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Abstract
Synthesis of amylase and protease by Aspergillus niger strain UO-1 was followed in media prepared with brewery (BW) and meat
(MPW) wastewaters supplemented with different starch concentrations. The highest amylase (70.29 and 60.12 EU/mL) and protease
(6.11 and 6.03 EU/mL) production were, respectively, obtained in the BW and MPW media supplemented with 40 g of starch/L of
medium after 88 h of fermentation. In addition, the initial chemical oxygen demand (COD) in both wastes was reduced by more
than 92%.
High amylase and protease activities were found in the BW medium supplemented with casaminoacids, peptone or yeast extract,
but ammonium nitrate and sodium nitrate were also good nitrogen sources for amylase production. The stabilities of amylase and
protease were higher at 50 C and pH 4.95 and at 53.4 C and pH 3.87, respectively, but they were highly sensitive at temperatures of
70 C or higher.
2005 Elsevier Ltd. All rights reserved.
Keywords: Aspergillus; Amylase; Protease; Meat processing wastes; Brewery wastes; Chemical oxygen demand (COD)
0260-8774/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2005.01.009
94 M.S. Hernández et al. / Journal of Food Engineering 73 (2006) 93–100
The Aspergillus species produce a large variety of at pH 6.8. The culture was incubated at 30 C/48 h
extracellular enzymes of which amylases and proteases (220 rpm).
are of significant industrial importance (Pandey et al., Brewery (BW) and meat processing (MPW) wastewa-
2000). Amylases have not only been used in fermenta- ters, which were used as a base of the culture media,
tion processes, but also in processed food industry were obtained from a local brewery and a local meat
and in the textile and paper industries (Ellaiah, processing plant. Both wastes were centrifuged at
Adinarayana, Bhavani, Padmaja, & Srinivasulu, 2002; 12,000·g/15 min to remove the solids in suspension.
Gigras, Sahai, & Gupta, 2002). Proteases have found a The supernatant obtained from BW contained (g/L):
wide application in several industrial processes such as COD, 3.40; total sugars, 1.98; reducing sugars, 1.46;
in cheese production, meat tenderization, in the baking total nitrogen, 0.095; total phosphorous, 0.034. The
industry, and in many other fields, including textiles composition of the supernatant obtained from MPW
and leather industries and as an additive to detergents (g/L) was: COD, 3.00; total sugars, 1.82; reducing sugars,
(Bai, Ge, & Zhang, 1999). 0.99; total nitrogen, 0.172; total phosphorous, 0.028.
Considering the substantial availability of highly rich The production media were obtained by supplement-
wastewasters at very low prices by local brewery and ing these supernatants with the following nutrients (g/
meat processing plants of Santiago de Cuba, the use of L): mycological peptone, 3.0; (NH4)2SO4, 6.6; CaCO3,
these wastes as culture media for different bioproduc- 8.0; NaCl, 5; KH2PO4, 3.5; FeSO4 Æ 7H2O, 0.15;
tions could provide a profitable substrate for a low cost MgSO4 Æ 7H2O, 0.10. To study the influence of the initial
production, with advantage of an effective reduction of concentration of starch on amylase and protease pro-
their initial COD. However, there is not information duction, the media were supplemented with soluble po-
available on the use of brewery and meat processing tato starch to obtain initial starch concentrations of 10,
wastes for the production of industrial enzymes by 20, 30 and 40 g/L. Media without starch were used as
Aspergillus niger. controls. The media were adjusted at pH 6.0 and steril-
The aim of this study was to examine the possible uti- ized (121 C/15 min), inoculated with a 2% inoculum
lization of both brewery and meat processing wastes of level (3 · 107 spores/mL) and incubated at 30 C/88 h.
Santiago de Cuba for amylase production by A. niger All submerged cultures were carried out in 250-mL
strain UO-1 under submerged culture conditions. Erlenmeyer with 50 mL of production media in an
Because Aspergillus strains are also capable of produc- orbital shaker (200 rpm), using eight flasks in each fer-
ing proteases (Punt et al., 2003), dynamic changes of mentation series.
these enzymes were followed along with amylase The culture samples, which comprises an experimen-
production in both media. The effects of the initial tal unit (one flask) were collected each 12 h. Mycelial
starch levels and the type of nitrogen source on the mass was harvested by paper filtration using a pre-dried
production of both enzymes were studied. The effects and pre-weighted Whatman filter paper No. 1, washed
of pH, temperature and metal ions on the stability of with distilled water and dried to constant weight at
both enzymes were also investigated. In addition, the 105 C. The growth of the organism was determined as
reduction in COD in both wastes after biological treat- dry weight. Paper-filtered media were used to perform
ment was determined. analytical determinations (pH, enzyme production, total
sugars, nitrogen and phosphorous and COD).
