidase production (Figure 3), with a maximum at 15 s milling time (Figure 4). BSG
varieties and polyphenols extraction runs were observed to exert 21.69%
and 19.82%, respectively. The lower influence of BSG varieties could be
interpreted because no statistically significant differences (p-value >0.05)
in the protein content among varieties were found.[5] Then it would be
not expected that there would be any substantial difference in the capacity
of fermentation from B. cereus. Therefore, a pool of BSG formed by the
same proportion of each variety was used for the further experiments.
As the polyphenols extraction cycle had a low contribution to the peptidase production, only one polyphenols extraction cycle was used for the
followed experimental procedures, taking into account the maximal average in the response. It is well known that polyphenols could inhibit some
matrix peptidases.[31] For that reason the option of treatment without polyphenols extraction cycle was discarded. In the other extreme, with two
extraction cycles, the extraction solution could affect the native protein
conformations, altering the active site so that the enzymatic hydrolysis
could be lower.

FIGURE 3 Contribution of the three factors on protease production by Bacillus cereus in a submerged
culture with BSG, using Taguchi experimental design.

420

C. E. Kotlar et al.

FIGURE 4 Effect of BSG grinding time in a submerged culture with BSG on protease production,
measured as the absorbance at 420 nm.

Mixture Design
The results using the proposed mixture design showed that peptidase
production varied within the range of 0.4260.601 U (Table 8). The highest
enzyme production (0.601 U) was observed in the eighth run in the experimental setup, which contained 60% BSG, 35% starch, and the rest FeSO4.
To obtain information concerning the media components interaction
over the enzyme production, the contours of constant height were plotted
on the two-dimensional (2D) triangle in a contour plot. Figure 5 illustrates
how different media components interact with each other, influencing the
enzyme production by B. cereus. Near the vertex where maximum iron content is present, a decreased in the enzyme production is predicted. This
could be attributed to the iron excess, which could create an oxidizing
environment, which can cause irreparable cell damage.[32]
Results of the analysis of variance using ADX Interface of SAS indicate
that X1 and X2 are significant and will be used in the response optimization, while X3 is not significant. Since the estimate parameters X1 and X2
are almost similar in magnitude, it can be concluded that both components
are effective in the medium formulation for peptidase production. BSG
and starch presented a positive influence on enzyme production
(Table 8). BSG had the lowest p-value (0.0001), indicating that it had a significant effect on the changes in its concentration on the peptidase production. On the contrary, Fe had the highest p-value, indicating that
peptidase production is not dependent on the concentration of this metal
source. Based on the preceding results it can be concluded that BSG and
starch are statistically significant factors and should be included in further

Statistical Optimization of a Novel Low-Cost Medium

421

TABLE 8 Mixture Design Used in This Study. Experimental Results Obtained for the Dependent Variables and Predicted Values by Linear Model
Proportion

Run
1
2
3
4
5
6
7
8
9
10

Design point
All
All
All
All
All
All
All
All
All
All

components
components
components
components
components
components
components
components
components
components

Proteolytic activity (U)

BSG X1 (g=L)

Starch X2 (g=L)

Fe X3 (g=L)

Coded

Real

Coded

Real

Coded

Real

Experimental

Predicted

0.50
0.50
0.50
0.50
0.50
0.57
0.60
0.60
0.70
0.70

12.00
12.00
12.00
12.00
12.00
13.68
14.40
14.40
16.80
16.80

0.25
0.25
0.35
0.45
0.45
0.32
0.25
0.35
0.25
0.25

6.00
6.00
8.40
10.80
10.80
7.68
6.00
8.40
6.00
6.00

0.25
0.25
0.15
0.05
0.05
0.12
0.15
0.05
0.05
0.05

6.00
6.00
3.60
1.20
1.20
2.88
3.60
1.20
1.20
1.20

0.426
0.439
0.476
0.512
0.502
0.548
0.584
0.601
0.530
0.518

0.430
0.430
0.474
0.518
0.518
0.506
0.494
0.538
0.558
0.558

Note. Each experimental result is the mean of three determinations; standard errors were less than
10% of the means.

experimentation for medium optimization in a pilot scale. The predicted

equation for the model based on the coded values is:
Y 0:64  X1 0:44  X2

where Y is the peptidase production, X1 the BSG content, and X2 the starch
content. Thus the predicted values obtained with Eq. (1) are shown in
Table 8.

