Optimization of Solid-state Fermentation for Acidophilic Pectinase Production by Aspergillus niger Jl-15 Using Response Surface Methodology and Oligogalacturonate Preparation
Polygalacturonases that hydrolyzed the α-(1,4) glycosidic linkages of
pectin is one of widely used industrial enzymes and applied in food, feed, paper
and pulp, fruit juice and textile industries. To improve the production of extracellular
pectinase (PgA) by a newly isolated Aspergillus niger JL-15 strain, the
conditions of solid-state fermentation (SSF) were optimized by response surface
methodology (RSM). The maximum pectinase activity (525.70 IU g-1
dry fermentation product) was obtained at 12.10% orange peel powder, 3.20% ammonium
sulfate employing wheat bran as the solid substrate, 51.10% moisture content
and 75.00 h fermentation and was 4.10 times as high as that of the basic medium
(125.80 IU g-1). SDS-PAGE analysis showed that the molecular mass
of PgA was about 40.0 kDa. The PgA was optimally active at 45°C and pH 4.0
and was stable over a broader pH range (4.0-8.0). The Michaelis-Menten constant
(Km) and maximal velocity (Vmax) of PgA for citrus pectin
was 4.04 mg mL-1 and 40.16 μmol min-1 mL-1,
respectively. The enzyme mediates a decrease in the viscosity of pectin associated
with a release of small amounts of reducing sugar. High performance liquid chromatography
(HPLC) analysis revealed that PgA liberated a series of oligogalacturonate from
pectin with the digalacturonate (G2) and trigalacturonate (G3) as major products.
The mode of action study showed that the enzyme was an endo-acting polygalacturonase.
to cite this article:
Ming-Qi Liu, Rong-Fa Guan, Xian-Jun Dai, Lan-Fang Bai and Lin Pan, 2012. Optimization of Solid-state Fermentation for Acidophilic Pectinase Production by Aspergillus niger Jl-15 Using Response Surface Methodology and Oligogalacturonate Preparation. American Journal of Food Technology, 7: 656-667.
September 25, 2012; Accepted: November 16, 2012;
Published: December 28, 2012
Pectin is a complex polysaccharide and one of the major components of plant
cell wall and fruit lamella. Pectin represents about 1.5-3.0% wet weight of
orange peel which is an important by-product of the can and beverage industry
(Voragen et al., 2003). Polygalacturonic acid and
rhamnogalacturonan are the two fundamental constituents of pectin. The former
can possess up to 200 galacturonic acid residues, while the later is a hetero-polysaccharide
having arabinans, galactans and highly branched arabinogalactans attached at
the C4 position of many of the rhamnosyl residues. The hydrolysis of the pectin
involve several enzymes, such as pectate lyase (EC 188.8.131.52), pectin lyase (EC
184.108.40.206) exo-polygalacturonase (EC 220.127.116.11) and endo-polygalacturonase (EC
18.104.22.168) and the later was the most important one (Nikolic
and Mojovic, 2007; Wei et al., 2010). Endo-polygalacturonase
have attracted considerable research interest in recent years mainly due to
their widely application in food, treatment of wastewater, animal feed, paper
and pulp, fruit juice, textile industries (Jayani et
al., 2005). For example, there are many researches that reported production
of POS by enzymatic hydrolysis pectin from pectin-rich materials (Kashyap
et al., 2001; Elboutachfaiti et al., 2008;
Yadav et al., 2008).
Filamentous fungi belonging to the genus Aspergillus, with the specific
reproductive and growth characteristics, are well adapted to a large variety
of substrates, being excellent decomposers of vegetal material. Aspergillus
niger is used throughout the world for production of hemicellulase, cellulase,
pectinase and other enzymes (Krengel and Dijkstra, 1996;
Martens-Uzunova and Schaap, 2009; Heerd
et al., 2012). The solid-state fermentation (SSF) that carried out
in absence or near-absence of free liquid water between the medium is a complex
heterogeneous three-phase (gas-liquid-solid) and generally defined as the growth
of microorganisms, on the surface of a porous and moist solid substrate particle
in which enough moisture is present to maintain microbial growth and metabolism
(Figueroa-Montero et al., 2011). The solid-state
fermentation gained renewed interest in recent years in the production of many
enzymes due to lower operation costs and energy requirements and simpler plant
and equipment projects compared to submerged fermentation (SmF) (Pandey,
2003; Couto and Sanroman, 2006). In SSF process,
byproducts of agro-industry are generally considered as suitable substrates
for growth of fungi and the production of enzymes (Bayoumi
et al., 2008). There has been an increasing trend towards efficient
utilization and value-addition of agriculture-industrial residues. In 2009,
the total orange production was about 18.5 million tonnes and the orange peel
and the orange pomace residue was over 5.2 million tonnes in China. The optimal
design of the culture medium and is a very important aspect in the development
of SSF processes. Response Surface Methodology (RSM) is a very useful tool for
the optimal selection of nutrient, which can provide statistical models aid
in understanding the interactions among the process parameters at different
levels and calculating the best level of each factor for the given target (Bas
and Boyaci, 2007).