Methods for determination of total sugars, nitrogen
2. Materials and methods and phosphorous were described or referred to in a pre-
vious work (Guerra & Pastrana, 2003). COD analyses
2.1. Microorganisms were carried out in the paper-filtered media at the end
of each fermentation using the closed reflux colorimetric
A. niger strain UO-1, was acquired from the Biotech- method as described previously (Greenberg, Connors, &
nology Center of the University of Oriente (Santiago de Jenkins, 1980). All determinations were carried out in
Cuba, Cuba) and it was maintained on potato dextrose triplicate.
agar slants at 4 C (Ellaiah et al., 2002).
2.3. Assays of amylase and protease activity
2.2. Inoculum preparation, culture media and
fermentation conditions Total amylase activity (TAA) was determined accord-
ing to Murado et al. (1993) mixing 80 lL of paper-
Inocula were prepared by transferring 2-mL of 60-h filtered medium (suitably diluted) with 400 lL of
old slant culture in 50 mL of medium (250 mL Erlen- 0.15 M citrate-phosphate buffer; pH 5.0 (1 volume)
meyer) composed by (g/L): glucose, 20; (NH4)2SO4, and 4% soluble starch (1.5 volumes) previously main-
6.6; KH2PO4, 3.5; FeSO4 Æ 7H2 O, 0.15; MgSO4 Æ 7H2O, tained at 40 C/15 min. The reaction mixture was incu-
0.10; MnCl2 Æ 2H2O, 0.45 and mycological peptone, 3.0; bated at 40 C for 10 min. The reaction was stopped
M.S. Hernández et al. / Journal of Food Engineering 73 (2006) 93–100 95
by addition of 480 lL of dinitrosalicylic acid, and the re- using the DeltaGraph sofware, version 4.0 (SPSS, Inc.,
leased glucose was determined by 3,5-dinitrosalicylic Chicago, IL, USA).
acid reaction (Bernfeld, 1951). One unit of amylase
activity (enzymatic units (EU)/mL) was defined as the 2.5. Effect of metal ions on amylase and protease activity
amount of enzyme that releases 1 mg/mL of reducing
sugars (glucose equivalents) under the assay conditions. Samples of cell-free medium were incubated with
Protease activity of cell-free medium was determined each compound (at 10 mM final concentration) for
by a modified Ansons method (Yang & Wang, 1999). 30 min in the optimum conditions of temperature and
The reaction mixture with 1 mL of 1% (w/v) casein in pH determined in the previous assay. After the incuba-
0.02 M NaOH and 2 mL of 0.4 M phosphate buffer tion the remaining activity was determined by the corre-
pH 6.0 and 1 mL of cell-free medium (suitably diluted) sponding enzyme assay. The residual enzyme activity
were incubated at 30 C/10 min. The reaction was was expressed as a percentage of the activity in control
stopped with 3 mL of 10% trichloroacetic acid, mixed samples without reagent (Aquino, Jorge, Terenzi, &
and after 5 min, centrifuged at 12,000· for 5 min, then Polizeli, 2003). Individual experiments were performed
0.5 mL of the supernatants were incubated with in triplicate. Data sets were analyzed by analysis of
2.5 mL of 0.1 M NaOH in 2% (w/v) Na2CO3 for variance (ANOVA) on SPSS 8.0 for Windows.
10 min. Thereafter 0.25 mL of Folin phenolic reagent
(commercial solution diluted 1:1 in distilled water) were
added, mixed and stayed for 30 min at room tempera- 3. Results and discussion
ture. The absorbance measured at 750 nm was converted
to mg of tyrosine/L using a calibration curve (mg tyro- 3.1. Amylases and proteases production in media prepared
sine/L vs absorbance). The tyrosine solutions dissolved with brewery and meat processing wastewaters
in 0.01 M HCl, were treated in the same conditions as supplemented with starch
the cell-free medium. One unit of protease (enzymatic
units (EU)/mL) was defined as the amount of enzyme Amylase production by A. niger strain UO-1 was fol-
that produced an absorbance at 750 nm equivalent to lowed in media prepared with brewery and meat pro-
1 lmol of tyrosine in 1 min under the assay conditions. cessing wastewaters supplemented with starch. The
time course of growth, pH, total sugars and enzymes
2.4. Effect of temperature and pH on stability of (amylases and proteases) production is shown in Fig. 1.
amylase and protease The levels of amylase and protease enzymes obtained
were markedly dependent on the concentration of initial
A second-order factorial plan, using a rotatable de- starch concentration in the culture media (Fig. 1).
