LWT - Food Science and Technology 43 (2010) 1132e1137

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LWT - Food Science and Technology

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Lipase production by solid fermentation of soybean meal with

different supplements
Elisandra Rigo a, Jorge Luiz Ninow a, Marco Di Luccio b, J. Vladimir Oliveira a, André Eliezer Polloni b,
Daniela Remonatto b, Francieli Arbter b, Renata Vardanega b, Débora de Oliveira b, Helen Treichel b, *
a
Departamento de Engenharia Química e de Alimentos, Universidade Federal de Santa Catarina, UFSC, SC, Brazil
b
Universidade Regional Integrada do Alto Uruguai e das Missões, Programa de Pós-Graduação em Engenharia de Alimentos, Food Engineering, Campus de Erechim,
Av. Sete de Setembro, 1621, 99700-000, Erechim, RS, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this work was to study the production of extracellular lipases by solid state fermentation in
Received 27 May 2009 soybean meal with different supplements. Lipase production by two microorganisms, screened by their
Received in revised form potential for lipase production, was followed in terms of hydrolytic activity at different pH values(4, 7
25 February 2010
and 9). The supplementation of the medium with urea and soybean oil significantly increased the
Accepted 3 March 2010
enzyme production for all studied pH and microorganisms. Microorganism Penicillium P58 and P74
showed the possibility of production of different lipases with alkaline and acidic characteristics. In
Keywords:
soybean meal supplemented with urea and soybean oil, this microorganism yielded 139.2 and 140.7U
Lipase
Penicillium sp.
lipase/g of dry substrate in 48 h of fermentation, in alkaline and acidic conditions, respectively. Different
Solid state fermentation behavior was observed when the enzyme extract was evaluated in neutral conditions, which yielded
Soybean meal 180.0U lipase/g in 72 h. A new promising lipase producer strain was testedon soybean meal with
different supplements. The strain produced a lipase with high activities by solid state fermentation of
soybean meal supplemented with urea and soybean oil.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Recently, lipases have assumed a prominent place in the world
market of enzymes, evidenced by the increase in the amount of
information reported in the literature, which reaches an average of
1000 publications per year. Following proteases and amylases,
lipases are considered as the third great group in volume of sales;
moving billions of dollars, showing their application versatility that
turns them especially attractive for industrial applications
(Contesini & Carvalho, 2006; Hasan, Shah, & Hameed, 2006; Jaeger
& Reetz, 1998; Snellman, Sullivan, & Colwell, 2002).
Lipases are enzymes belonging to the group of the hydrolases,
whose main biological function is to catalyze the hydrolysis of
insoluble triacylglycerols to free fatty acids, mono and diac-
ylglycerols and glycerol. Besides its natural function, lipases can
catalyze esterification, interesterification and transesterification
reactions in non-aqueous media (Freire, 1996; Houde, Kademi, &
Leblanc, 2004). Microbial lipases are biocatalysts that have inter-
esting characteristics, as action under mild conditions, stability in
* Corresponding author. Tel.: þ55 54 35209000; fax: þ55 54 35209090.
E-mail address: helen@uricer.edu.br (H. Treichel).
organic solvents, high substrate specificity and regio- and enan-
tioselectivity (Snellman et al., 2002).
The potential for industrial applications of lipases comprises the
industry of additives (modification of aromas), fine chemistry (ester
synthesis), detergents (hydrolysis of fats), wastewater treatment
(decomposition and removal of oleaginous substances), leather (fat
removal from animal skin), pharmaceutical and the medical area
(medicines, digestive and enzymes for diagnosis) (Burkert, Maugeri,
& Rodrigues, 2004). However, the high costs of production of these
biocatalysts often restrict their use (Snellman et al., 2002). Con-
cerning these aspects, studies based on the use of different micro-
organisms, supplements and substrates for lipase production can
contribute in finding ideal combinations to obtain lipases with high
value, using substrate and operational conditions that facilitate the
reduction of the production costs in industrial scale.
In this sense, solid state fermentation (SSF) seems to be an
interesting alternative to microbial enzymes production, due to the
possibility of using agro-industrial residues and/or by-products as
nutrients source as well as support to microorganism development.
The use of these substrates for lipases production, besides making
possible to aggregate value to materials of low cost can also
decrease the final cost of the enzyme (Pandey, 2003; Rodríguez
et al., 2006; Soccol & Vandenberghe, 2003).

