STATISTICAL OPTIMIZATION OF A NOVEL LOW-COST MEDIUM BASED

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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. Agüero,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 2National 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 bio- chemical 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 brewer’s 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. cereuswas 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, taguchidesign

INTRODUCTION

Microbial enzymes are more advantageous than enzymes derived fromplants or animals because of their great variety of catalytic activities,

Address correspondence to Catalina Elena Kotlar, Food Engineer, Grupo de Investigación enIngenierı́a en Alimentos, Faculty of Engineering, National University of Mar del Plata, Juan B. Justo

4302, Mar del Plata 7600, Argentina. E-mail: [email protected]

Preparative Biochemistry & Biotechnology , 42:406–425, 2012Copyright # Taylor & Francis Group, LLCISSN: 1082-6068 print/1532-2297 onlineDOI: 10.1080/10826068.2011.635739


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possible high yields, stability, easy of genetic manipulation, regular supply due to absence of seasonal fluctuations, rapid microorganisms growth, andmore convenient and safer protection methods.[1] Bacterial strains are gen-

erally more used as they offer higher activities compared to yeast.[2]

Of themicrobial enzymes, peptidase is of particular interest due to its primary appli-cation. Peptidases account for approximately 60%of all enzyme sales becauseof their varied applications.[3] Among bacteria, Bacillus sp. is a specific pro-ducer of peptidases.[4] Peptidase production from this genus using variousagricultural residues has been widely described in the literature.[5] In Argen-tina, a livestock agricultural country, a wide range of agro-industrial by-products is available in large quantities and these by-products have consider-able nutritional potential. These wastes, which represent an environmentalproblem to the industry, constitute an important protein source.

The selection of an ideal agro-biotech waste for enzyme productiondepends upon several factors, mainly related to cost and availability of the substrate material, and thus may involve screening of severalagro-industrial residues.

The expansion of biotechnology has created an increasing demand fornew and low-cost microbial growth substrate. In most instances, the growthmedium account for approximately 40% of the production cost of indus-trial enzymes.[6] Searching for cheap substrates that are effective on bac-terial 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 proce-dures are time-consuming, cumbersome, require more experimental datasets, and cannot provide information about the mutual parameters interac-tions.[8] As an alternative to conventional optimization procedure, designof experiments (DOE) methods and statistical tools helps to gain moreinformation about the optimization conditions in a few trials. DOE meth-ods have been widely employed in bioprocess optimization because thesemethods 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 enzymeproduction involves a stage of screening the critical medium componentsand process parameters that influence the desired products production.The primary goal in this step is to study the statistical significance of aneffect exerted on a particular factor on the dependent variable of interest.Once the critical components to the production are screened, the secondstage of media optimization is to find the optimum concentration of each component for maximum product formation. While developing anindustrial process, it is imperative to carry out the optimization studies that

can be scaled up at larger scale easily.

Statistical Optimization of a Novel Low-Cost Medium 407


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In a previous work, a Bacillus cereus strain producing alkaline peptidase was isolated from fermented cabbage.[9] The crude extracellular peptidaseextract exhibited the ability to hydrolyze proteins of low and high molecu-

lar weight. This fact leads us to look for the formulation of media bases oncheap substrate, and it was the objective of this study to investigate the pro-duction of Bacillus cereus peptidase in a low-cost medium, usingagro-industrial by-products provided from the local industries. To achievethis objective, we focused first on the application of the Taguchi methodto test the relative importance of medium components (i.e., organic sub-strate, inorganic nitrogen, metal ions) and environmental factors (i.e., agi-tation speed and initial pH) in the peptidase production by this strain.Then we used mixture design experiments to select the best combinationof 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 pu ´ tida, and Enterococcus hirae , isolated

from fresh cabbage, and Lactococcus lactis subsp. lactis , which was isolatedfrom fermented cabbage.[9] These organisms were identified in theCERELA center (CONICET [Tucumán, Argentina], 2008). The culture

was routinely maintained on soft brain and heart agar (3.5% w = v of agar-agar) at 18C. 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 wasinoculated in 8 mL BHI; after that 2 mL of culture was centrifuged at 1,000 rpm for 3 min at 4C. The precipitate was then added to 25 mL freshBHI and statically incubated at 32C 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% bacterio-logical glucose (w = v) (Britannia, lot 095, Buenos Aires, Argentina) and0.25% yeast extract (w = v) (Acumedia, lot 66-22, Maryland), buffered at pH 8 and at 32C, which represents the optimal conditions for peptidaseproduction.[10] The bacterial cells were grown in MB in a 250-mL

Erlenmeyer flask on an orbital shaker (TS-1000, Zhejiang, China).