The purpose of the present study was to optimize pectinase production by Aspergillus
niger JL-15, a new enzyme producer, in SSF employing wheat bran as substrate
and orange peel powder as an inducer. The pectinase was partially purified and
characterized. The hydrolytic products released from citrus pectin and oligogalacturonides
by the pectinase were determined and quantified specifically.
MATERIALS AND METHODS
Materials: Citrus pectin, D-(+)-galacturonic acid, the standard oligogalacturonides
and Bovine Serum Albumin (BSA) were from Sigma Chemical Company in 2008. The
protein molecular weight marker was obtained from Transgen Biotech Company,
Beijing in 2008. Sephadex G-25 and SephacrylTM S-100 HR were from Amersham Biosciences
(2008). Pellicon-2 Mini Holder and hollow fibre ultra-filtration membrane modules
were from Millipore in 2007. All other chemicals used in the present study were
of analytical grade.
Microorganism and solid-state fermentation: The enzyme producer, Aspergillus
niger JL-15 strain, was isolated from Hangzhou Botanical garden soil. For
pectinase production Aspergillus niger JL-15 was cultivated in solid-state
fermentation (SSF). The basic medium contained wheat bran (9.80 g), MgSO4
(0.05 g), KH2PO4 (0.15 g) and H2O (12.00
g) and then sterilized at 121°C for 20 min. Aspergillus niger JL-15
was cultivated in a 250 mL flask containing 25.00 g medium at 30°C for 72
Optimization of PgA production via response surface methodology (RSM):
Optimization of SSF for PgA production focused on the concentration of orange
peel powder (inducer) and ammonium sulfate (added nitrogen source) employing
the wheat bran of basic medium, moisture content, fermentation time and their
interactions between each other. According to central composite design (CCD,
4-variable, 5-level), total 31 experiments were employed to fit the polynomial
Y = β0+β1X1+β2X2+β3X3+β4X4+β11X12+β12X1X2+β13X1X3+β14X1X4+β22X22+β23X2X3+β24X2X4+β33X32+β34X3X4+β44X42
where, Y is the dependent or response variable, β is the regression coefficient
and X is the coded level of the independent variables. Corresponding coefficients
of both variables and interaction variables were estimated by SAS 9.0 (SAS Institute
Inc., Beijing, China) while their response surface graphs were drawn by MATLAB
6.5 (Math Works, USA) (Dai et al., 2011; Bai
et al., 2011).
Statistical analysis on the significance of coefficient estimations was performed
via Students t-test and Fisher F-test. The optimum values of the selected
variables were obtained by solving the regression equation and also by analyzing
the response surface plots.
Partially purification and SDS-PAGE analysis of PgA: Three hundred grams
fermentation products were suspended in 3.0 L of McIlvaines
buffer (0.2 M sodium phosphate, 0.1 M citric acid, pH 6.0) with constant stirring.
The 3.0 L crude enzyme was concentrated to 500 mL using ultra-filtration module
with a polyethersulfone membrane (Millipore Biomax, 100 and 10 kDa cut-off)
and the trans-membrane pressure of 0.8 kg cm-2. The 500 mL concentrated
sample was treated with ammonium sulfate to 65% saturation to precipitate enzyme.