0023-6438/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2010.03.002
 E. Rigo et al. / LWT - Food Science and Technology 43 (2010) 1132e1137
The residual cakes of oil extraction processes are usually used as
animal feed, since they can be used as good sources of proteins.
Many studies have evaluated the use of these agro-industrial resi-
dues as substrates in bioprocesses. The biotechnological application
of sunflower, soybean, palm, olive, coconut, mustard, cotton and
canola cakes has permitted the production of enzymes, antibodies,
biopesticides, vitamins, etc. In the enzymes production, the cakes
are used as substrates for SSF or as carbon and nitrogen supple-
ments to production medium (Ramachandran, Singh, Larroche,
Soccol, & Pandey, 2007).
Lipids are generally essential inducers to lipase production. As
an example, the lipase production by Yarrowia lipolytica resulted in
activities of 69 U/g when using ground peanut as substrate
(Domínguez, Costas, Longo, & Sanromán, 2003). The use of sugar-
cane bagasse as substrate in the fermentation of Rhizopus homo-
thallicus was evaluated using different supplements for lipase
production. The optimized experimental conditions led to a lipase
activity of 826 U/g in 12 h of fermentation, using urea (4 g/L) and
olive oil (40 g/L) as nitrogen and carbon sources, respectively
(Rodríguez et al., 2006). On the other hand, some authors have
shown that the supplementation has no positive effect on lipases
production (Di Luccio et al., 2004; Kamini, Mala, & Puvanakrishnan,
1998).
Based on these aspects, the objective of this work was to eval-
uate the lipase production using soybean meal as substrate and
different supplements, as soybean oil (SO) and urea (UR), the
mixture of them (urea and soybean oil) (URSO). The effect of using
complex supplements as agro-industrial residues (sugarcane
molasses e SM; corn steep liquor e CSL and yeast hydrolysate e
YH), which contain macro and micronutrients, was also evaluated
in lipases production. The behavior of two Penicillium strains,
previously isolated and screened as good lipase producers, was
observed in terms of hydrolytic activity, measured in pH 4, 7 and 9,
so as to pre-identify the lipases obtained in each substrate used.
2. Experimental
2.1. Microorganism and inoculum
The microorganisms used in this work were previously isolated
from samples of soybean meal and cheese and pre-identified as
Penicillium sp (Rigo et al., 2009). The isolation of microorganisms
and cultivation was carried out in Petri dishes using PDA (potato
dextrose agar) medium at 30  C. All stock cultures have been stored
at 10  C.
Erlenmeyer flasks (500 mL) with 100 mL of sterile PDA (potato
dextrose agar) medium were inoculated with 0.5 mL of a spore
suspension obtained from stock cultures. The flasks were incubated
for seven days at 30  C. Spore collection was carried out by adding
20 mL of an aqueous 0.1 mL/100 mL Tween 80 solution and sterile
glass beads to the flasks. The resulting suspension was then used as
inoculum, after adjusting the amount of spores to achieve a final
concentration of 108 spores/g of dry meal (Freire, Teles, Bon, &
Sant'Anna, 1997).
Table 1
Different supplements added to the soybean meal (used as substrate) and the respective C/N ratios. All supplements were added at 1 g/100 g concentration except when stated
otherwise.
Code Component
1 (SO) Soybean meal þ soybean oil