408 C. E. Kotlar et al.


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In order to assess the ability of B. cereus to use any growth factorsexcreted by other proteolytic strains, the seed was prepared by mixingequal 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 1.2by adding sterile water to each culture under sterile conditions.[11]

Preparation of Fermentation Medium

Peptidase production by Bacillus cereus was carried out in a mediumconsisting of the following composition of constant constituents:Na3C6H5O7 2H2O (Alun Metroquı́mica, Argentina), 4 g=L; K 2HPO4(Biopack, Argentina), 4 g=L; CaCl2 (Anedra, Argentina), 0.002 g=L; andMgSO4 7H2O (Timpar, Argentina), 0.5 g=L. A 10-g=L Na2CO3(Lennox,England) solution was sterilized separately and added to the rest of themedium after cooling.[12] This was the base level ingredient concentrationused for the experiments, called mineral based medium (MBM).

Brewer’s spent grains (BSG), sunflower cake (SFC), soybean cake(SBC), and wheat bran (WB) were probed as organic substrate in the fer-mentation medium. In a previous work,[10] the peptidase productionmedium, 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 andnucleic acids. Considering the protein content in MM and in these fouragro-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, followingthe experimental matrix presented in Table 1.

The importance of the inorganic nitrogen source for the peptidaseproduction by Bacillus cereus strain was evaluated adding 0.5% w = v of differ-ent 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 PeptidaseProduction by Bacillus cereus

Organic nitrogen source

VariablesBrewer’s spent

grains (1)Sunflowercake (2)

Soybeancake (3)

Wheat bran (4)

Inorganic nitrogensource (0.5%)

(NH4)2SO4 (1) NO3K (2) NO3Na (3) NH4Cl (4)

Metal ions Mn (1) Fe (2) Cu (3) Zn (4)

Agitation speed (rpm) 0 (1) 40 (2) 80 (3) 120 (4)Initial pH 5 (1) 7 (2) 9 (3) 11 (4)



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USA), and ammonium chloride (Merck, Germany), according to theexperimental 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 Metroquı́mica, 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 asexogenous carbon source.

Substrate Preparation

After an initial screening, the more suitable organic substrate waschosen for further study in terms of the effect of their size reduction, poly-

phenols 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 shakerat 50 rpm at room temperature for 60 min. The mixture was filtratedand the retained was collected. This procedure was followed none, one, ortwo times.

Three batches of brewer’s spent grain from different raw material vari-eties comprised of (1) 77.8% Pilsner malt, 17% caramel malt, 4.5% choc-

olate malt, and 0.7% black malt; (2) 93% Pilsner malt and the remainingpart caramel malt; and (3) 100% Pilsner malt were kindly supplied by

Antares S.A. (Mar del Plata, Argentina). These treatments were combinedaccording to Table 2.

Submerged Fermentation

Organic substrate and inorganic nitrogen source and metallic ions wereadded 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 Pro-duction by Bacillus cereus with BSG as Organic Substrate

BSG variety

Variable

77.8% Pilsner malt. 17% caramelmalt. 4.5% chocolate malt and

0.7% black malt (1)93% of Pilsner malt and

7% caramel malt (2)100% of Pilsner

malt (3)

Grinding time (s) 0 (1) 15 (2) 30 (3)Polyphenols

extraction runs

0 (1) 1 (2) 2 (3)



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flask separately, sterilized at 121.5C 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 for36 hr in an orbital shaker at 0, 40, 80, and 120 rpm, depending on theexperimental matrix.

