Appl Biochem Biotechnol

DOI 10.1007/s12010-013-0506-6

Solid-State Fermentation of Rapeseed Meal

with the White-Rot Fungi Trametes versicolor
and Pleurotus ostreatus
Jerzy uchowski & ukasz Pecio & Magdalena Jaszek &
Anna Stochmal

Received: 25 July 2013 / Accepted: 30 August 2013

# Springer Science+Business Media New York 2013

Abstract Rapeseed meal is valuable high-protein forage, but its nutritional value is significantly reduced by the presence of a number of antinutrients, including phenolic compounds.
Solid-state fermentation with white-rot fungi was used to decrease the sinapic acid concentration of rapeseed meal. After 7 days of growth of Trametes versicolor and Pleurotus ostreatus,
the sinapic acid content of rapeseed meal was reduced by 59.9 and 74.5 %, respectively. At the
end of the experiment, sinapic acid concentration of T. versicolor cultures decreased by 93 % of
the initial value; in the case of cultures of P. ostreatus, 93.2 % reduction was observed.
Moreover, cultivation of white-rot fungi on rapeseed meal resulted in the intensive production of extracellular laccase, particularly strong during the late phases of growth of T.
versicolor. The obtained results confirm that both fungal species may effectively be used to
decompose antinutritional phenolics of rapeseed meal. Rapeseed meal may also find use as an
inexpensive and efficient substrate for a biotechnological production of laccase by white-rot
fungi.
Keywords Solid-State Fermentation . Rapeseed Meal . Sinapic Acid . White-Rot Fungi .
Trametes versicolor . Pleurotus ostreatus

Introduction
With a world production of 62 million tons in 2011 (FAOSTAT), rapeseed and canola are
among the most important oil crops. The meal obtained after oil extraction is a rich source of
nutrients. A high-protein content which may exceed 40 %, the presence of vitamins (B group
and E), and significant amounts of calcium, magnesium, zinc, and copper, make it a valuable
J. uchowski (*) : . Pecio : A. Stochmal
Department of Biochemistry, Institute of Soil Science and Plant CultivationState Research Institute,
ul. Czartoryskich 8, 24-100 Puawy, Poland
e-mail: jzuchowski@iung.pulawy.pl
M. Jaszek
Department of Biochemistry, Maria Curie-Skodowska University, ul. Akademicka 19,
20-033 Lublin, Poland

Appl Biochem Biotechnol

feed additive for livestock and poultry [14]. However, the high nutritional value of rapeseed
and canola meals is remarkably decreased by high levels of fiber and antinutrients such as
glucosinolates, phytic acids, and phenolic compounds. The last group comprises of condensed tannins, and most significantly, phenolic acids, which may constitute up to 2 % of the
meal. Among phenolic acids, sinapic acid is present in the highest amounts. The free acid
constitutes a minor part of the total sinapic acid and most of the compound is present mainly
in the form of esters. The most abundant of them is sinapine, the choline ester of sinapic
acid, a dominant phenolic compound of rapeseed meal. Phenolic compounds contribute to
its dark color, astringency, and bitter taste. Moreover, rapeseed meal containing feeds may
cause a fishy taint of hen eggs because of the high content of sinapine, which may be
metabolized to trimethylamine. Phenolic compounds can also form complexes with proteins,
influencing their nutritional value [5, 6].
The presence of antinutrients significantly impedes the usage of rapeseed meal as forage or a
potential source of protein for human nutrition, and many attempts were undertaken to remove
these substances. Different methods of removal of phenolic compounds have been developed,
most of them based on chemical extraction methods, chemical transformation, or a combination
of both processes; however, most of these methods are not efficient enough [7]. Another
possibility is to carry out enzymatic or biological transformation of rapeseed phenolics.
White-rot fungi are considered to be the most efficient lignin-degrading organism. Their
extracellular ligninolytic enzymes, including laccase and peroxidases, can decompose different kinds of phenolic substrates, and have found practical use in bleaching and pulping
processes, as well as in the removal of environmental pollutants [8, 9]. White-rot fungi or
their enzymes may also be used to improve the digestibility of lignocellulosic waste
materials, such as wheat straw or rice husk, which may be utilized in ruminant feeding [10].
A preparation of ligninolytic enzymes of a white-rot fungus, Trametes versicolor, was
successfully used by Lacki and Duvnjak [7] to decrease the phenolic content of canola meal.
Biotransformation of rapeseed meal with the use of white-rot fungi may also be applied [11].
This article describes the effect of a solid-state fermentation with two white-rot fungi,
T. versicolor and Pleurotus ostreatus, on the sinapic acid level of rapeseed meal. The extracellular laccase activity was also determined. Both fungal species are efficient lignin degraders and
potent producers of laccase, used in many laboratories investigating ligninolytic organisms and
their biotechnological application. P. ostreatus, known as oyster mushroom, is a popular edible
fungus. T. versicolor is a medicinal mushroom with anticancer properties [12].