2 (SM) Soybean meal þ sugarcane molasses
3 (CSL) Soybean meal þ corn steep liquor
4 (YH) Soybean meal þ yeast hydrolysate
5 (PSB) Pure soybean meal
6 (UR) Soybean meal þ urea
7 (URSO) Soybean meal þ (1 g/100 g) urea þ (0.33 g/100 g) soybean oil
1133
2.2. Solid state fermentation
The substrate used in all experiments was soybean meal of
a same batch obtained from a local soybean oil industry. The meal is
the residue of soybean oil extraction after cold press and solvent
extraction. The meal was sieved and the three major fractions based
on the particle diameter were: 1e2 mm (Tyler Standard Sieve
Series of 9e16), 0.5e1 mm (Tyler Standard Sieve Series of 16e32)
and 0.25e0.50 mm (Tyler Standard Sieve Series of 32e60). The
0.5e1 mm (Tyler Standard Sieve Series of 16e32) fraction was
collected and stored at 18  C until the moment of utilization.
Fermentations were carried out with 10 g of dry meal in cylindrical
tray reactors covered with hydrophobic fabric. The moisture content
of the meal was adjusted to 55 mL/100 g. Supplements were added as
shown in Table 1. The effect of different nitrogen and carbon sources
(C/N ratio approximately at 6.11) were evaluated in terms of lipase
production using the two microorganisms selected before.
After sterilization (121  C/15 min) each reactor was inoculated
with a suspension of spores to a final count of 108 spores/g and
incubated in climatic chamber at a temperature of 27  C, with
humid air injection.
After 48 h of fermentation, phosphate buffer (100 mmol/L, pH
7.0) at a proportion of 4:1 (v/w) was added to the fermented cake
and the enzyme extraction was carried out in an orbital shaker at
150 rpm, 35  C for 30 min. After filtration of solids, the supernatant
was used for analytical assays. The pH determination was carried
out weighting 0.5 g of fermented soybean meal in assay tube and
adding 5 mL of distilled water. This content was homogenized and
the measurement was performed by inserting the sensor in the
liquid media. All experimental conditions were determined in
previous works (Di Luccio et al., 2004; Kempka et al., 2008; Vargas
et al., 2008).
2.3. Determination of lipase activity
Lipase hydrolysis was assayed using an emulsion of olive oil
(10 mL/100 mL) in arabic gum (5 mL/100 mL) in different buffers to
evaluate the specificity of lipase extract to pH. The buffers used
were sodium acetate 0.1 mol/L pH 4.0, sodium phosphate 0.1 mol/L
pH 7.0 and triseHCl 0.1 mol/L pH 9.0. After incubation for 15 min at
37  C and 160 rpm, the reaction was stopped by addition of
a solution of acetone/ethanol (1:1). The fatty acids produced due to
the hydrolysis were titrated with NaOH 0.05 mol/L in a pH-stat
(Mettler DL50). Control assays (blanks) were carried out adding the
acetone/ethanol solution for enzyme inactivation. One unit of
lipase activity was defined is the amount of enzyme preparation
necessary to produce 1 mmol of free acid per minute in the assay
conditions. The results are expressed in terms of units per gram of
dry substrate (U/g) (Freire et al., 1997).
2.4. Determination of protease activity
Protease activity was determined based on colorimetric method
using azocasein. One unit of protease activity was defined as the
Carbon (g/100 g) Nitrogen (g/100 g) C/N Ratio
43.34 6.96 6.23
42.87 6.96 6.15
42.73 6.99 6.11
42.92 7.05 6.09
42.55 6.96 6.11
42.75 7.43 5.76
45.37 7.43 6.11
1134 E. Rigo et al. / LWT - Food Science and Technology 43 (2010) 1132e1137

