Food Bioprocess Technol (2011) 4:578–586
DOI 10.1007/s11947-008-0176-5
ORIGINAL PAPER
Isolation and Screening of Lipase-Producing Fungi
with Hydrolytic Activity
Nara Griebeler & André E. Polloni & Daniela Remonatto &
Francieli Arbter & Renata Vardanega &
Jeferson L. Cechet & Marco Di Luccio &
Débora de Oliveira & Helen Treichel &
Rogério L. Cansian & Elisandra Rigo & Jorge L. Ninow
Received: 9 September 2008 / Accepted: 11 December 2008 / Published online: 8 January 2009
# Springer Science + Business Media, LLC 2009
Abstract Lipases are enzymes that can be secreted by
several microorganisms, making interesting the biodiversity
exploration for searching new microorganisms able to
produce these enzymes. Many agro-industrial residues can
be used as potential substrates for production of enzymes.
The main objective of this work was the isolation and
screening of microorganisms with potential to produce
lipases. Among 24 fungi, five were selected as good lipase
producers using tributyrin on agar plates and solid state
fermentation of soybean bran. Two of them were isolated
from soil samples, another two from soybean bran, and one
from dairy products. These fungi were identified by
microcultivation technique as from Penicillium and Asper-
gillus genera. Through random amplified polymorphic
DNA technique, the most promising strains could be
genetically discriminated, selecting two fungi as good
lipase producers but genetically different. One isolated
from soybean bran could hydrolyze efficiently triglycerides
with fatty acids with different chain length. Another
isolated from dairy products was only effective to hydro-
lyze triglycerides with long-chain fatty acids. Two distinct
N. Griebeler : A. E. Polloni : D. Remonatto : F. Arbter :
R. Vardanega : J. L. Cechet : M. Di Luccio (*) : D. de Oliveira :
H. Treichel : R. L. Cansian
Programa de Pós-graduação em Engenharia de Alimentos,
Universidade Regional Integrada do Alto
Uruguai e das Missões-Campus de Erechim,
Av. Sete de Setembro, 1621,
99700-000 Erechim, RS, Brazil
e-mail: helen@uricer.edu.br
E. Rigo : J. L. Ninow
Departamento de Engenharia Química e de Alimentos,
Universidade Federal de Santa Catarina, UFSC,
Florianópolis, SC, Brazil
groups could be verified by means of this technique,
comprising the most productive strains and the lowest or
nonproductive ones in terms of hydrolytic activity.
Keywords Lipase . Microorganism screening .
Filamentous fungi . Microcultivation . RAPD
Introduction
Lipases (E.C. 3.1.1.3) can be used in fat and oil
modification by reactions of hydrolysis, esterification,
and/or interesterification, being important catalysts in the
production of specific fatty acids or glycerides from
vegetable oils (Kumar et al. 2005; Bradoo et al. 2002).
Several techniques have been developed to obtain higher
conversions, as highly specific enzymes for each applica-
tion, improving the possibility of industrial applications
for these biocatalysts (Franken et al. 2008; Soccol &
Vandenberghe 2003).
Microorganisms able to produce lipases can be found in
several habitats, including wastes of vegetable oils and
dairy product industries, soils contaminated with oils,
seeds, and deteriorated food (Sharma et al. 2001). The soil
has a great variety of microorganisms that can be isolated
and evaluated for their potential as enzyme producers (Ko
et al. 2005). The isolation and screening of microorganisms
can lead to the production of lipases with stability to
different temperatures and pH ranges, specificity to certain
fatty acids, and enantioselectivity (Hernalsteens & Maugeri
2008; Björkling et al. 1991).
Several methods can be used for microorganism screening
based on the determination of the presence of extracellular
lipases. The use of a solid medium with inducer substrates
Food Bioprocess Technol (2011) 4:578–586
such as vegetable oils, standard triglycerides (tributirin,
triolein), Tween 80, and dyes (Ko et al. 2005; Cardenas et
al. 2001; Wang et al. 1995) has been widely described in the
literature. However, some of these substrates may not be
adequate for lipase detection, turning of extreme importance
the verification of the specificity of the analysis for lipases
and esterases (Jaeger & Reetz 1998). Both enzymes catalyze
the hydrolysis and synthesis of bound ester. The esterases
hydrolyze esters with carboxylic acids of short chain, while
lipases prefer long-chain fatty acids (Kim et al. 2007).
The solid state fermentation (SSF) is an interesting
alternative for microbial enzyme production due to the
possibility of using residues and by-products of agro-
industries as nutrient sources and support for microorgan-