Materials and Methods

Fungi and Growth Conditions
T. versicolor (ATCC 44308; strain FCL 7) and P. ostreatus (strain FCL 247) were obtained from
the culture collection of the Department of Biochemistry, M. Curie-Skodowska University in
Lublin, Poland. Both fungi were grown at 25 C under stationary conditions, in 250-mL
Erlenmeyer flasks containing the growth medium prepared according to Lindeberg and Holm
[13], with glucose (10 g L1) as a carbon source and L-asparagine (2.5 g L1) as a nitrogen
source. Fungal cultures used to inoculate rapeseed meal were grown for 10 days in Erlenmeyer
flasks containing glass balls and 30 mL of the appropriate medium. Before inoculation of
rapeseed meal, the cultures were shaken firmly to free fragments of mycelium and hyphens.
Portions of rapeseed meal obtained from a local feed shop (3,000 g) were weighted into
100-mL Erlenmeyer flasks, then 15 mL of distilled water was added, and the mixture was

Appl Biochem Biotechnol

autoclaved for 30 min at 121 C. The sterile rapeseed meal was inoculated with 2 mL of
mycelium suspension. Non-inoculated rapeseed meal was used as control. In a single
experimental cycle, white-rot fungi were grown in duplicates on rapeseed meal for up to
28 days. Fungal cultures were collected and frozen after every 7 days of cultivation.
Experimental flasks were divided into two equal parts. One group was subsequently
freeze-dried and weighed. Dried mycelia were separated from rapeseed meal and their mass
was determined. After weight measurements, the mycelia and the medium were combined
again, powdered in mortar, and used for analyses of sinapic acid. The second group of fungal
cultures was used for determination of extracellular laccase activity. In this case, fungal
cultures grown on Lindeberg and Holm liquid medium, without any rapeseed meal supplementation, were used as control.
Isolation of Phenolic Acids
The procedure of isolation of phenolic compounds from rapeseed meal was loosely based on
the method described by Thiyam et al. [14]. The powdered cultures were extracted under
reflux (three times) with 30 mL of 70 % methanol (v/v), for 20 mi. The pooled extract was
centrifuged (6,500g, 7 min), and the obtained supernatant was topped up to 100 mL with
70 % methanol (J.T. Baker, Deventer, Netherlands). A 50-mL aliquot of the extract was
evaporated to dryness in a rotary evaporator. The residue was dissolved in 25 mL of Milli-Q
water and subjected to 2-h alkaline hydrolysis in the dark, after addition of 1.25 mL 4-M
NaOH. The hydrolyzate was subsequently acidified with ice-cold 6-M HCl, to a pH value of
about 2. The mixture was extracted three times with 30 mL of ethyl acetate (POCH S.A.,
Gliwice, Poland). The organic phase was collected, filtered, and evaporated to dryness in a
rotary evaporator, and the residue was dissolved in 4 mL of 50 % acetonitrile (v/v; J.T.
Baker, Deventer, Netherlands) and stored in a freezer.
HPLC Analysis
The sinapic acid content in rapeseed meal extracts was determined by reversed-phase ultrahigh pressure liquid chromatography, performed on a ACQUITY UPLC Systems chromatograph (Waters Corporation, Milford, CT, USA), equipped with a photodiode array
detector, and Waters ACQUITY UPLC BEH C18 column (502.1 mm, 1.7 m). Samples
were separated using a mobile phase gradient of solvent A (0.1 % formic acid in water, v/v)
and solvent B (40 % acetonitrile and 0.1 % formic acid in water, v/v). The solvent gradient
was programmed as follows: 5 min, 0 % B; 0.5 min, 1 % B; 2.5 min, 10 % B; 10 min, 10
100 % B; 1 min, 100 % B; and 1 min, 1000 % B. The flow rate was kept at 0.3 ml/min and
the column temperature was maintained at 50 C. The concentration of sinapic acid in
samples was calculated on the base of standard curve (Sigma Chemical Co. St. Luis, USA).
Laccase Activity Assay
Determination of extracellular laccase activity was based on the oxidation of syringaldazine
(4-hydroxy-3,5-dimethoxybenzaldehyde; Sigma Chemical Co. St. Luis, USA) [15]. Laccase
activity was calculated with the extinction coefficient for the reaction product (65,000 M1
cm1) and expressed in microkatals per liter [16]. Portions of rapeseed meal overgrown
with mycelium were gently mixed with phosphate buffer (pH 7.4), in a 1:1 ratio (w/v), and
centrifuged at 4 C. The obtained supernatants were used as a source of extracellular
laccase.

