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Vol.52, n. 3: pp.567-572, May-June 2009 BRAZILIAN ARCHIVES OF

ISSN 1516-8913 Printed in Brazil
BIOLOGY AND TECHNOLOGY
A N I N T E R N A T I O N A L J O U R N A L

Production of Pectinases by A. niger: Influence of

Fermentation Conditions
María A. Martos1, Francisco Martinez Vazquez1, Fernando O. Benassi1* and Roque A.
Hours2
1
Centro de Investigación y Desarrollo Tecnológico; Facultad de Ciencias Exactas, Químicas y Naturales;
Universidad Nacional Autónoma de México; Félix de Azara 1552, (3000); Posadas - Misiones - Argentina. 2 Centro
de Investigación y Desarrollo de Fermentaciones Industriales; Facultad de Ciencias Exactas; Universidad
Nacional de La Plata; Calle 47 y 115, (1900) La Plata – Argentina

ABSTRACT

Response surface methodology was used for optimization of polygalacturonase (PG) and pectinesterase (PE)
production in submerged fermentation by A.niger. A Central Composite Experimental Design was applied,
consisting of 22 experiments, including eight central points. Variables studied were: fermentation time (24 to 120
h), pH (3.5 to 6.5) and initial concentration of pectin (5 to 20 g/l). Maximum PE production was 220 U/l, after 74 h
of culture, in a medium containing 20 g/l of pectin (pH 6.5). The optimal conditions for PG production were pH:
4.1, 20 g/l of pectin and 94 h of fermentation with a maximum value of 1032 U/l. Under these conditions, the PE
production was low (15 U/l). A liquid extract with high PG activity and low PE activity could be suitable to be used
in food processing in order to reduce the production of methanol.

Key words: Pectinases; Polygalacturonase; Pectinesterase; Aspergillus niger; Response surface methodology;
Citrus pectin

INTRODUCTION Aspergillus niger strains are widely used in several

fermentation processes for the production of pectic
Pectinases are widely used in industrial processing enzymes. The synthesis of pectinases is induced
of fruits and vegetables, because they reduce the by pectin or for some of its derivatives (Solís
viscosity of juices and facilitate extraction, Pereira et al., 1993). The cell growth, sporulation
maceration, liquefaction and clarification and production of the enzymes can be affected by
processes (Naidu and Panda, 1999). the composition of the medium and fermentation
Polygalacturonase (PG) is a depolymerizing conditions (Maldonado and Callieri, 1989; Costa
enzyme that cleaves glycosidic bonds of pectins by et al., 2007).
means of hydrolysis. Pectinesterase (PE) is the Most reported studies are carried out varying one
pectolytic enzyme that catalyzes the hydrolysis of factor at a time while keeping the others at a
ester links of the pectin molecule. Action of both constant level. This approach does not depict the
enzymes is needed in order to obtain complete effects of the interactions between the variables.
hydrolysis of the pectin molecule (Taragano and In the present work, response surface methodology
Pilosof, 1999). was applied to optimize pH, initial pectin
concentration and fermentation time conditions on
*
Author for correspondence: amartos@arnet.com.ar

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568 Martos, M. A. et al.