ism development. The use of by-products as substrates for
lipase production, adds high value, and low-cost substrates
may reduce the final cost of the enzyme (Menoncin et al.
2008, Rodriguez et al. 2006; Soccol & Vandenberghe 2003;
Pandey 2003; Castilho et al. 2000).
The random amplified polymorphic DNA (RAPD)
technique has been shown to be extremely useful in
evaluation of genetic variability of microbial strains.
Several authors cite the RAPD as ideal in the study of
genomic polymorphism. This method has been used to
compare intra- and interspecific differences in bacteria. The
purified DNA or the cell extracts cultivated in agar can be
used (Williams et al. 1990; Silveira et al. 2000). The
adoption of RAPD markers in detection, diagnostics, and
determination of genetic diversity is related to the simplic-
ity of the method, high sensibility, low cost, promptness,
safety, and amplitude of the generated results. The RAPD
technique has been proved to be useful for measuring and
characterizing the genetic variability of microorganisms,
plants, or animals (Ikeh 2003).
The objective of this work was the isolation and
screening of microorganisms with potential to produce
lipases and the evaluation of their ability to produce these
enzymes using tributyrin on agar plates and solid state
fermentation of soybean bran. Genera were identified, and
RAPD technique was used to evaluate genetic differences
between microorganisms. This work is directed as an initial
step to the isolation of new lipases producers able to
produce enzymes with different catalytic properties of
commercial interest.
Materials and Methods
Isolation of Microorganisms
The microorganisms were isolated from several sources as
soil, olive oil, cheese, tomato extract, soybean oil, milk
cream, meat, soybean bran, and contaminated culture media.
579
The microorganism isolation and cultivation was carried out
in Petri dishes using potato dextrose agar (PDA) medium at
30 °C. All stock cultures were stored at −10 °C.
Screening of Lipolytic Microorganisms Using Tributyrin
The screening of microorganisms that produce lipases with
hydrolytic activity was carried out in Petri dishes using a
medium constituted by (% m/v in distilled water): peptone
0.5%; yeast extract 0.3%; tributyrin 0.1%; agar 2%, pH 6.0
(Freire 1996). Culture plates inoculated with the fungal
strains were incubated at 30 °C for 168 h, and diameter (d)
of the colonies and the diameter (D) of total clear hydrolytic
halos including the colonies were determined. The strains
that yielded higher halos (D−d) were selected as potential
microorganisms for lipase production using tributyrin as
substrate.
Screening of Lipolytic Microorganisms by Solid State
Fermentation
All isolated microorganisms were also screened by solid
state fermentation using a fixed amount (10 g) of soybean
bran (residue of soybean oil industry obtained after
grinding of the seeds) as substrate. The inoculum grown
in PDA medium was incubated at 30 °C for 7 days. The
resultant suspension was used as inoculum for SSF,
standardizing the concentration at 108 spores/g of dry
soybean bran (Freire 1996).
The experiments for extracellular lipase production were
carried out aseptically in conical reactors covered with
hydrophobic fabric. An aqueous solution containing the
supplements was added to the substrate (soybean bran) and
the resulting medium sterilized at 121 °C for 20 min.
Temperature of 27.5 °C and moisture of 55% were used in
all experiments, fixing a fermentation time of 48 h, as
previously optimized by Kempka et al. (2008).
The fermented medium was weighed, added to 45 mL of
0.1 mol L−1 sodium phosphate buffer at pH 7.0, and
incubated at 35 °C and 200 rpm for 30 min for enzyme
extraction. Following extraction, the liquid fraction was
separated by filtration and assayed for lipase activity.
The hydrolytic activity of the crude enzymatic
extracts was determined by titrimetric method. An
emulsion of olive oil (100 g kg−1) and Arabic gum
(50 g kg−1) in sodium phosphate buffer 0.1 mol L−1,
pH 7.0 was incubated with a sample of the enzyme extract
at 37 °C and 160 rpm for 15 min. The reaction was
stopped, and the fatty acids extracted with a solution of
acetone and ethanol (1:1). The fatty acids produced were
titrated with NaOH 0.05 mol L−1 (Vargas et al. 2008). One
unit of lipase activity was defined as the amount of
enzyme required to release 1 μmol of fatty acid per minute
580
under the specified conditions. All assays were performed
in duplicate runs.
Microorganism Identification
The microorganisms screened as promising for lipase
production in terms of hydrolytic activity were identified