Appl Biochem Biotechnol

Native Polyacrylamide Gel Electrophoresis

In order to estimate the changes of relative laccase activity, the 12 % native polyacrylamide
gel electrophoresis (PAGE) was used. The samples were ultrafiltrated using Microcon
Centrifugal Filter Units 3000 NMWL (Millipore, Bedford, MA, USA), 25 L of each of
sample was deposited per lane. Electrophoretic separations were performed in the cold, at
145 V. The laccase activity was visualized by reaction with guaiacol (Sigma Chemical Co.
St. Luis, USA) in 0.1-M citrate-phosphate buffer (pH 5.2), at 25 C.
Statistical Analysis
The experiments were performed at least in triplicate. Results were subjected to ANOVA
analysis (Statgraphics Online), and means were compared using Tukey's multiple range test.
Statistical significance was declared at P=0.05.

Results and Discussion

Water-soaked rapeseed meal was a good cultivation medium for T. versicolor and
P. ostreatus; the surfaces of 1-week cultures were wholly covered with mold-like mycelia.
Both species differed in the pattern of growth; P. ostreatus more intensively penetrated the
whole medium, while T. versicolor grew mainly, though not solely, on the surface of
rapeseed meal. As a result, masses of 4-week mycelia of P. ostreatus were higher than in
the case of T. versicolor, though the initial rate of growth of P. ostreatus was distinctly lower.
On the other hand, metabolic processes seem to have been more intensive in the cultures of
T. versicolor, where the decrease of culture mass during cultivation was visibly greater, as
compared to P. ostreatus (Fig. 1). Moreover, T. versicolor caused more intensive darkening
of the medium as compared to P. ostreatus, which may reflect differences in composition or
functioning of their ligninolytic apparatus. Cultivation of both fungi led to a rapid lowering
of sinapic acid content in rapeseed meal (Table 1). P. ostreatus was visibly more efficient
during the first 2 weeks of cultivation, causing a 74.5 and 82.3 % (the initial concentration of
sinapic acid in the rapeseed meal was 6.150.52 mg g1) decrease in the concentration of
this compound, after 1 and 2 weeks of cultivation, respectively. In the case of T. versicolor,
the respective reduction of sinapic acid levels reached 59.9 and 79.1 %. During the