the production of PG and PE by A. niger in acetate buffer (pH 4.5) with 1 ml of the enzyme
submerged fermentation. extract. Reaction was carried out at 37 ºC for 60
min. The release of reducing groups was
determined by the dinitrosalicylic acid method
MATERIALS AND METHODS (Miller, 1959). A calibration curve was made
using galacturonic acid (Sigma) as standard. One
Fermentation medium unit of PG was defined as the amount of enzyme
The liquid medium used contained (g/l): KH2PO4, that released 1 µmol of galacturonic acid per
4; Na2HPO4, 6; FeSO4 7H2O, 0.01; CaCl2, 0.01; minute (Maldonado and Strasser de Saad, 1998).
MgSO4 7H2O, 0.2; (NH4)2 SO4, 2; H3BO3, 10 µg/l; PE activity was measured by adding 2 ml of the
MnSO4, 10 µg/l; ZnSO4 7H2O, 70 µg/l; citrus enzyme extract to 10 ml of 0.5 % citrus pectin
pectin (Parafarm), 5-20 g/l (Maldonado et al., (Sigma) in 0.1 M NaCl, the pH was adjusted to 4.5
1996). with 0.1 M NaOH. The reaction was carried out at
Pectin agar: Idem fermentation medium, citrus 35 ºC for 60 min. The amount of equivalents due
pectin 15 g/l; agar 15 g/l. to the carboxyl groups released was measured by
titration with 0.02 N NaOH. One unit of PE was
Microorganism defined as the amount of enzyme that released 1
A. niger Nº 300, isolated in our laboratory from µmol of carboxyl groups per minute (Maldonado
mouldy citrus fruit peels, was maintained in pectin et al., 1996).
agar slants. The inoculum was prepared by stirring
one-week-old slants with a sterile solution of Experimental design
0.05% Tween 80 in water to obtain a 1.25 107 The experimental design selected analyzed three
spores/cm3 suspension. variables at five levels (design type CCD = 22
runs, including eight central points for
Enzyme production experimental error detection) (Freund and Wilson,
Two hundred and fifty millilitres Erlenmeyers 1997). Variables studied were: fermentation time
flasks with 45 ml of medium were inoculated with (24 to 120 h), initial pH (3.5 to 6.5) and initial
5 ml of the inoculum and then incubated at 30 ºC pectin concentration (5 to 20 g/l).
at different times on a rotary shaker at 200 rpm. Real and transformed variables according to the
The biomass was separated by filtration through a experimental design selected are presented in
paper filter (Whatman Nº1). The filtrate was stored Table 1.
at –18 ºC until assayed. Regression linear analysis was used to eliminate
terms of P > 0,05.
Enzyme assays All the experiments were conducted in triplicate
PG activity was determined by incubating 4 ml of and the results showed the mean values of the
0.5% polygalacturonic acid (Sigma) in 0.2 M activities.

Table 1 - Experimental variables in real and transformed values

Time (h) PH Pectin (g/l)
+α* 120 6.5 20.0
+1 100.54 5.892 16.956
0 72 5.0 12.5
-1 43.46 4.108 8.040
-α* 24 3.5 5.0
* α = 1.68179.

RESULTS AND DISCUSSION variables studied and the respective levels of

significance are shown in Table 3.
Table 2 presents the treatment combinations and Figs 1 to 4 show the response surfaces and contour
responses. The regression coefficients of the plots for PG and PE production as a function of
equations relating the enzymes production with the two independent variables with the other one at a

Braz. Arch. Biol. Technol. v.52 n.3: pp. 567-572, May/June 2009
 Production of Pectinases by A. niger: Influence of Fermentation Conditions 569

given constant level. production was observed when pectin

All the equations presented a very good concentration was increased (Fig. 1).
adjustment with experimental data (R2 coefficients The negative values of quadratic effects for pH
> 0.8) (Table 3). and time indicated the existence of a maximum as
The three independent variables had significant a function of these variables (Table 3).
linear effects on PG production (Table 3). Pectin Optimum PG production was observed for pH 4.1
had a linear and positive effect: a high PG and 94 h of culture (Fig. 2).

Table 2 - Treatment combinations and mean responses.

Run Pectin (g/l) Time (h) pH PG (U/l) PE (U/l)
1 -1 -1 -1 654 33
2 0 -α 0 726 23
3 -1 +1 -1 915 51
4 -1 -1 +1 650 57
5 0 0 0 799 58
6 0 0 0 863 49
7 0 0 0 881 51
8 0 +α 0 852 33
9 0 0 0 958 60
10 +1 +1 +1 829 142
11 0 0 +α 608 210
12 +1 -1 +1 676 72
13 0 0 0 872 59
14 +α 0 0 988 50
15 0 0 0 808 57
16 -α 0 0 680 7
17 -1 +1 +1 685 33
18 0 0 0 898 57
19 +1 +1 -1 923 42
20 +1 -1 -1 862 37
21 0 0 -α 934 69
22 0 0 0 898 43
PG: polygalacturonase activity. PE: pectinesterase activity.

Table 3 - Regressions coefficients and levels of significance.

PG activity PE activity
Constant Level of significance Constant Level of significance
Independent term -461.541 857.421
Time 7.65078* 0.0035 1.92157*
pH 405.149* 0.0001 -378.546* 0.0001
Pectin 14.8432* 0.0006 -3.68687* 0.0153
pH2 -49.2359* 0.0158 35.8914* 0.0000
t2 -0.0402687* 0.0380 -0.0133442* 0.0390
Pectin2 ** -0.537691* 0.0419
pH * pectin ** 4.0541* 0.0292
R2 0.81 0.86
* Significant (P < 0,05), ** not significant (P> 0,05)

Braz. Arch. Biol. Technol. v.52 n.3: pp. 567-572, May/June 2009
570 Martos, M. A. et al.