The saturated solution was left overnight at 4°C, centrifuged and precipitate
was resuspended in 25 mL of McIlvaines
buffer (pH 6.0). The fraction was loaded onto Sephadex G-25 column (Pharmacia,
20.0x2.4 cm) and eluted with McIlvaines
buffer (pH 6.0). The fractions containing polygalacturonase activity were pooled
and concentrated. The concentrated enzyme solution was then applied to SephacrylTM
S-100 HR column (Pharmacia, 60.0x1.6 cm) and eluted with the same buffer at
a flow rate of 1.0 mL/min. The active fractions were pooled for subsequent assays.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis
of the partially purified sample was carried out by using Bio-Rad electrophoresis
system. In the Laemmli system (Laemmli, 1970), the stacking
and separating gels consisted of 5 and 12% polyacrylamide, respectively. Proteins
were visualized with Coomassie brilliant blue R-250 straining. Molecular weight
of test protein was compared with standard protein marker.
Enzyme assays: The pectinase activity was assayed with 0.5% citrus pectin
(w/v) as substrate and the liberation of reducing sugars was estimated by the
dinitrosalicylic acid (DNS) procedure (Miller et al.,
1959) using the D-(+)-galacturonic acid as standard. Protein concentration
was measured by the dye-binding method of Bradford and Bovine Serum Albumin
(BSA) was used as the standard (Bradford, 1976). The
kinetic parameters for pectinase were calculated from initial velocities using
concentrations ranging from 1 to 10 mg mL-1 citrus pectin.
One unit of pectinase activity was defined as the amount of the enzyme that
catalyzed the formation of 1.0 μmol of reducing sugar from pectin in l
min under its optimal conditions (at 45°C, pH 4.0). For each assay in this
study, triplication measurements were conducted to obtain a mean value of activity.
Effect of temperature on the activity and thermal stability of PgA:
Effect of temperature on pectinase activity was measured from 30 to 80°C
at pH 4.5 (McIlvaines buffer).
Thermal stability of xylanase was determined by assaying residual activity after
incubation from 30 to 80°C at pH 4.5 for 4 min, respectively.
Effect of pH on the activity and stability of PgA: Effect of pH on pectinase
activity was measured over a range of pH 2.2 to 7.0 (McIlvaines
buffer) and 8.0 to 9.0 (Glycine-NaOH buffer) at 45°C. To determine the pH
stability, PgA was incubated in various pH buffers at 25°C for 1 h and the
residual activities were measured at 45°C and pH 4.5.
Changes in viscosity and reducing sugars concentration during hydrolysis:
The relationship between reducing sugars and viscosity of citrus pectin solution
in the presence of the PgA was determined as follows: a 250 mL reaction mixture
containing 3.0% pectin and purified enzyme (400.0 U) was incubated at 30°C
(Liu et al., 2010). The viscosity and the amount
of reducing sugar released were estimated at different time intervals.
Oligogalacturonate released by PgA from pectin: The 1.0% (w/v) citrus
pectin in McIlvaines buffer (pH 4.5) was hydrolyzed by the partially purified
PgA at 45°C for 2 h with constant shaking (100 rpm). The hydrolysis products
were analyzed by HPLC with Sugar-pakTM 1 column (6.5 mm diameter
and 300 mm length, Waters), pure water as mobile phase (0.5 mL/min) and injection
volumes of 10 μL. The column was maintained at 80°C. Sugar peaks were
screened using Waters 2410 refractive index detector (Sun
et al., 2007). The hydrolysates were quantified based on their own
The mode of action of PgA on oligogalacturonate: The mode of action
of PgA was determined using digalacturonate (G2) and trigalacturonate (G3) as
substrates. The standard oligosaccharides solutions (water, pH 7.0) were incubated
with purified PgA at 30°C. The samples at different intervals were determined
and quantified by HPLC. The injection volume was 10 μL.
RESULTS AND DISCUSSION
Optimization of PgA production by RSM: The concentration of added orange
peel powder and ammonium sulfate to basic medium, moisture content and fermentation
time had a significant effect on the PgA production (p<0.05). The Fisher
F-test with a very low probability value (p<0.0001) showed the high statistical
significance of the regression model. The goodness of fit of the model was checked
by the determination coefficient (R2 = 0.929), which indicates that
the following second order polynomial model could explain 92.9% of the total
variation (Table 1, 2). The model can be
shown as follows:
Y = 529.731+9.711X1+11.221X2+8.479X3+13.539X4+0.390X1xX2+14.155X1xX3+18.628X1xX4-
where, Y is the pectinase yield and Xi is the coded independent
variables (X1 and X2, concentration of added orange peel
powder and ammonium sulfate, respectively; X3, moisture content;
X4, fermentation time). Statistical optimization method for fermentation
process could overcome the limitation of classic empirical methods and was proved
to be a powerful tool for the optimization of the production of enzyme.
|| The variables in central composite design (CCD) and the PgA
|Coded levels (+2, +1, 0, -1, -2) and actual values (in parentheses)
of the independent variables in CCD. X1 and X2, the
concentration of orange peel powder and ammonium sulfate added to basic
medium, respectively, X3: moisture content; X4: fermentation
The application of RSM yielded the regression equation showing positive linear
and negative quadratic effect and expressing a relationship between the PgA
production and the independent variables.