by microcultivation technique. The fungi were inoculated
on a slice of agar laid on a sterile glass slide and covered by
a sterile coverslip. The slide was then placed in a Petri dish,
and the setup was then incubated for 5 days at 25 °C.
The coverslip with the adhered hyphae was withdrawn
and stained with cotton blue dye. The same procedure was
adopted for examining spores and hyphae bound to the
slide.
The identification of the fungi genus was based on the
macroscopic morphology of the colonies and on the study
of fructification structures of the strains, following the key
of investigation of genera proposed by Barnett et al. (1998).
Genetic Characteristics of the Microorganisms by RAPD
The RAPD technique was used to correlate the results
obtained in the fermentative screening with the genetic
characteristics of the isolated microorganisms. Firstly, the
extraction and quantification of the DNA was carried out,
followed by amplification, electrophoresis in agarose gel,
and the analysis of the data obtained.
The DNA was extracted from grown cells in liquid
medium (yeast malt for yeasts and potato dextrose for
fungi) for 24 h, according to the methodology proposed by
Raeder & Broda (1985). Details about the experimental
procedure used for genomic DNA extraction can be found
in the work of Toniazzo et al. (2006).
For DNA concentration, 980 μL of sterile ultrapure
water (MilliQ) were added in a test tube with 20 μL of
sample. DNA concentration was estimated by measuring
the optical density at 260 nm, using spectrophotometer
(Agilent 8453) and checked by electrophoresis on a 0.8%
agarose gel (Gibco BRL, Carlsbad, California, USA) in 1×
TBE (0.89 M Tris, 0.89 M of H3BO3, and 0.08 M
ethylenediamine tetraacetic acid).
Amplification followed the procedure reported by
Williams et al. (1990), with some modifications. The
reactions were carried out in 25 μL. The reaction mixture
contained buffer (50 mM Tris–HCl pH 9.0, 50 mM KCl),
deoxyribonucleotide triphosphates mix (200 mM of each
nucleotide and 0.2 mM of primer, 3 mM MgCl2 (Life
Technologies, São Paulo, Brazil), 0.25 mM, 1.5 U of Taq
DNA polymerase recombinant (Invitrogen Life Technolo-
gies, São Paulo, Brazil, Triton-X-100 (Merck, Germany)
and 40 ng of DNA of each microorganism previously
isolated.
Food Bioprocess Technol (2011) 4:578–586
The kits OPA, OPF, OPH, OPW, and OPY from the
Operon Technologies (Nattermannallee, Cologne, Ger-
many), with 20 primers of each one, were used to identify
the primers that yielded the best results, estimating the
number, intensity, size of each band, as well as reproduc-
ibility, and the polymorphism.
The amplification was carried out in a Programmable
Thermal Controller (MJ Research, Watertown, MA, USA,
model PTC100TM) as follows: one initial cycle of 3 min at
92 °C, followed by 40 cycles comprising denaturation
(1 min at 92 °C); annealing (1 min at 35 °C); and extension
(2 min at 72 °C), and then a final extension for 3 min at
72 °C.
Amplification products were separated by electrophore-
sis in 1.4% agarose gels in buffer 1× TBE using a
horizontal electrophoresis apparatus. The run was carried
out at constant voltage of 90 V. DNA lambda digested with
EcoRI and HindIII of the Gibco BRL was included as
molecular size marker. Gels were visualized by staining
with ethidium bromide, and band patterns were photo-
graphed under UV light using a charge-coupled device
camera (GEL-PRO, Media Cybernetics, Silver Spring, MD,
USA).
The results were obtained by determination of the
presence or absence of band yielding a matrix that was
analyzed with help of the software NTSYS version 1.7
(Numerical Taxonomy System of Multivariate Analysis
System). The dendrogram was built by the algorithm
unweighted pair group method using arithmetic averages
(UPGMA) using the Jaccard coefficient of similarity.
Statistical Analysis
The analysis of variance followed by Tukey test was
applied for the statistical analysis of the obtained data. The
data were treated with the aid of Statistica 6.0 (Statsoft Inc.,
Tulsa, OK, USA). All analyses were performed considering
a level of 95% of confidence (p<0.05).
Results and Discussion
Isolation and Screening of Lipolytic Microorganisms Using
Tributyrin
The microorganism plate screening on solid medium
containing tributyrin yielded 24 fungi with visible halos
after 3 days of incubation. The selected fungi were isolated
from samples of soil (T1F1, T1F2, T1F3, T1F3C2, T1F4,
T1F7, T2F1, T2F2, T2F3), dairy products (RMF6, RMF4,
RMF2, RMF3, RMF2C4, RF3, RQF2, CLF1C3, CLF1),
butter (MF1), olive oil (OOF6), meat (CMB1C2), tomato
extract (ETF2), and soybean bran (F0F1, F0F2).
Food Bioprocess Technol (2011) 4:578–586 581