Table 2
Evolution of pH with time in fermentations with different supplements using Penicillium P58.

Mediumb pH of pure supplement pH of fermentation medium

0h 48 h 96 h 120 h
1 (SO) 6.42  0.06a 6.60  0.03a 7.20  0.14a 7.79  0.02a 7.63  0.16a
2 (SM) 6.09  0.55a 6.53  0.01a 7.11  0.01a 7.94  0.06a 7.85  0.09a
3 (CSL) 4.26  0.03a 6.49  0.02a 7.49  0.07a 7.85  0.06a 7.99  0.05a
4 (YH) 6.22  0.01a 6.43  0.01a 7.05  0.37a 7.65  0.16a 7.67  0.06a
5 (PSB) 5.87  0.03a 6.51  0.01a 7.16  0.16a 7.65  0.02a 7.75  0.01a
6 (UR) 5.85  0.46a 6.79  0.06a 7.35  0.03a 7.86  0.18a 7.86  0.08a
7 (URSO) 6.00  0.11a 6.70  0.01a 7.38  0.09a 7.85  0.03a 7.69  0.07a
a
Standard deviation of n ¼ 3.
b
SO Soybean meal þ soybean oil; SM Soybean meal þ sugarcane molasses; CSL Soybean meal þ corn steep liquor; YH Soybean meal þ yeast hydrolysate; PSB Pure soybean
meal; UR Soybean meal þ urea; URSO Soybean meal þ (1 g/100 g) urea þ (0.33 g/100 g) soybean oil.

amount of enzyme that produces a unitary change in absorbance
(428 nm)/min under assay conditions (Charney & Tomarelli, 1947).
3. Results and discussion
The effect of substrate supplementation on lipase production by
fermentation of soybean meal of the two screened microorganisms
(P58 and P74) was evaluated by a kinetic study. In order to evaluate
the different sources of macro and micronutrients added to soybean
meal, the C/N ratio was kept constant at 6.11. The pH of the medium
was followed during the fermentative process for each evaluated
microorganism, as presented in Table 2 and 3. These tables show that
the pH increased in all supplementations tested. Rodríguez et al.
(2006), using urea as supplement and pH 5.5 for lipase production
by R. homothallicus obtained a hydrolytic activity of 594 U/g after
10 h of fermentation. Different behavior was observed when pH
values of 2.5 were used. In this sense, the lipase activity showed an
abrupt decrease. Based on the results obtained in literature and in
the present work, the pH seems to be an important parameter to be
controlled in solid state fermentation, but the non homogeneity of
the system can be considered a difficulty.
3.1. Lipase production by solid state fermentation of soybean meal
using Penicillium P58
The behavior of lipase production by solid state fermentation
using soybean meal as substrate and Penicillium P58 is presented in
Fig. 1. The results showed that the hydrolytic activity at pH 4.0
(Fig. 1A) was lower than in other 2 pH for all supplements (Fig. 1B
and C). After 48 h of fermentation, activities of 139.2 U/g, 144.0 U/g
and 134.4 U/g were obtained at pH 4.0, 7.0 and 9.0, respectively.
However, the analysis of the results of the other supplements tested
evidences differences between the behaviors, suggesting the
production of different enzymes, expanding the potential applica-
tions of the enzymes produced.
The use of Tukey's test permitted us to evaluate which supple-
ment led to higher activities in each fermentation time (24, 48, 72, 96
and 120 h), considering each pH separately. The evaluation of lipase
activity at pH 4.0 showed that the supplementation with a mixture
of soybean oil and urea (URSO) significantly improved lipase activity
(p < 0.05) when compared to the other tested supplements. When
activity at pH 7.0 is taking into account, the medium supplemented
with URSO is statistically (p < 0.05) equal to that supplemented with
SM, YH, CSL and UR. The activity of the extract at pH 9.0 presented
some particularities depending on the fermentation time consid-
ered. In general, the use of URSO as supplement led to higher lipase
activities followed by UR, SO, YH and CSL.
The use of pure soybean meal (Di Luccio et al., 2004) and
babassu cake (Gutarra, Godoy, Castilho, & Freire, 2007) showed that
lipase activities of 21.0 U/g and 30.0 U/g, could be obtained at 48
and 36 h of fermentation, respectively. The use of wheat meal as
substrate and Y. lipolytica as microorganism yielded lipase activities
of 69 U/g (Domínguez et al., 2003).
In the present work, the use of soybean oil could not be
considered an important inductor for lipases production. This result
is in disagreement with some works presented in the literature that
affirm that fats and oils are promising inductors for hydrolytic
enzymes production (Azeredo, Gomes, Sant'Anna, Castilho, &
Freire, 2007; Hasan et al., 2006; Rodríguez et al., 2006).
Production of thermostable lipases by Rhizopus rhizopodiformis
and Rhizomucor pusillus, using sugarcane bagasse supplemented
with olive oil could yield 79.6 U/g and 20.2 U/g after optimization
with suitable inducers (Cordova et al., 1998). Rodríguez et al. (2006)
also report an improvement in lipase yield by fermentation of
sugarcane bagasse by R. homothallicus caused by the addition of
olive oil and urea.
However, medium supplementation could eventually not
improve lipase production. For instance, no improvement in
supplementation of sesame seed cake and soybean cake could be
observed in the production of lipases by SSF using Aspergillus niger
MTCC 2594 (Kamini et al., 1998) and Penicillium simplicissimum
(Di Luccio et al., 2004).
Generally, the high concentration of nitrogen sources in the
media is effective in enhancing the production of lipases by