FIGURE 5 Contour plot of linear model predicted protease production values.

422

C. E. Kotlar et al.

FIGURE 6 Plot of variables marginal means for the response.

Finally, the profile in Figure 6 displays the optimal setting rounded of

0.5667, 0.3167, and 0.1167% of BSG, starch, and FeFO4, respectively, which
gives an estimated response of 0.514.
To further validate the proposed experimental methodology, fermentation experiments were performed in triplicate for peptidase production
by employing the obtained optimized culture composition. The experimental data showed an enhanced peptidase yield of 0.531 U (48% improvement in peptidase production) with the modified culture conditions.
Mixed Inoculums
The possible interaction of Bacillus cereus with other proteolytic
microbial strains was evaluated. Figure 7 presents the peptidase production

FIGURE 7 Proteolytic activity in optimized fermentation medium with mixed inoculum at 32 C in a
rotatory shaker at 60 rpm.

Statistical Optimization of a Novel Low-Cost Medium

TABLE 9

x1
x2
x3

423

Estimated Parameters for the Linear Model

Estimate

Std. error

t Ratio

p-Value

0.63917
0.44362
0.09363

0.086299
0.1394
0.17036

7.4064
3.1823
0.54959

0.0001
0.0154
0.5997

Note. Asterisk indicates significant.

in fermentation medium inoculated with a mixed inoculum. A notorious

synergistic effect was observed when B. cereus was inoculated either with
Pseudomonas putida or Pseudomonas not classified.
The interaction BacillusPseudomonas strains incremented by 5060%
the peptidase production obtained only with Bacillus. The results presented
suggested the possible start of substrate hydrolysis by Bacillus cereus and
then the Pseudomonas strains complete the degradation processes through
endopeptidase activity. Similar results were found by Oyama et al.[33] and
Gobbetti et al.,[34] who purified an endopeptidase in the cell free extract
of a Pseudomonas sp. Odagaki et al.[35] isolated a pyroglutamyl peptidase
I, a group of exopeptidases responsible for the hydrolysis of N-terminal pyroglutamate residues, from Bacillus spp. Furthermore this result could indicate that B. cereus strain is able to exploit growth factors and=or vitamins
secreted by Pseudomonas sp.
On the other hand, the interaction with Enterococcus hirae strain reduced
by 2040% the peptidase activity, indicating a possible antagonistic effect
between the two strains. Two possible effects could be responsible for the
lower protein hydrolysis when the two strains work together: a possible competition for the catalytic site, and=or bacteriocin-like substance production
by E. hirae that inhibits the growth of Bacillus cereus. Some bibliographic data
reinforce this last fact. Lasagno et al.[36] reported a bacteriocin produced by
Enterococcus hirae that inhibited the growth of Bacillus cereus, Listeria monocytogenes, Clostridium perfringes, and Staphylococcus aureus.
A similar result was found with the interaction of Lactococcus lactis subsp.
Lactis, reducing the peptidase activity by 5060%. It is well known that
this genus produced bacteriocin with antimicrobial activity against Bacillus
cereus.[37]
CONCLUSIONS
In this preliminary study, the production of peptidase by B. cereus was
investigated; the possible interaction with other proteolytic microbial
strains was also evaluated. It was possible to optimize the formulation of
a medium for the production of proteolytic enzymes at low cost through
the utilization of regional by-products of the brewing industry. This

424

C. E. Kotlar et al.

brought a triple benefit, first, opening the possibility to produce technical

enzymes at low cost, second, giving greater value to a food industry
by-product, and third, reducing the environmental impact caused by the
product removal directly into the environment. The use of these cheaper
and readily available sources of both carbon and nitrogen suppliers instead
of commercial substrate are the key attraction for the cost-effective production on an extracellular peptidases. Additional experiments should be
conducted to identify bioactive substances or inducers present in brewers
spent grains.

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