Statistical Optimization of Media Components for Xylanase Production by Aspergillus spp. Using Solid State Fermentation and its Application in Fruit Juice Clarification

Xylanases are enzymes that convert xylan into xylose, xylobiose, and xylotriose. The present study deals with the production and optimization of xylanase through Solid-State Fermentation (SSF) using different agricultural wastes by Aspergillus spp. The Plackett Burman (PB) design was used to screen significant media components affecting the xylanase production. The carbon sources screened were wheat bran, rice bran, sugarcane bagasse, corn cob, and orange peel. The nitrogen sources screened were yeast extract, peptone, (NH4)2SO4, Na2NO3, and urea. Also, nine different salts such as KCl, MgSO4, Na2HPO4, CaCl2, FeSO4, ZnSO4, Na2CO3, KH2PO4, and NaH2PO4 which act as trace elements were screened. The results showed that wheat bran, yeast extract, Na2NO3 and KCl are the significant factors that affect xylanase production. A 3 3 Full Factorial Design (FFD) was performed to optimize the significant media components (wheat bran, KCl, yeast extract) obtained from PB design using Response Surface Methodology (RSM). Statistical analysis of results showed that wheat bran, KCl, yeast extract, and interaction between wheat bran and Original Research Article Patil et al.; JPRI, 33(54A): 151-166, 2021; Article no.JPRI.77068 152 yeast extract were found to be significant. The optimum concentration of wheat bran, KCl, yeast extract was 8 g/L, 0.1 g/L and 3 g/L. The Partial purification of xylanase was carried out using ammonium salt precipitation and dialysis. Gel filtration chromatography was performed to optimize the elution time, which was found to be 6 minutes. Application of xylanase in orange juice clarification was studied at 40 °C, 50 °C, and 60 °C. The optimum temperature obtained was 60 oC.


INTRODUCTION
Xylan is the key structural polysaccharide found in the plant cell walls, and it is the next most widespread in the environment after cellulose. Xylan is a heterogeneous polymer that consists of a linear β-(1,4)-D-xylose backbone, with side chains of α-D-glucuronosyl and α-L-arabinosyl units [1,2,3,4]. Xylanase (EC 3.2.1.8) is the key enzyme that depolymerizes the xylan molecules into monomers. Microbes utilize these monomers as a major carbon source [5,6,7]. Xylanase is of immense research importance because of its important applications in the field of biotechnology such as the production of ethanol from lignocellulosic waste materials, clarification of juices and beverages, and bread-making [8,9]. Xylanases are useful in the bio-bleaching of wood pulp and rayon production [10,11,12,13].
The enzyme production cost can be reduced by optimizing the process parameters and media components during the fermentation process [14,15,16]. The conventional approach of optimization is costly and time-consuming [17,18]. Hence statistical methods such as PB design are advantageous to study the effect of various parameters by performing a minimum set of experiments [19,20,21].
Agro-industrial waste is highly nutritious in nature and facilitates microbial growth. Most agricultural wastes are lignocellulosic in nature. Agricultural residues can thus be used for the production of various value-added products, such as industrially important enzymes. Agricultural waste can be therefore productively harnessed as a raw material for fermentation [22].
Solid-state fermentation (SSF) is preferred over Submerged Fermentation (SmF) because it requires less investment, less wastage of water, more product recovery, high product concentration, lower production cost, and simple cultivation equipment [23]. In SSF, various agricultural wastes such as orange peels, sugar cane bagasse, wheat bran, lemon peels and soya bran are used as substrates for fermentation [24,25]. SSF is used to produce various commercially important products, such as enzymes, fuels, pesticides, organic acids, secondary metabolites, and aromatic compounds [26,27,28].
The present study deals with screening of media components by PB design, statistical optimization of xylanase production by FFD and RSM, partial purification of the enzyme, and application of enzyme in orange juice clarification.

Isolation of Xylanolytic Microbes
Several soil samples from different locations of Hubballi and Dharwad were collected and processed to isolate microorganisms using the standard microbiological spread plate method. The media used for isolation was xylan agar consisting of (g/L): Xylan from Birchwood, 5.0; yeast extract, 5.0; peptone, 5.0; K 2 HPO 4 , 1.0; MgSO 4 .7H 2 O, 0.2 and agar,20.0. Different bacterial and fungal strains were isolated and those capable of producing xylanase were screened on xylan agar media for xylanolytic activity.