1100
1000

PG (U/l)
900
800
700 124
104
600 64 84
5 7 9 44
11 13 15 17 19 24
21 Time (h)
Pectin (g/l)

Figure 1 - Effects of fermentation time and initial pectin on PG production (pH: 5.0).

1100
1000
PG (U/l)

900
800
700 124
104
600 84
64
3.5 3.8 4.1 4.4 4.7 44 Time (h)
5 5.3 5.6 5.9 6.2 24
6.5
pH

Figure 2 - Effects of fermentation time and initial pH on PG production (initial pectin

concentration: 20 g/l).

No significant interactions were observed among The pH showed positive quadratic effects,
the variables (Table 3). indicating a minimum as a function of this variable
Low PG values obtained at about pH 6.5 were in at the range 4.2 - 4.8 (Fig. 3).
agreement with those reported by Solís Pereira et Interaction between pH and pectin was significant
al. (1993). Acuña-Argüelles et al. (1995) studied and positive (Table 3), and low pH values did not
the stability of Aspergillus niger CH4 PG in affect PE production, but the effect was strongly
submerged fermentation as a function of pH and positive at higher pH values (Fig. 3). The highest
observed that the enzyme was quickly denatured PE production was obtained at pH 6.5 and 20 g/l
for pH values higher than 5.0. of pectin.
Low initial pectin concentrations yield low Table 4 showed that PE production slightly
pectinases production, since pectinases are increased at about 74 h of fermentation.
generally inducible enzymes (Naidu and Panda, At pH higher than 5.0, PE production increased
1998; Malvessi and Silveira, 2004). with pectin since the synthesis of this enzyme was
The three variables studied had significant linear induced by this substrate(Maldonado and Callieri,
effects on PE production (Table 3). 1989; Maldonado et al., 1994).

Braz. Arch. Biol. Technol. v.52 n.3: pp. 567-572, May/June 2009
 Production of Pectinases by A. niger: Influence of Fermentation Conditions 571

210
180
150

PE (U/l)
120
90
60
30 21
1719
0 111315
3.5 3.8 4.1 4.4 4.7 5 9
5.3 5.6 5.9 6.2 6.5 57 Pectin (g/l)
pH

Figure 3 - Effects of initial pectin concentration and pH on PE production (fermentation time: 72 h).

240
210
PE (U/l)

180
150
120
90
60
30 124
104
0 84
64
5 7 9 44 Time (h)
11 13 15 17 24
19 21
Pectin (g/l)

Figure 4 - Effects of fermentation time and initial pectin concentration on PE production (pH: 6.5).

CONCLUSIONS RESUMO

Maximum PE production was 220 U/l at 74 h of A metodologia de superfície de resposta foi

culture, 20 g/l of pectin and pH 6.5. utilizada para a otimização da produção de
Optimum conditions for PG production were 94 h poligalacturonasa (PG) e pectinesterasa (PE), por
of incubation, 20 g/l of pectin and pH 4.1. Under A. niger em fermentação submergida. Foi aplicado
these conditions PG production was 1032 U/l and um Desenho Experimental Composto Central
PE production was only 15 U/l. abrangendo 22 experiências, incluindo oito pontos
A liquid extract with a high PG activity would centrais. As variáveis estudadas foram: tempo de
accelerate the depolymerization of the pectin fermentação (24 a 120 h), pH (3.5 a 6.5) e
molecule while a low PE activity could be concentração inicial de pectina (5 a 20 g/l). A
advantageous for processing juices taking into produção máxima de PE foi de 220 U/l, após 74h
account PE liberated methanol that would damage de cultivo, 20 g/l de pectina e pH 6.5. As
the volatile ester content responsible for the condições ótimas para a produção de PG foram pH
specific scent of fruits. 4.1, 20 g/l de pectina e 94 h de fermentação, com
um valor máximo de 1032 U/l. Sob estas
condições, a produção de PE foi baixa (15 U/l).
Um extrato líquido com alta atividade PG e baixa
atividade PE poderia ser conveniente para ser
utilizado no processamento e alimentos, visando
reduzir a produção de metanol.

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572 Martos, M. A. et al.

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