After incubation the crude enzyme was obtained by centrifuging theculture broth at 10,000 rpm for 10 min at 4C. 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 minat 32C. The reaction was stopped by the addition of 480 mL trichloroaceticacid (TCA) to a final concentration of 100 g=L and incubated for 30 min at 4C before being centrifuged at 10,000 rpm for 10 min; 800mL of the super-natant from the centrifuged reaction was added to 200 mL of 1.8 N sodiumhydroxide and the absorbances at 420 nm were measured in a SpectrumSP-2000 ultraviolet (UV) spectrophotometer (Zhejiang, China). For thecontrol, the reaction was stopped with TCA immediately after the super-natant 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 nmunder the assay conditions (120 mL of enzyme source, 30 min at 32C).

Experimental Design

Taguchi Method The Taguchi experimental design allowed us to determine the most

suitable organic substrate, the metal ion that promotes the hydrolysis andthe initial pH optimum to start the fermentation process (Tables 1 and 3).

Having established the main effects, it was determined also through theTaguchi 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 fourlevels and three factors in three levels, respectively. As already mentioned,the levels of the factors studied and the layout of the Taguchi’s array areshown in Tables 1 and 2.

The results were analyzed to extract independently the main factorseffects; the variance technique analysis was then applied to determine

which factors were statistically significant. The controlling factors were



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identified, through the magnitude of the quantified effects, and the statisti-cally significant effects were determined. Accordingly, the optimal con-ditions were determined by combining the factors levels that had thehighest main effect value. All calculations were performed using SAS soft-

ware (version 8.0, Cary, NC).

TABLE 4 L9 Orthogonal Arrays of Taguchi Experimental Design and Corresponding PeptidaseProduction by Bacillus cereus in a Submerged Culture With BSG as Organic Substrate

Run Variety Grinding time

Polyphenols

extraction runs

Proteolytic activity in a

submerged medium (U)

1 1 1 1 0.4892 1 2 2 0.6933 1 3 3 0.6414 2 1 2 0.6155 2 2 3 0.6616 2 3 1 0.330

7 3 1 3 0.2078 3 2 1 0.6479 3 3 2 0.536

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

means.

TABLE 3 L16 Orthogonal Array of Taguchi Experimental Design and Corresponding PeptidaseProduction by Bacillus cereus in a Submerged Culture

RunOrganicsubstrate

Inorganicnitrogen

Metalions

Agitationspeed

InitialpH

Proteolytic activity in a submerged

medium (U)

Without exogenouscarbon source

With exogenouscarbon source

1 1 1 1 1 1 0.113 0.1882 1 2 2 2 2 0.351 0.3593 1 3 3 3 3 0.075 0.211

4 1 4 4 4 4 0.097 0.3465 2 1 2 3 4 0.062 0.196

6 2 2 1 4 3 0 0.0507 2 3 4 1 2 0.028 0.0188 2 4 3 2 1 0 0.062

9 3 1 3 4 2 0.093 0.05910 3 2 4 3 1 0 0.031

11 3 3 1 2 4 0.05 0.03212 3 4 2 1 3 0.176 0.035

13 4 1 4 2 3 0 0.01914 4 2 3 1 4 0 0.03715 4 3 2 4 1 0.004 0.11616 4 4 1 3 2 0 0

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

means.



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Mixture Design The mixture design method was used to obtain the possible propor-

tions of the selected solid substrates. In the mixture experiment, the inde-

pendent factors were proportions of different components in a blend andthe total proportions of the different factors had to be 100%.Thus, the component concentrations cannot be independently chan-

ged.[13] However, one cannot design experiments where the concentrationof each media component equals one. To overcome this, the concentrationof each medium component in a given experiment was expressed in termsof a fraction of the maximum value. These calculated proportions arenamed as coded values.

An augmented simplex centric design from Scheffé’s special cubicmodel (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 responsesthan the pure components, it was necessary to use constraints. In a prelimi-nary experiment that was carried out without establish constraints con-ditions, a maximum response was not found in the boundary region(data not shown).

Algebraically, the experimental design constraints are as follows. Lets X idenote the proportion of i in the mixture and n the number of components:

Mixture design constraints were:

Xn

i ¼1

X i ¼ 1

0:5 X 1 0:7

0:25 X 2 0:45

0:05 X 3 0:25

where X1, X2, and X3 are the BSG, starch, and FeSO4 proportions, respect-

ively. For each component the equations established low and highconstraints.