The relation between factors and response can be understood by examining three-dimensional
response surface and contour plots as a function of two factors at a time and
holding all other factors at fixed levels. It was evident from the plots that
the addition of higher concentration of orange peel powder to wheat bran, the
middle levels of the ammonium sulfate and moisture content and the longer fermentation
time is responsible for the enhancement of PgA production (Fig.
1). Based on the analysis of regression equation and plots, optimum of the
four variables were found to be, the orange peel powder 12.10%, ammonium sulfate
3.20%, moisture content 51.10% and fermentation time 75.00 h, where the production
of 531.20 IU g-1 (dry fermentation products).
|| Analysis of variance and coefficient estimates of central
|| Response surface/contour plot showing effect of independent
variables (a) Orange peel powder (X1) and fermentation time (X4)
and (b) Initial moisture content (X3) and fermentation time (X4)
on PgA production with the other two variables at 0 level
The predicted yield was verified by performing an experiment with the optimized
variables in basic medium and the recovery of pectinase was 525.70 IU g-1
(dry fermentation products), which was close to the predicted one and was 4.10
times as high as that of the basic medium (125.80 IU g-1).
The RSM method was used for some similar topic. The Aspergillus niger
Aa-20 was cultured in solid-state bioreactor using lemon peel pomace as support
and carbon source. The maximum pectinase activity obtained at 96 h was 2181
IU L-1 maximum biomass (Ruiz et al., 2012).
||SDS-PAGE analysis of PgA, Note: Lanes M: standard protein
marker; Lane 1: crude enzyme; Lane 2: partially purified PgA after ultrafiltration
and chromatography on Sephacryl S-100 HR
The optimum operational conditions for maximum pectinase yield by Bacillus
subtilis RCK in SSF were moistened wheat bran (1:7 solid substrate-to-moisture
ratio), 1.5% (v/w) of 24 h old strain, 48 h fermentation and the predicted pectinase
value was 1610.6 IU g-1 dry substrate (Gupta
et al., 2008). Effects of cultivation time, pH and substrate concentration
on production of xylanase by A. niger AN-3 were studied. The optimal
fermentation parameters for enhanced xylanase production were cultivation time
(53.3 h), pH (7.92) and wheat bran concentration (54.2 g L-1). Under
these conditions the xylanase production was 127.12 IU mL-1 (Cao
et al., 2008).
Purification and kinetic parameters of PgA: The ultrafiltration, ammonium
sulfate precipitation and gel filtration resulted in a 9.10-fold increase in
specific activity of pectinase (from 27.60 to 251.20 U mg-1) with
25.20% recovery. SDS-PAGE revealed that the molecular mass of PgA was about
40.0 kDa (Fig. 2). Kinetic parameters that reflect the effect
of substrate concentration on the reaction velocity were depicted. The rate
dependence of the enzyme reaction on citrus pectin concentration followed the
Michaelis-Menten kinetics. The values for Km an Vmax were
of 4.04 mg mL-1 and 40.16 μmol min-1 mL-1,
respectively, which were lower than those of pectinase from Aspergillus oryzae
JL-14 (Liu et al., 2010) and higher than those
of pectinase from Bacillus subtilis JL-13 (Bai et
al., 2011). These values are consistent with the reported range of kinetic
values for microbial pectinase (Jayani et al., 2005).
Effect of temperature on the activity and stability of PgA: The pectinase
activity increased with the rise of temperature, reached the maximum at 45°C
and then decreased with the rise of temperature. Over 40°C, the PgA was
much less stable (Fig. 3a). The Tm of PgA was 51.9°C,
which was lower than many reported pectinase (Celestino
et al., 2006).
|| Relative and residual activities of optimum and stable (a)
Temperature and (b) pH of PgA, respectively
Effect of pH on the activity and stability of PgA: The PgA showed high
activity in a pH range of 4.0-5.0, with the optimal pH at 4.0 (Fig.