Figure 1 presents the size of halos produced by the
tributyrin hydrolysis after 3 to 5 days of incubation by
lipases produced by the fungi isolated from soil samples
(Fig. 1a), dairy products (Fig. 1b), and other samples
(Fig. 1c). The analysis of the results presented in Fig. 1a
indicates the fungus T1F3C2 as a good lipase producer
comparing to the other fungi isolated from soil samples.
The halo of this strain after 4 days (22.4 mm) was similar to
the fifth day (26.5 mm), suggesting the existence of a
maximum degradation in the first days of incubation. The
lipase produced by the fungus T1F3 resulted in a halo
diameter of 18.7 mm after 3 days of incubation and
22.2 mm after 5 days, also showing a good potential for
lipase production.
The fungus CLF1C3 isolated from milk cream was
identified as the most promising in terms of tributyrin
hydrolysis, presenting halo diameters of 22.0 and 26.7 after
4 and 5 days of incubation, respectively (Fig. 1b).

Fig. 1 Evaluation of the halos

produced by hydrolysis of trib- a 30
utirin on the third, fourth, and
fifth days of growth of fungi 25

Halo Hydrolysis (mm)

isolated from: soil (a), dairy
products (b), and other samples 20
from animal and vegetable
sources (c) 15

10

0
3 4 5
Time (days)

T1F1 T1F2 T1F3 T1F3C2 T1F4 T1F7 T2F1 T2F2 T2F3

b
30
Halo Hydrolysis (mm)

25

20

15

10

0
3 4 5
Time (days)

CLF1C3 RMF4 RMF6 CLF1 RF3 RMF2 RMF3 RQF2 RMF2C4

c 30

25
Halo Hydrolysis (mm)

20

15

10

0
3 4 5
Time (days)