The model that represents the response as a function of the mixture variables consists of a first-order function (linear), described as follows:

Y ¼X3

i ¼1bi x i

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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Validation

To validate the proposed experimental methodology, fermentationexperiments 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 productionoptimization by Bacillus cereus spp. strain.

Table 3 shows the proteolytic activity ranging from 0 to 0.359 Ucorresponding to the combined effect of the five factors in their specificranges. The experimental results suggested that these factors at optimumlevel strongly support the peptidase production. In run 16, with wheat branas 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 pro-duction 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 brewer’s 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 exogenouscarbon source, using Taguchi experimental design. A: organic substrate, B: inorganic nitrogen source;

C: metal ions; D: agitation speed; E: initial pH.



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presents the contribution of selected factors on the peptidase productionin 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 majorcontribution 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 BSGcontains iron, supplementing it with exogenous Fe2þ led to a peptidaseactivity increase.[16] Probably this metallic ion acts as an inducer in theenzymatic hydrolysis by B. cereus .

FIGURE 2 Effect of organic substrate (a and b), metal ion (c and d) and initial pH (e) in a submergedculture 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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Furthermore, peptidase production by microbial strains depends onthe extracellular pH because culture pH strongly influences many enzy-matic processes and various components transport across the cell mem-

branes, which in turn support the cell growth and product production.[17] Bacillis cereus strain exhibited its maximum peptidase pro-duction at pH 7 (Figure 2) regardless of the addition of exogenous starchas carbon source. This is in complete accordance with the findings of many

workers. The optimum pH values for the maximum peptidase production were 7–7.5 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 productionmedia 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 thesame factor and level numbers, to reach the same results as those of theorthogonal array method, at least 625 experiments are necessary. There-fore, the Taguchi experimental design is a better option for the optimiza-tion of biotechnological processes for microbial enzymes production.

The fermentation medium was formulated limiting the energy sourcefrom sugars and relatively poor in protein content. This poor growing con-dition would be expected to enhance the microbial extracellular peptidasesproduction.[9]

Conflicting results regarding the effects of organic substrate on alkalinepeptidase production by Bacillus sp. have been reported in the literature.Do Nascimento and Martins reported maximum enzyme activity by thermo-philic Bacillus sp . strain SMIA-2 with wheat and fish powders enhancing sig-nificantly the peptidase production with comparison with commercialsubstrate.[15] The main chemical compositions of the organic substratesused by other authors are given in Table 5.

Of interest was the fact that B. cereus strain was successful in producingpeptidase 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

Organicsubstrate Water Protein Fat

Carbo-hydrate Fiber

Iron(mg=100g)

Ca(mg=100g)

P(mg=100 g) Reference

BSG 78.00 6.38 1.82 9.21 4.31 NA 160 650 [23]SFC 6.72 25.54 6.28 55.34 NA NA 400 1000 [24]SBC 6.11 36.00 14.03 40.67 0.21 2.63 13.33 300 [25] WB 2.17 15.00 4.08 28.1 45.6 15.1 78.3 560 [26]

Note. NA: not analyzed.



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

it should be noted that the fermentation medium included BSG as uniquesource 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 accord-ing to the factors with contributions higher than 10%.[23] In the Taguchiapproach, ANOVA is used to analyze the results of the orthogonal array (OA) experiments and to determine how much variation each factor hascontributed. By studying the main effects of each factor, the general trendsof the factor’s 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 cal-culated ratios ( F ), it can be inferred that the organic substrate is statistically significant at 95% confidence limit. The ANOVA of peptidase productionhas a model F value of 4.78, which implies the model is significant. The modelobtained from ANOVA indicated that the multiple correlation coefficient of R 2 is 0.8776 — that 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 carbonsource (potato starch) in the fermentation medium on the peptidase pro-duction. Bacillus cereus has the ability to hydrolyze starch enzymatically.[30]

Figure 1b shows that the organic substrate and metal ions presented thehigher contributions (74.96 and 13.04%, respectively) on the enzymeproduction. 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 sourceof starch. The presence of a quick energy source may alter the cellular

metabolic demands for the release of proteolytic enzymes, making highly

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

Source DF Sum of squares Mean square F value Pr > F

Model 9 0.11726656 0.01302962 4.78 0.0352

A 3 0.06132519 0.02044173 7.50 0.0187

C 3 0.03807669 0.01269223 4.66 0.0522E 3 0.01786469 0.00595490 2.18 0.1909Error 6 0.01635837 0.00272640Total 15 0.13362494 0.00595490

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



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significant in this case the organic nitrogen source and metal ions versusthe pH fermentation medium.