3b), which suggested it was a acidophilic pectinase. PgA was very stable
from pH 4.0 to 8.0. Over 80% of xylanase activity was retained after treatment
of the enzyme by preincubation over a pH range of 4.0-8.0 for 1 h at 25°C.
Decrease in viscosity and the release of reducing sugars from citrus pectin:
The PgA rapidly decreased the viscosity of pectin solution by 30.2 and 80%,
at 5 min and 45 min, respectively, but released a small amount of reducing sugars
(Fig. 4), which was similar to the endo-polygalacturonase
from the psychrophilic fungus Mucor flavus (Gadre
et al., 2003). The pectinase decreased the viscosity of pectin associating
with a release of only small amounts of reducing sugar.
Oligogalacturonate released by PgA from pectin: Pectin represents about
1.5-3.0% wet matter of orange peel, which is an important by-product of the
can and beverage industry (Voragen et al., 2003).
The major hydrolysis products released by PgA from citrus pectin were digalacturonate
and trigalacturonate (Fig. 5), which was similar to those
pectinase from Mucor flavus and Mucor rouxii NRRL 1894, respectively
(Gadre et al., 2003; Saad
et al., 2007). After 2 h reaction, the concentration of digalacturonate
and trigalacturonate in hydrolysis products from citrus pectin by PgA were 0.375
and 0.575 mg mL-1, respectively.
Oligogalacturonate that used as a functional food in many countries can suppress
the activity of entero putrefactive bacteria, shown to inhibit toxicity of Shiga-like
toxins from Escherichia coli O157:H7 and can selectively be used by the
beneficial gastrointestinal microflora, Bifidobacterium species. (Tomomatsu,
1994; Sako et al., 1999; Olano-Martin
et al., 2003). Reported beneficial effects of Bifidobacterium
spp. on human health include: preventing the proliferation of pathogenic intestinal
bacteria and facilitating the digestion and absorption of nutrients (Van
Loo et al., 1999).
||The changes in viscosity and reducing sugars from pectin solution,
The viscosity of reaction mixture was measured at 5 min interval by DV-II
prime viscometer (Brookfield, spindle: sp-2, speed: 100 rpm), 1.0 mL reaction
mixture withdrawn and the reducing sugars released were quantified using
||HPLC profiles of pectin degradation, HPLC analysis of hydrolysis
product from citrus pectin released by the PgA for 2 h, The positions of
galacturonic acid (G), digalacturonate (G2), trigalacturonate (G3) are shown
Oligogalacturonate can increase the Eubacterium rectale population
and butyrate levels, which is of potential benefit to the host (Mussatto
and Mancilha, 2007).
The mode of action of PgA on oligogalacturonate: The mode of action
of PgA was determined using digalacturonate and trigalacturonate as substrate,
which could be further hydrolyzed by the pectinase. The PgA showed very low
activity on digalacturonate and trigalacturonate (Fig. 6a,
|| Hydrolysis products of PgA incubated with (a) 1.05 mg mL-1
digalacturonate and (b) 1.5 mg mL-1 trigalacturonate at 40°C
for different time, At regular time intervals, aliquots of the reactions
were analyzed by HPLC for examining hydrolysis products
The presence of trace amounts of galacturonic acid from digalacturonate and
trigalacturonate by PgA revealed that enzyme preferentially cleaved the internal
glycosidic bonds of oligogalacturonate and it was an endo-acting pectinase.
In the study, the optimum parameters of SSF for pectinase production by A.
niger JL-15 were obtained using RSM method. The verifiable pectinase yields
were 525.70 IU g-1 dry fermentation products, which was 3.10 times
higher than that of the basic medium (125.80 IU g-1). Digalacturonate
and trigalacturonate were main hydrolysis products released from citrus pectin
by PgA. These results indicate the present method can be successfully used to
improve the pectinase production by A. niger Jl-15 and a potentiality
of PgA for the production of oligogalacturonate.
This study was supported by the foundation of the National Natural Science
Foundation of China (No. 31201831), the Science Technology Department of Zhejiang
Province (No. 2012C22079), the Zhejiang Provincial Natural Science Foundation
of China (No. Y3090503) and the Science Technology Department of Hangzhou city
(No. 20110232B81). The authors thank Dr. Shang-Wei Chen for his kind assistance
in HPLC analysis.
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