Fig. 1 Changes of fungal biomass and the mass of culture during solid-state fermentation of rapeseed meal
with T. versicolor (a) and P. ostreatus (b). Data are means SD from at least three independent experiments

Appl Biochem Biotechnol

Table 1 The effect of cultivation of T. versicolor and P. ostreatus on the sinapic acid content of rapeseed
meal. Data are expressed as means SD from at least three independent experiments
Fungi

Time of cultivation (days)

Sinapic acid content (mg g1)

T. versicolor
P. ostreatus

Control

14

21

28

6.150.52

2.460.14d

1.280.24c

0.620.08ab

0.430.05a

1.570.33d
*

1.030.34cb

0.620.16ab

0.420. 07a

Within each row, means with the same letter are not significantly different (P0.05)
*Means within a column marked with asterisk differ significantly (P0.05)

remaining weeks of cultivation, the rate of decomposition of phenolic compounds was much
slower, and cultures of both fungal species practically did not differ in concentration of
sinapic acid. After 28 days of cultivation, the sinapic acid content of cultures of T. versicolor
and P. ostreatus decreased to 7.0 and 6.8 % of the original value, respectively. The results
correspond well with those obtained for P. ostreatus by Hu and Duvnjak [11], who
investigated the influence of solid-state fermentation on the total concentration of sinapic
acid esters in canola meal of varying moisture content, degree of the medium pulverization,
and the inoculum size. The maximum moisture of the medium used in their experiment was
lower than in this work (75 and 85 %, respectively), and the reduction in the percentage of
sinapic acid esters after the first week of cultivation seems to be slightly lower than the one
reported by us for sinapic acid. Rapeseed meal seems to be very well suited for biotransformation by white-rot fungi, as sinapic acid, present in high amounts in rape seeds, is an
excellent substrate for laccase, one of the most important ligninolytic enzymes [1719]. It is
very probable that many other antinutritional phenolics, such as condensed tannins, were
also efficiently decomposed during the experiment.
The solid-state fermentation of canola meal by both investigated fungi was accompanied by
a very significant increase of the extracellular activity of laccase (Figs. 2 and 3). The highest
activities of the enzyme were observed on the 21 and 28 day of growth of P. ostreatus and
T. versicolor, respectively. Our results concerning the dynamic of changes of laccase activity in

Fig. 2 Activity of extracellular laccase in cultures of T. versicolor and P. ostreatus. a cultures grown on
rapeseed meal; b control cultures, grown on Lindeberg and Holm liquid medium. Data are means SD from at
least three independent experiments

Appl Biochem Biotechnol

Fig. 3 Native PAGE of extracellular laccase from 14-day cultures
of T. versicolor (a) and P.
ostreatus (b) collected 14 days
after inoculation. Lane 1 a control
culture, grown on Lindeberg and
Holm liquid medium; Lane 2 a
culture with rapeseed meal

the cultures of P. ostreatus are quite similar to those noted by Hu and Duvnjak [11]. Moreover,
the maximum activity of laccase obtained in the cultures of T. versicolor was about 5 times
higher than in the case of P. ostreatus.
Fungal solid-state fermentation of rapeseed meal was also effectively applied to remove
other antinutrients. Rhizopus sp. can efficiently decompose glucosinolates, and species of
Aspergillus were used to decrease phytic acid level [2023].

Conclusion
From the presented results, it may be concluded that both investigated fungi, P. ostreatus and
T. versicolor, effectively degrade the antinutritive phenolics of rapeseed meal in solid-state
fermentation, causing a 6075 % reduction of sinapic acid content after 7 days of cultivation.
Moreover, the fungal growth was accompanied by the strong induction of extracellular laccase
activity, which makes rapeseed meal potentially useful in the biotechnological production of
this enzyme. Our experiment and the literature data [11] clearly suggest that rapeseed meal may
be used as an easily available, non-toxic and inexpensive substrate for a solid-state production
of ligninolytic enzymes by white-rot fungi. According to our best knowledge, this is the first
report concerning the application of T. versicolor in solid-state fermentation of rapeseed meal.
P. ostreatus appears potentially more useful in animal feed preparation, as it is an edible
mushroom and it decomposed distinctly more sinapic acid in the initial stage of cultivation
on rapeseed meal. On the other hand, T. versicolor may be more applicable in the production of
laccase, being a much more efficient producer of this enzyme.