Several studies have reported that proteins and peptides are necessary for effective peptidase production, while carbohydrates repressed peptidaseformation.[24–26] However, some works reported better peptidase synthesisin the presence of a carbon source.[27,28]

The data analysis for the determination of significant parameters on pep-tidase 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 themodel significance. The minor significance of metallic ions could attributeto 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 beuse as an immediate energy source. Thus, in the further experiments themedium 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, withBSG 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 brewingindustry.[30]

In order to investigate how preliminary pretreatments applied on theorganic substrate affect the enzyme production, an L9 (33) experimentaldesign 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

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

Source DF Sum of squares Mean square F value Pr > F

Model 6 0.0994 0.1650 4.36 0.0244

A 3 0.0613 0.0204 5.38 0.0214

C 3 0.0381 0.0126 3.34 0.0698Error 9 0.0342 0.0036Total 15 0.1336

Note. R -squared: 0.988; coefficient of variation: 41.627; root MSE: 0.046; mean: 0.110. A: Organic sub-

strate; C: metal ions; asterisk indicates significant terms.



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(Table 2). Table 4 also shows the experimental results for the response(peptidase production). Pretreatments applied on BSG organic substratesignificantly affected the peptidase activity from B. cereus (Table 4).

The maximal peptidase production (0.693 U) was reached in run 2 withthe following operating conditions: variety 1, 15 s of milling time and onepolyphenols extraction run (2) (Table 4), which gave twice the peptidaseproduction 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 pro-duction (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 beinterpreted because no statistically significant differences ( p -value >0.05)

in the protein content among varieties were found.[5]

Then it would benot expected that there would be any substantial difference in the capacity of fermentation from B. cereus . Therefore, a pool of BSG formed by thesame proportion of each variety was used for the further experiments.

As the polyphenols extraction cycle had a low contribution to the pep-tidase production, only one polyphenols extraction cycle was used for thefollowed experimental procedures, taking into account the maximal aver-age in the response. It is well known that polyphenols could inhibit somematrix peptidases.[31] For that reason the option of treatment without poly-phenols extraction cycle was discarded. In the other extreme, with two

extraction cycles, the extraction solution could affect the native proteinconformations, altering the active site so that the enzymatic hydrolysiscould 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.



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Mixture Design

The results using the proposed mixture design showed that peptidaseproduction varied within the range of 0.426–0.601 U (Table 8). The highest enzyme production (0.601 U) was observed in the eighth run in the experi-mental setup, which contained 60% BSG, 35% starch, and the rest FeSO4.

To obtain information concerning the media components interactionover the enzyme production, the contours of constant height were plottedon the two-dimensional (2D) triangle in a contour plot. Figure 5 illustrateshow different media components interact with each other, influencing theenzyme production by B. cereus . Near the vertex where maximum iron con-tent is present, a decreased in the enzyme production is predicted. Thiscould be attributed to the iron excess, which could create an oxidizingenvironment, 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 optimiza-tion, while X3 is not significant. Since the estimate parameters X1 and X2are almost similar in magnitude, it can be concluded that both componentsare effective in the medium formulation for peptidase production. BSGand starch presented a positive influence on enzyme production(Table 8). BSG had the lowest p -value (0.0001), indicating that it had a sig-nificant effect on the changes in its concentration on the peptidase pro-duction. On the contrary, Fe had the highest p -value, indicating that peptidase production is not dependent on the concentration of this metalsource. Based on the preceding results it can be concluded that BSG and

starch are statistically significant factors and should be included in further

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



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experimentation for medium optimization in a pilot scale. The predictedequation for the model based on the coded values is:

Y ¼ 0:64 X 1 þ 0:44 X 2 ð1Þ

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

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

Proportion Proteolytic activity (U)

