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Abstract Response surface methodology based on application in starch saccharification and in the brewing
mixture design was employed for statistical optimization and distilling industries(Souza and Magalhes, 2010;
of medium components for the growth and production of Gashtasbi et al., 2014).
-amylase by Escherichia coli pAC92. The combined In the recent decades, the demand for industrial -amylase
effects of media constituents (peptone, yeast extract and has been on the rise because of their economic and
extruded beans) were analyzed using a cubic model, environmental benefits(Hellmuth and Van-Den-Brink,
which was developed by3-factor simplex lattice mixture 2013; Kim et al., 2014). In addition, due to rising prices
design in predicting the optimum yield of growth and - of nitrogen and carbon sources, the bio-based enzymes
amylase activity. Results evidenced that extruded common production has been emerged as a promissory technology
bean was more effective as a nutrient source for in biotechnology field. It is known that the development
E.colipAC92 growth. On the other hand, the completely of a bio-based industrial enzymes production requires a
substituted medium with extruded common bean resulted low-cost medium and a versatile producing organism able
in 68% of increase in the growth of Escherichia coli to utilize a wide range of low-cost feedstock(Carneiro et
pAC92. In addition, the culture medium containing 0.5% al., 2013). In this scenario, hardened beans, an agro-
of extruded bean and 0.5% of peptone reached a - industrial by-product that contains high amount of
amylase activity of 44.59 U. The optimal medium carbohydrates and proteins could be a promising nutrient
composition was determined by a numerical method source for -amylase production.
based on desirability function, by which the optimal However, the microbial production of amylases can be
composition for maximum optical density and enzyme affected by certain factors, including microbial strain,
activity was found using 0.5% peptone and 0.5% extruded culture medium formula and physicochemical
common bean as media constituents. Therefore, these conditions(Hii et al., 2012). Considering the composition
results evidenced that extruded common bean can be of culture medium, the nature of nitrogen and carbon
successfully used as substitute of peptone and yeast source could influence the rate of amylase production
extract in culture media for production of -amylase and, therefore the enhancement of target enzyme
byE.colipAC92. productivity in any fermentation system could be
Keywords extruded bean; mixture design; -amylase achieved through the improvement of the culture medium
production; culture medium. composition(Ye et al., 2010; Hortsch and Weuster-Botz,
2011; Boumba et al., 2013; Carneiro et al., 2013).
I. INTRODUCTION Conventional change one-factor-at-a-time approach has
Amylases are enzymes widely used in biotechnology been applied to optimize the enzyme production using
processes, constituting a class of industrial enzymes different medium constituents. However, this technique is
having of the world enzyme market. -Amylases have time-consuming, expensive, and often leads to
potential application in food, fermentation, textile, paper, misinterpretationof results, once the interactions among
detergent, and pharmaceutical industries. In addition, with parametersare not taken into account(Hii et al., 2012). An
the advances in biotechnology, the amylase application alternative to overcome this technical limitation is the use
has expanded in many fields such as clinical, medicinal of statistical designs coupled to response surface
and analytical chemistry, as well as their widespread methodology, which allows the study of several factors
where X1, X2 and X3 denotes peptone, yeast extract and extruded bean, respectively. The value of adjusted r2 indicates that
the proposed experimental model can determine 95.8% of the response variability.
Results of the multivariate analysis for the media using was the content of extruded cowpea, which presented a
cowpea as nitrogen source evidenced that although the positive correlation with the bacterial growth(r=0.78).
interaction between peptone and extruded bean (X1X3) Using regression analysis, the polynomial equation that
had no effect on the response, all other variables describes the correlation between the response and the
significantly affected the growth of E.colipAC92. In significant variables can be represented by the following
addition, the variable that most influenced the response equation (adj-r2=0.979):
3.2. Response surface analysis to a further growth by increasing its concentration.In this
Multivariate design of experimentscoupled to response scenario, the mathematical prediction of an optimum
surface methodologyis a widely used method in composition is presented as 0.25% yeast extract and
optimization processes, once it consumes less time, 0.75% of extruded bean. With this formulation, is
requires a smaller set of experimental procedures and expected to achieve an optimal density of 1.385 at 600
resources, allows the obtainment of large quantities of nm, a value very close to that previously observed in the
data in a single step process and enables the discoverof culture medium containing extruded bean as unique
the most desirable conditions, or desirability(Yin et al., nitrogen source (run 3).
2009; Mohamad et al., 2011; Boumba et al., 2013; Cheng This fact can be explained in terms of the nutritional
et al., 2013).Hence, a response surface methodology requirements of the bacteria under study,which needs
(RSM) was used to determine the influence of nutrient beyond the protein from peptone, also minerals and
sources (peptone, yeast extract and both extruded bean) in vitamins present in yeast extract. Thus, the improvement
the E.coli growth. is probably due toa better nutritional balance betweenthe
Despite the good results of optical density in the media extruded bean and the yeast extract, encompassing not
containing common bean and yeast extract or peptone in only a more complete source of nitrogen, but also
equivalent proportions, the surface response confirms the essential vitamins and active compounds(Watson, 1976;
highest growth profile using extruded common beanas Pepler, 1982; Batista et al., 2013).
sole nitrogen source(Figure 1a).The projection evidenced The effect of inter-relations and interactions of the
a negative effect of peptone (run 1) and yeast extract (run proportion of peptone, yeast extract and extruded cowpea
2) as isolated sources, while common bean shows a trend on E.coligrowth are depicted in Figure 1b. The response
Table.2: Mixture design matrix used for optimization of -amylase production by E.coli pAC92 using extruded common bean
as nutrient source.
Run Peptone Yeast Extract Extruded Bean -amylase activity (U)
(X1) (X2) (X3)
Observed Predicted
Figure captions
Figure 1. Mixture contour maps showing the effect of three variables on the optical density of E.coli pCA92. (a) yeast
extract, peptone and extruded common bean or (b) yeast extract, peptone and extruded cowpea.
Figure 2. Effect of different nitrogen sources in the growth of Escherichia coli: (a) mixture design using common bean as
nitrogen source; (b) mixture design using extruded cowpea as nitrogen source. As the control, the microorganism was grown
in a commercial Luria-Bertani medium. Results are means standard deviation of triplicate samples.
Figure 3. Pareto chart (a) and mixture contour map (b) for the -amylase activity in the mixture design experiments. In the
Pareto chart, the horizontal bar represents the ratio between the effects of variables and their respective standard error. All
tests were conducted in triplicate.
Figure 4. Response surface plot of desirability as a function of the proportion of yeast extract, peptone and extruded common
bean.
Figure 2
Figure 3