OOF6 ETF2 CMB1C2 MF1 F0F1 F0F2

582
From Fig. 1c, one can see that the fungi F0F2 and F0F1,
isolated from soybean bran, were the most promising for
lipase production in terms of tributyrin hydrolysis. In this
case, it is important to observe the particular behavior of the
halo diameter (23.5 and 21.0 mm) after 5 days of
incubation, indicating slow and progressive degradation of
the triglyceride.
The screened fungi having good potential for lipase
production in tributirin, after 5 days of incubation, can be
summarized as:
T1 F3 C2 > CLF1 C3 > F0 F2 > T1 F3 > F0 F1
The selected fungi had hydrolysis halos that varied from
1.4 to 26.7 mm in initial pH=6.0. Colen et al. (2006)
presented the screening of fungi isolated from soil samples
and considered good alkaline-lipase producers. In the
referenced work, the higher halo diameters were found as
12 and 14 mm after 18 h of incubation. The authors
selected the fungus Colletotrichum gloesporioides (14 mm
of halo diameter) as the main lipase producer. The five
fungi selected in this work presented halo diameters higher
than those presented in literature.
Screening of Lipolytic Microorganisms in Solid State
Fermentation
The objective of this study was to validate the agar plate
screening, finding those strains able to produce lipases with
potential specificity for long-chain triglycerides.
Among the 203 isolated strains, the fungi presented
higher potential for lipase production both in tributyrin and
in SSF. The hydrolytic activity of the lipases produced by
the isolated strains using SSF is presented in Fig. 2. It is
worth to mention that some microorganisms that presented
hydrolysis halos did not present hydrolytic activity when
inoculated in soybean bran. The lipase production by solid
state fermentation pointed out some potential fungi isolated
from soil (T1F3 e T1F3C2) and meat (CMB1C2) samples
(Fig. 2a) with hydrolytic activities of 15.9, 10.7, and
17.5 U/mL, respectively. Microorganisms isolated from
soybean bran (F0F2 and F0F1) samples (Fig. 2b) showed
lipase activities of 15.9 and 19.2 U/mL, respectively, while
the strains CLF1C3 and RMF2C4 (Fig. 2c) presented
activities of 10.4 and 21.9 U/mL also considered potential
for lipase production.
Table 1 presents the comparative evaluation among the
screened fungi promising for lipase production using both
tributyrin and soybean bran as substrates. The analysis of the
results indicates that fungus RMF2C4 presented the highest
hydrolytic activity (21.89 U/mL) using soybean bran as
substrate in solid state fermentation. However, the same
fungus presented the lowest hydrolysis halos (1.17 mm).
Distinct behavior was observed for F0F1, F0F2, CLF1C3,
Food Bioprocess Technol (2011) 4:578–586
T1F3, and T1F3C2 fungi, all selected as good lipase producers
by solid state fermentation and using tributyrin as substrate.
This aspect should be emphasized since the fungus RMF2C4
was not screened by tributyrin but present relevant lipase
activities in solid state fermentation.
A possible explanation for this is that esterases are able to
hydrolyze esters with low and long-chain fatty acids, while
lipases hydrolyze long-chain acylglycerides (≥10 carbon
atoms; Kim et al. 2007). In this way, the difference in results
obtained in tributyrin (C4:0) and by solid state fermentation
of soybean bran (C18 as major constituents) can be related
both to production process, where different enzymes can be
produced in agar plate and SSF, and to the assay method
used for enzymatic activity determination. In this way, the
same enzyme could be produced in both systems but with
different specificity, i.e., the fungus RMF2C4 produces an
enzyme that prefers long-chain acylglycerides and have
better activity in the titrimetric assay (with olive oil
substrate), than in the agar plate. The inverse can also occur.
Colen et al. (2006) selected the C. gloesporioides (14 mm
of halo diameter) as a promising microorganism for lipase
production by submerged (SmF) and solid state fermentation.
The fungus presented hydrolytic activity of 18.8 U/mL after
72 h of incubation using olive oil as supplement. The activity
was measured in alkaline medium. Penicillium restrictum
grown in media supplemented with olive oil yielded
activities of 12.1 U/mL and 17.4 U/g in SmF and SSF,
respectively (Azeredo et al. 2007). SSF is increasingly being
used for lipase production. However, the results presented in
the literature still reveal a great diversity of information. The
use of alternative media, a great advantage of SSF, makes it
possible to obtain different levels of enzyme production.
Babassu cake, when fermented by Penicillium simplicissi-
mum, resulted in activities around 26.4 U/g in optimized
conditions (Gutarra et al. 2005; Cavalcanti et al. 2005).
Similar results were obtained using P. restrictum also in
babassu cake (around 30.3 U/g; Palma et al. 2000; Gombert
et al. 1999). The use of soybean meal as substrate showed
good results in terms of lipase activities in SSF by P.
simplicissimum (around 30 U/g; Vargas et al. 2008; Di
Luccio et al. 2004). Using Penicillium verrucosum, 40 U/g
of hydrolytic activity was obtained in optimized conditions
(Kempka et al. 2008). It is worth to mention that the
hydrolytic activities obtained in the present work were not
obtained in optimized conditions, demonstrating the potential
of microorganisms isolated and screened here.
Microorganism Identification by Microcultivation
The analysis of the results indicated that the isolated fungi
presented higher potential for lipase production than
bacteria and yeasts. The best lipase-producing strains were
identified by microcultivation technique, based on the
Food Bioprocess Technol (2011) 4:578–586 583