sign (Box, Hunter, & Hunter, 1989) based on five levels Increased amylase and protease levels were obtained in
and two variables was used to study the combined influ- the media supplemented with starch when compared
ence of pH and temperature on the stability of both to the control cultures (BW and MPW media without
amylase and protease. The design consisted of 13 exper- starch).
iments with four (22) factorial points, four axial points The high initial concentration of substrate (40 g of
to form a central composite design with a = 1.267 and starch/L) in the media favored cell growth, as the pro-
five center points for replication. duction of both enzymes. Although the maximum level
Samples of cell-free medium containing the enzymes of amylase was reached in BW medium (70.29 EU/
were buffered at different pH with the appropriate buffer mL), which was higher than in MPW medium
(0.1 M potassium hydrogen phthalate-HCl buffer for pH (60.12 EU/mL), the maximum protease levels (ffi6 EU/
2.3, 3.0 and 5.5; 0.1 M Tris–HCl buffer for pH 8.0 and mL) were similar in both media.
8.7). The buffering agents were prepared as concentrated During fermentation, the pH initially dropped from
stock solutions. Then, appropriate volumes of them 6.0 to 4.0 after 24 h followed by its increase to 7.4
were mixed with the supernatant samples to obtain a approximately (48 h), and remained constant thereafter.
final buffer concentration of 0.1 M. The samples were The concentration of total sugars, nitrogen and phos-
incubated for 10 min (for protease) and 5 min (for amy- phorous decreased during fermentation, coinciding with
lase) at the corresponding temperature according to the an increase of biomass and enzyme production leading
experimental matrix defined by the design used (Table to a final COD of 0.24 g/L (in the media supplemented
2). The enzyme activities obtained were corrected with with 40 g of starch/L) or lower (in the media with lower
the corresponding dilution factor. initial starch concentrations). This indicates that at least
Results were analyzed by Experimental Design 92% and 93% of the initial COD were removed from the
Module of the Statistica sofware package (Statistica brewery and meat processing wastes, respectively.
for Windows computer program manual; StatSoft Inc. Although the culture media prepared with both
Tulsa, OK, USA). The response surfaces were plotted wastes were capable of supporting the growth and
96 M.S. Hernández et al. / Journal of Food Engineering 73 (2006) 93–100
Fig. 1. Time course of growth and enzyme synthesis by A. niger strain UO-1 grown in media prepared with brewery (BW) and meat processing
(MPW) wastewaters supplemented with different initial concentrations of total sugars ((d) 0, (s) 10, (h) 20; (n) 30; () 40 g/L). TS: total sugars;
TAA: total amylolytic activity; PA: protease activity; Nt: total nitrogen; P: total phosphorous. The cultures were carried out at 30 C/88 h in an
orbital shaker at 200 rpm.
enzyme production by A. niger strain UO-1 (Fig. 1), To determine whether increasing concentrations of
probably the starch was not the best carbon source for starch caused substrate inhibition on enzyme produc-
protease production (Prasad, Malik, & Mathur, 1984; tion, the experimental data of total amylolytic and pro-
Walker & Campbell, 1983). These authors reported that tease activities obtained at the end of each fermentation
the use of glucose and maltose as carbon and energy were adjusted to an Andrews-type model:
source resulted in good growth but low protease produc-
EAm S
tion, suggesting the use of slow metabolisable carbon EA ¼ ð1Þ
sources to improve protease production. K s þ S þ K I S2
M.S. Hernández et al. / Journal of Food Engineering 73 (2006) 93–100 97
Fig. 2. Total amylolytic (TAA) and protease (PA) activity levels produced by A. niger strain UO-1 after 88 h of incubation as a function of initial
concentrations of total sugars on BW and MPW media.
Table 2 lated to be 0.977 and 0.969 for models (2) and (3),
Experimental domain and codification of the variables used in the respectively (Fig. 3), which indicated a high significance
factorial design analysis for the combined influence of temperature and
pH on the stability of amylases and proteases produced in BW medium
of both models. All of the above considerations indicate
an excellent adequacy of the quadratic models to the
Codified values Natural values
experimental data.
