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Optimization of Solid-state Fermentation for Acidophilic Pectinase Production by Aspergillus niger Jl-15 Using Response Surface Methodology and Oligogalacturonate Preparation



Ming-Qi Liu, Rong-Fa Guan, Xian-Jun Dai, Lan-Fang Bai and Lin Pan
 
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ABSTRACT

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.

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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.

DOI: 10.3923/ajft.2012.656.667

URL: https://scialert.net/abstract/?doi=ajft.2012.656.667
 
Received: September 25, 2012; Accepted: November 16, 2012; Published: December 28, 2012

INTRODUCTION

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 4.2.2.2), pectin lyase (EC 4.2.2.10) exo-polygalacturonase (EC 3.2.1.67) and endo-polygalacturonase (EC 3.2.2.15) 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 h.

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 model:

Y = β01X12X23X34X411X1212X1X213X1X314X1X422X2223X2X324X2X433X3234X3X444X42

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 Student’s 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 McIlvaine’s 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 McIlvaine’s buffer (pH 6.0). The fraction was loaded onto Sephadex G-25 column (Pharmacia, 20.0x2.4 cm) and eluted with McIlvaine’s 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 (McIlvaine’s 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 (McIlvaine’s 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 McIlvaine’s 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 standard curves.

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-
13.087X2xX3-17.733X2xX4-26.073X3xX4-33.254X12-51.925X22-33.744X32-55.763X42

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.

Table 1: The variables in central composite design (CCD) and the PgA production
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 time

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).

Table 2: Analysis of variance and coefficient estimates of central composite design
*p<0.05, **p<0.005

Fig. 1(a-b): 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).

Fig. 2: 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).

Fig. 3(a-b): 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).

Fig. 4: 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 DNS method

Fig. 5: 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, b).

Fig. 6(a-b): 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.

CONCLUSION

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.

ACKNOWLEDGMENTS

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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