Fig. 2 Evaluation of hydrolytic

activity on olive oil for lipases a 30
obtained by SSF and different

Hydrolysis activity (U/mL)

microorganisms isolated from 25
soil (a), soybean bran (b), and
dairy product (c) samples 20

15

10

1
C

L1
F1

F3

F6
2
F3

F6

C
C
C

F1

M
ET

T1

T2
F3

F3
O

L1
B1

1N

C
O

BF

T1

T1
M

AN
C
b
30

Hydrolysis activity (U/mL)

25

20

15

10

-
F1NF1C1 F0F1 F32NF4 F0F2 F32NF3 F14NF8 F1NF1 F14NF7
c
30
Hydrolysis activity (U/mL)

25

20

15

10

-
3

F1
F1
6

F1

F3
1C

C
C

Q
R

M
F0

F2

F2
F3

F3

LF

R
R

R
M

M
M

R
Q

Table 1 Comparative evaluation among the filamentous fungi screened as lipase producers in solid state fermentation of soybean bran and in agar
plates using tributyrin

Filamentous fungi Hydrolytic activitya (U/mL) Halo diametera (D−d)b (mm)

T1F3 15.98±0.98 b 22.20±0.55 b

T1F3C2 10.67±1.10 c 26.50±0.65 a
CLF1C3 10.36±0.86 c 26.70±0.79 a
F0F2 15.91±0.79 b 23.35±0.87 b
F0F1 19.23±1.13 a 20.95±0.90 b
CMB1C2 17.50±1.45 a 2.03±0.87 c
RMF2C4 21.89±1.95 a 1.17±0.43 c
a
Means±standard deviations within rows without a common letter are significantly different at a 5% level (Tukey Test)
b
d, diameter of the colonies and D, the total diameter of clear hydrolytic halos including the colonies
584 Food Bioprocess Technol (2011) 4:578–586

Table 2 Total and polymorphic number of fragments obtained for each primer used for the isolated microorganisms