Table 3
Evolution of pH with time in fermentations with different supplements using Penicillium P74.

Medium** pH of pure supplement pH of fermentation medium

0h 48 h 96 h 120 h
1 (SO) 6.18  0.28a 6.65  0.01a 7.54  0.01a 7.71  0.08a 8.06  0.01a
2 (SM) 5.5  0.11a 6.47  0.07a 7.56  0.09a 6.90  0.21a 7.68  0.13a
3 (CSL) 4.38  0.19a 6.50  0.01a 7.58  0.06a 7.68  0.29a 8.08  0.08a
4 (YH) 6.11  0.13a 6.46  0.05a 6.79  0.02a 7.78  0.03a 7.50  0.30a
5 (PSB) 5.38  0.22a 6.50  0.21a 7.33  0.08a 7.79  0.19a 7.78  0.06a
6 (UR) 5.28  0.33a 6.70  0.05a 7.35  0.03a 7.95  0.03a 7.63  0.23a
7 (URSO) 6.05  0.17a 6.69  0.01a 7.38  0.09a 7.88  0.03a 7.62  0.02a

**SO Soybean meal þ soybean oil; SM Soybean meal þ sugarcane molasses; CSL Soybean meal þ corn steep liquor; YH Soybean meal þ yeast hydrolysate; PSB Pure soybean
meal; UR Soybean meal þ urea; URSO Soybean meal þ (1 g/100 g) urea þ (0.33 g/100 g) soybean oil.
a
Standard deviation of n ¼ 3.
 E. Rigo et al. / LWT - Food Science and Technology 43 (2010) 1132e1137 1135

a 220 improved six-fold lipase activity when compared to the enzyme

yield in the medium supplemented with yeast extract. Similar
200
observation has been reported using babassu cake supplemented
180 with nitrogen sources (Gombert, Pinto, Castilho, & Freire, 2000). The
160
effects of organic and inorganic nitrogen sources on lipase
Lipase activity (U/g)

140

120
a 220
100 200
80 180
60 160

Lipase Activity (U/g)

40 140
20 120

0 100
0 20 40 60 80 100 120
80
Time (h)
b 220 60

40
200
20
180
0
160 0 20 40 60 80 100 120
Lipase Activity (U/g)

140 Time (h)

120

100 b 220

200
80
180
60
160
Lipase Activity (U/g)

40
140
20
120
0
0 20 40 60 80 100 120 100

Time (h) 80

60
c 220
40
200
20
180
0
160 0 20 40 60 80 100 120
Lipase Activity (U/g)

Time(h)
140

120
c 220
100
200
80
180
60
160
Lipase activity (U/g)

40
140
20
120
0
100
0 20 40 60 80 100 120

Time (h) 80

60
Fig. 1. Lipase production by Penicillium P58 in soybean meal with different supple-
ments, measured at pH 4 (a), pH 7 (b) and pH 9 (c). OS ¼ soybean oil, SM ¼ sugarcane 40
molasses, CSL ¼ corn steep liquor, YH ¼ yeast hydrolysate, PSB ¼ pure soybean meal,
20
UR ¼ urea, URSO ¼ urea þ soybean oil. Where: C SO; - SM; : CLS; YH; þ PSB; >
UR; A UROS. 0
0 20 40 60 80 100 120

microorganisms. Supplementation of Jatropha curcas seed cake with Time (h)