BSG X1 (g=L) Starch X2 (g=L) Fe X3 (g=L)

Experimental PredictedRun Design point Coded Real Coded Real Coded Real

1 All components 0.50 12.00 0.25 6.00 0.25 6.00 0.426 0.4302 All components 0.50 12.00 0.25 6.00 0.25 6.00 0.439 0.4303 All components 0.50 12.00 0.35 8.40 0.15 3.60 0.476 0.4744 All components 0.50 12.00 0.45 10.80 0.05 1.20 0.512 0.5185 All components 0.50 12.00 0.45 10.80 0.05 1.20 0.502 0.5186 All components 0.57 13.68 0.32 7.68 0.12 2.88 0.548 0.5067 All components 0.60 14.40 0.25 6.00 0.15 3.60 0.584 0.494

8 All components 0.60 14.40 0.35 8.40 0.05 1.20 0.601 0.5389 All components 0.70 16.80 0.25 6.00 0.05 1.20 0.530 0.558

10 All components 0.70 16.80 0.25 6.00 0.05 1.20 0.518 0.558

Note. Each experimental result is the mean of three determinations; standard errors were less than10% of the means.

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



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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, whichgives an estimated response of 0.514.

To further validate the proposed experimental methodology, fermen-tation experiments were performed in triplicate for peptidase productionby employing the obtained optimized culture composition. The experi-mental data showed an enhanced peptidase yield of 0.531 U (48% improve-ment in peptidase production) with the modified culture conditions.

Mixed Inoculums

The possible interaction of Bacillus cereus with other proteolyticmicrobial strains was evaluated. Figure 7 presents the peptidase production

FIGURE 6 Plot of variables marginal means for the response.

FIGURE 7 Proteolytic activity in optimized fermentation medium with mixed inoculum at 32C in a

rotatory shaker at 60 rpm.



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in fermentation medium inoculated with a mixed inoculum. A notorioussynergistic effect was observed when B. cereus was inoculated either withPseudomonas pu ´ tida or Pseudomonas not classified.

The interaction Bacillus –Pseudomonas strains incremented by 50–60%the peptidase production obtained only with Bacillus . The results presented

suggested the possible start of substrate hydrolysis by Bacillus cereus andthen the Pseudomonas strains complete the degradation processes throughendopeptidase activity. Similar results were found by Oyama et al.[33] andGobbetti et al.,[34] who purified an endopeptidase in the cell free extract of a Pseudomonas sp. Odagaki et al.[35] isolated a pyroglutamyl peptidaseI, a group of exopeptidases responsible for the hydrolysis of N-terminal pyr-oglutamate residues, from Bacillus spp. Furthermore this result could indi-cate that B. cereus strain is able to exploit growth factors and=or vitaminssecreted by Pseudomonas sp.

On the other hand, the interaction with Enterococcus hirae strain reduced

by 20–40% the peptidase activity, indicating a possible antagonistic effect between the two strains. Two possible effects could be responsible for thelower protein hydrolysis when the two strains work together: a possible com-petition for the catalytic site, and=or bacteriocin-like substance productionby E. hirae that inhibits the growth of Bacillus cereus . Some bibliographic datareinforce this last fact. Lasagno et al.[36] reported a bacteriocin produced by Enterococcus hirae that inhibited the growth of Bacillus cereus, Listeria monocyto- genes, Clostridium perfringes, and Staphylococcus aureus .

A similar result was found with the interaction of Lactococcus lactis subsp.Lactis , reducing the peptidase activity by 50–60%. 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 wasinvestigated; the possible interaction with other proteolytic microbialstrains 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

TABLE 9 Estimated Parameters for the Linear Model

Estimate Std. error t Ratio p-Value

x1 0.63917 0.086299 7.4064 0.0001

x2 0.44362 0.1394 3.1823 0.0154

x3 0.09363 0.17036 0.54959 0.5997

Note. Asterisk indicates significant.



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brought a triple benefit, first, opening the possibility to produce technicalenzymes 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 cheaperand readily available sources of both carbon and nitrogen suppliers insteadof commercial substrate are the key attraction for the cost-effective pro-duction on an extracellular peptidases. Additional experiments should beconducted to identify bioactive substances or inducers present in brewer’sspent grains.

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