Primer Sequence (5′ to 3′) Total fragments Polymorphic fragments

OPY 17 GACGTGGTGA 11 11
OPF 05 CCGAATTCCC 09 09
OPW 19 CAAAGCGCTC 12 11
OPA 20 GTTGCGATCC 14 14
OPA 13 CAGCACCCAC 9 9
OPA 05 AGGGGTCTTG 6 6
OPA 03 AGTCAGCCAC 15 15
OPH 19 CTGACCAGCC 14 14
OPF 09 CCAAGCTTCC 10 10
OPW 11 CTGATGCGTG 11 11
Total fragments 111 110
Polymorphism (%) 99

shape of fructification body. By this procedure and by
comparison of macro- and microscopic aspects, 86.7% of
the fungi were identified as belonging to the Penicillium
genera (presence of paintbrush-like conidia). From all the
isolated fungi, 13.3% were classified as Aspergillus, based
on the presence of hyaline and dark septated mycelium
(Samson et al. 1995).
Species of Aspergillus are considered initiators of
deterioration of seeds and grains, since they could grow at
low water content. In a second step follows the contami-
nation by Penicillium, at higher water content, as a result of
metabolic activity of the first microorganisms (Meronuck
1987). Penicillium and Aspergillus are common contami-
nants of tropical and subtropical regions, predominating
among other fungi (Rossetto et al. 2003).
Genetic Characteristics of the Microorganisms by RAPD
The genetic analysis of the isolated microorganisms by
RAPD markers was carried out with the main objective of
identifying possible duplications of same isolated strains
and determining the genetic distance between the strains.
Table 2 presents the ten primers used in the RAPD
analysis and the relation between the total number of
fragments and polymorphic fragments generated by RAPD
markers. A total of 111 fragments, with average number of
fragments per primer of 11.1, were obtained. A high
polymorphism (99%) was observed among the isolated
fungi. Similarity indices (Jaccard coefficient) among the
filamentous fungi evaluated in this work varied from 0.1 to
0.98 with a mean value of 0.39. These results indicate a
high variability among the strains, related to distinct species
or genera, although a clear separation between Aspergillus
and Penicillium genera was not observed.
From the dendrogram presented in Fig. 3, it is possible
to note that, despite the high polymorphism, the RAPD
markers were efficient for identifying the F0F1 and F0F2
fungi as genetically equal. This observation is important,
since it avoids the execution of the same work in the
optimization and purification steps.
The hydrolytic activities obtained in the fermentative
screening could be related to the genetic analysis,
pointing out the fungi RMF2C4 (Penicillium sp) and
F0F1 (Penicillium sp) as similarly productive but geneti-

Fig. 3 Dendrogram generated

by UPGMA method using
Jaccard coefficient, from RAPD
markers of the isolated
filamentous fungi
Food Bioprocess Technol (2011) 4:578–586
cally different. The use of fungi F0F2 (Penicillium sp),
CMB1C2 (Penicillium sp), and T1F3 (Penicillium sp)
would possibly lead to similar results in the optimization
step, since high degree of similarity was observed. Other
fungi selected as good lipase producers in Table 1, like
T1F3C2 (Penicillium sp) and CLF1C3 (Penicillium sp),
also were considerably similar to RMF2C4, suggesting the
selection of this strain for the continuity of the work.
The existence of two different groups in the dendrogram
could be observed in Fig. 3. This separation shows a
tendency of grouping the filamentous fungi in relation to
their productivity. The first group includes the filamentous
fungi with a higher ability to produce lipases with
hydrolytic activity (78.6% of the strains in that group),
while in the second, only 50% of the fungi are able to
produce lipases. All the fungi producers of lipases with
hydrolytic activities higher than 10 U/mL could be found in
the same genetic group (good producers).
Conclusions
Among the isolated microorganisms, the filamentous fungi
presented the best results in terms of lipase production by
solid state fermentation.
The most promising fungi in terms of lipase activity
were identified by microcultivation technique as belonging
to Penicillium and Aspergillus genera. The random ampli-
fied polymorphic DNA technique identified and classified
the good lipase producers, selecting two fungi (F0F1 and
RMF2C4) as similar producers but genetically different.
Two distinct groups in the dendrogram were observed,
comprising the most productive strains and the lowest or
nonproductive ones based on hydrolytic activity.
Acknowledgments The authors thank CAPES/PROCAD, CNPq,
and Intecnial for the financial support of this work and scholarships.
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