T (C) pH The response surfaces obtained from the empirical
1.267 22 2.3 models (2) and (3) are depicted in Fig. 3. Optimal amy-
1 30 3.0 lase activity was at pH 4.95 (coded value of 0.22) and
0 60 5.5
+1 90 8.0
50 C (coded value of 0), with visible decrease toward
+1.267 98 8.7 low or high values of both variables (Fig. 3). However,
Increments 1 30 2.5 the maximal protease activity was obtained at pH 3.87
(coded value of 0.65) and 53.4 C (coded value of
0.22).
PA ¼ 6:12 0:25T 1:11 pH 1:13T 2 0:98 pH2 At optimal conditions both enzymes retained approx-
imately 85% of initial activities after 1 h of incubation.
ð3Þ
Nevertheless, when the amylase and protease samples
where TAA and PA are the residual total amylolytic were adjusted for optimum pH value but incubated at
activity and proteolytic activity (EU/mL), respectively 70, 90 and 100 C, their initial activities were completely
and T is the temperature (C). In both cases, the model lost at 30, 22 and 18 min, respectively (Fig. 4).
terms T, pH, T2 and pH2 were found to be significant
according to the Student t-test (a = 0.05) and P-values, 3.4. Effects of metal ions on stability of amylase and
meanwhile the interaction term between T and pH vari- protease
ables (T pH) was found to be non-significant in both
equations (Table 3). Since the P-values in the ANOVA Table 5 shows the effect of various metal ions on the
table (Table 4) are less than 0.05 for both models, there enzymic activity of amylase and protease. Amylase
is a statistically significant relationship between the activity was slightly activated by metal ions such as
variables at the 95% confidence level. The value of the Ca2+ and Na+, but was strongly inhibited by Cu2+,
adjusted determination coefficient (adj. R2) was calcu- Hg2+ and Zn2+ (P < 0.05). Similar results have been
Table 3
Comparison of the observed and the predicted responses (amylase and protease activity after pH and heat treatment) and coefficients of regression
analysis
T pH Amylase activity (EU/mL) Protease activity (EU/mL)
Observed response Predicted value Observed response Predicted value
1 1 39.81 39.87 2.54 2.64
1 1 26.43 25.61 4.94 4.86
1 1 29.15 32.75 3.59 3.15
1 1 18.93 18.49 5.22 5.37
1.267 0 36.37 37.16 4.02 3.97
1.267 0 30.43 28.14 4.40 4.61
0 1.267 51.09 48.40 2.90 3.14
0 1.267 29.12 30.32 6.03 5.95
0 0 60.53 63.78 5.94 6.12
0 0 62.36 63.78 6.12 6.12
0 0 67.53 63.78 6.33 6.12
0 0 63.48 63.78 6.23 6.12
0 0 64.40 63.78 6.02 6.12
Significance of factors
Factor Model (2) Model (3)
Coefficient t-value P-value Coefficient t-value P-value
*
Constant 63.78 55.34 0.000000 6.11 66.98 0.000000*
T 3.56 3.67 0.010328* 0.25 3.27 0.013601*
pH 7.13 7.36 0.000219* 1.11 14.45 0.000002*
T Æ pH 0.79 0.61 0.583885 1.13 12.49 0.103900
T2 19.39 16.92 0.000001* 0.98 10.75 0.000005*
pH2 15.21 13.28 0.000005* 0.19 1.87 0.000013*
*
Significant at P < 0.05 or t (a = 0.05; t = 4) > 2.78.
M.S. Hernández et al. / Journal of Food Engineering 73 (2006) 93–100 99
Table 4
Summarized data of analysis of variance (ANOVA) of the empirical models obtained for amylase and protease stability after pH and temperature
treatment according to the experimental design defined in Table 2
Source Sum of squares Degrees of Mean of squares F-value P-value
freedom
TAA PA TAA PA TAA PA TAA PA
*
Regression 3617.65 21.22 8 452.21 2.65 66.82 106.66 0.0005 0.0002*
Residual 27.07 0.10 4 6.77 0.03
Total 3644.72 21.32 12
TAA: total amylase activity, PA: protease activity.
*
Significant at P < 0.05.
Table 5
Effect of metal ions on amylase and protease activity
Chemical Relative amylase Relative protease
activity (%) activity(%)
None (control) 100 100
CaCl2 105.3 116.5
CuCl2 8.6 15.3
HgCl2 6.2 42.5
MgCl2 93.8 111.3
MnCl2 87.1 98.9
NaCl 107.9 102.4
ZnCl2 12.6 34.9
4. Conclusions