Appl Biochem Biotechnol

Acknowledgments The authors would like to thank Prof. Jerzy Rogalski for his kind consent for the use of
fungal strains from the culture collection of the Department of Biochemistry, M. Curie-Skodowska University
in Lublin, Poland.

References
1. Bell, J. M. (1984). Journal of Animal Science, 58, 9961010.
2. Burel, C., Boujard, T., Tulli, F., & Kaushik, S. J. (2000). Aquaculture, 188, 285298.
3. Vioque, J., Snchez-Vioque, R., Clemente, A., Pedroche, J., & Milln, F. (2000). Journal of the American
Oil Chemists' Society, 77, 447450.
4. Vuorela, S., Meyer, A. S., & Heinonen, M. (2004). Journal of Agricultural and Food Chemistry, 52,
82028207.
5. Shahidi, F., & Naczk, M. (1992). Journal of the American Oil Chemists' Society, 69, 917924.
6. Khattab, R., Eskin, M., Aliani, M., & Thiyam, U. (2010). Journal of the American Oil Chemists' Society,
87, 147155.
7. Lacki, K., & Duvnjak, Z. (1999). Biotechnology and Bioengineering, 62, 422433.
8. Kirk, T. K., & Cullen, D. (1998). In R. A. Young & M. Akhtar (Eds.), Environmentally friendly
technologies for the pulp and paper industry (pp. 237307). New York: Wiley.
9. Shah, V., & Nerud, F. (2002). Canadian Journal of Microbiology, 48, 857870.
10. Isroi, Millati, R., Syamsiah, S., Niklasson, C., Cahyanto, M. N., Lundquist, K., & Taherzadeh, M. J.
(2011). BioResources, 6, 52245259.
11. Hu, J., & Duvnjak, Z. (2004). Engineering in Life Sciences, 4, 5055.
12. Smith, J. E., Rowan, N. J., & Sullivan, R. (2002). Biotechnology Letters, 24, 18391845.
13. Lindeberg, G., & Holm, G. (1952). Physiologia Plantarum, 5, 100114.
14. Thiyam, U., Stckmann, H., & Schwarz, K. (2006). Journal of the American Oil Chemists' Society, 83,
523528.
15. Lundell, T., Leonowicz, A., Rogalski, J., & Hatakka, A. (1990). Applied and Environmental Microbiology, 56, 35153520.
16. Jaszek, M., uchowski, J., Dajczak, K., Cimek, M., Grz, M., & Grzywnowicz, K. (2006). International
Biodeterioration & Biodegradation, 58, 168175.
17. Kwang-Soo, S., & Chang-Jin, K. (1998). Biotechnology Techniques, 12, 101104.
18. Lacki, K., & Duvnjak, Z. (1998). Biotechnology and Bioengineering, 57, 694703.
19. Koroleva, O. V., Gavrilova, V. P., Stepanova, E. V., Lebedeva, V. I., Sverdlova, N. I., Landesman, E. O.,
Yavmetdinov, I. S., & Yaropolov, A. I. (2002). Enzyme and Microbial Technology, 30, 573580.
20. Bau, H.-M., Villaume, C., Lin, C.-F., Evrard, J., Quemener, B., Nicolas, J.-P., & Mjean, L. (1994).
Journal of the Science of Food and Agriculture, 65, 315322.
21. Vig, A. P., & Walia, A. (2001). Bioresources Technology, 78, 309312.
22. Al-Asheh, S., & Duvnjak, Z. (1995). World Journal of Microbiology and Biotechnology, 11, 228231.
23. El-Batal, A. I., & Abdel Karem, H. (2001). Food Research International, 34, 715720.