NaNO3 was found to improve lipase production by Pseudomonas Fig. 2. Lipase production by Penicillium P74 in soybean meal with different supple-
aeruginosa (Mahanta, Gupta, & Khare, 2008). Rodríguez et al. (2006) ments, measured at pH 4 (a), pH 7 (b) and pH 9 (c). Where: C SO; - SM; : CLS;
verified that the supplementation of sugarcane bagasse with urea YH; þ PSB; > UR; A UROS. Symbols as for Fig. 1.
1136 E. Rigo et al. / LWT - Food Science and Technology 43 (2010) 1132e1137

production by Candida sp. showed that (NH4)2SO4 was the optimal
nitrogen source, which increased lipase activity (Palma et al., 2000).
Considering that, in most cases in the present work, the supple-
mentation with urea and soybean oil presented the more significant
effect on lipase yield; the results of the evolution of lipase production
with time using these supplements were statistically analyzed. The
highest lipase yield at pH 4.0 was observed in 72 h of fermentation,
followed by a small decrease and remaining constant until 120 h.
Similar behavior was observed at pH 9.0, where the maximum
activity was reached in 48 h. An activity peak occurred after 72 h of
fermentation at pH 7.0, and another maximum was reached in 120 h
(203.7 U/g). These results permit us to observe that probably
different enzymes are produced by this strain, which could be suit-
able for applications in acidic, neutral or alkaline media.
3.2. Lipase production by solid state fermentation of soybean meal
using Penicillium P74
The behavior of lipase production by SSF of soybean meal and
Penicillium P74 is presented in Fig. 2. No significant difference
(p < 0.05) between the results obtained with different supplements
were observed for all tested pH. However, when the lipase activity
in 48 h of fermentation is considered, the best result was found in
pH 7.0 (148.8 U/g), suggesting that this microorganism tends to
produce considerable amounts of neutral lipases.
3.3. Kinetics of protease production
Monitoring protease production is of great importance during
lipase production by SSF. In general, the increase in pH is caused by
the increase in the levels of protease. Proteolytic activity leads to
release of amino acids and ammonia to the medium. The kind of
supplement is directly related to the production of different
enzymes (Palma et al., 2000). Protease production by Penicillium
P58 and P74 is presented in Fig. 3. The proteolytic activity was low
(<1.0 U/g) in all studied conditions. Maximum protease activity
obtained here was 0.05 U/g, which is much lower than those values
reported in literature with Penicillium restrictum (Tan, Zhang, Wang,
Ying, & Deng, 2003) grown in babassu cake. Similar results were
obtained by our group with Penicillium verrucosum (Kempka et al.,
2008) (0.65 U/g) and P. simplicissimum (Vargas et al., 2008) (0.46 U/
g) also grown in soybean meal. These results show that the species
of the fungus can show very different behavior, even when similar
media is used. The observed increase in pH with time could be
attributed to the de-amination of amino acids from degraded
proteins in the medium (Nagel, Oostra, Tramper, & Rinzema, 1999).

4. Conclusions
a 0.07
Solid state fermentation for lipase production, using soybean
0.06 meal as substrate was studied. High lipase activity (up to 200 U/g)
was achieved depending on the medium supplementation and pH
considered. The best results were obtained using supplementation
Proteasic Activity (U/g)

0.05
with 1 g/100 g of soybean oil and 3 g/100 g of urea, keeping C/N
ratio at 6.11.
0.04
Penicillium P58 presented the most promising results since it
produced a crude enzymatic extract that shows high activity in
0.03
a pH range from 4 to 9 (146.8, 140.7 and 203.72 U/g, at 48, 96 and
120 h of fermentation, respectively).
0.02

Acknowledgements
0.01

The authors thank CAPES/PROCAD, CNPq and Intecnial for the

0.0 financial support of this work and scholarships.
0 20 40 60 80 100 120

Time (h)
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