Screening for Xylanase Producing Strain
Screening of the bacterial and fungal strains for their ability to produce xylanase was carried out by the point inoculation method. The media used was Czapek's agar which contains xylan as the main carbon source. After inoculation the plates were kept for incubation for 48 h at 37 ᵒC for bacterial cultures and seven days at 30 ᵒC for fungal cultures. To observe zone of clearance formed, 0.1% (w/v) Congo Red was flooded on the plates and incubated for 30 min. Further it was washed with 1 M NaCl. The results indicated that fungal strains showed a higher xylanase activity when compared to bacterial strains and therefore were selected for the production of xylanase by SSF.

Selection of Fungal Strain
Two fungal strains P11 and P15, were considered for SSF. SSF was carried out using wheat bran (substrate) and varying incubation periods. The results indicated, P15 fungal strain was a potent producer of xylanase compared to P11 and therefore was considered for further studies.

Xylanase Production by SSF
SSF was conducted by adding carbon sources, nitrogen sources, and salts as per the high and low values generated in the PB design ( Table 1). The moisture content was set to 80% using salt solution. After sterilizing the flasks, an inoculum of 1% was added aseptically, and it was incubated at 27ᵒC for five days. Substrates in SSF: Wheat bran, sugarcane bagasse, rice bran, corncob and orange peel were taken as substrates. These substrates were cut into small pieces and ground to fine powder of maximum particle size limit of 2 mm and dried at 60±5ºC for 24 h.

Enzyme Extraction and Enzyme Activity
The incubated flasks were treated with a volume equal to ten times the substrate mass of 0.1% tween 80 solution and kept in an orbital shaker for 3h at 100 rpm. It was then filtered with a muslin cloth. The filtrate obtained was then centrifuged at 8000 rpm for 12 minutes. The supernatant collected was used for the estimation of xylanase activity and protein concentration. The enzyme assay was done using DNS (3, 5-dinitrosalicylic acid) method. Birchwood xylan (1%) added in 0.1M Phosphate buffer (pH 5.0) was used to determine crude enzyme activity. The substrate along with the enzyme was incubated at 37˚C for one hour. The color developed was estimated at 540 nm using a Spectrophotometer (make: Elico). One unit (U) of enzyme activity is expressed as the amount of enzyme releasing 1mmol of reducing sugar equivalent per minute under the assay conditions. The Protein concentration was estimated using Lowry's method. The standard used was Bovine Serum Albumin (BSA). The color developed was measured at 660 nm with the help of a Spectrophotometer.

Optimization by Response Surface Methodology
The significant factors obtained from PB design were selected and used for optimization using RSM [30]. The optimal conditions for the enzyme production were determined using 3 3 full factorial design with 27 experimental runs. Enzyme activity (U/mL) was chosen as a dependent variable for this study, whereas wheat bran, yeast extract and KCl were considered independent variables whose levels are shown in Table 2.

Partial Purification of Xylanase
Ammonium sulfate precipitation: Ammonium sulfate (85% saturation) was added to 50ml of the supernatant and left overnight. The next day, the supernatant was centrifuged at 8000rpm for 15 minutes to get the precipitate. The above procedure was performed at 4°C [31].

Dialysis:
The precipitate obtained after ammonium sulfate precipitation was suspended in 0.05M potassium phosphate buffer (pH: 7.0) and then dialyzed against the same buffer at 4ºC for about 3 h on a magnetic stirrer by changing the buffer every hour [31].
Gel filtration Chromatography: The dialyzed sample (5 mL) was chromatographed on a sephadex G-50 column equilibrated, and eluted with 0.05 M phosphate buffer of pH 7.0, flowing at 0.375 mL/h. The protein content of the fractions (1.5 mL) was estimated using spectrophotometer at 280 nm. The DNS method was used to determine the xylanase activity [31].

Application of Xylanase in Fruit Juice Clarification
The clarification process of orange juice was performed using xylanase. A comparative fruit juice clarification study was performed using crude enzyme, purified enzyme, and standard commercial xylanase enzyme. The extracted orange juice was filtered using a muslin cloth. To determine % clarification at various temperatures, the enzyme, and the orange juice were mixed in the ratio of 1:10. The clarification was studied at 40, 50 and 60 °C for 90 minutes. Further, the samples were heated in a water bath at 100 o C for 5 minutes for inactivation of the enzyme. After boiling, the samples were centrifuged at 8000 rpm for 15 min. The supernatants were studied for yield, clarity and reducing sugar. The juice clarity was determined using a spectrophotometer at 660 nm. The juice yield was determined by measuring its volume after centrifugation. DNS assay was used to determine the reducing sugars. Untreated fruit juices were considered as control [32].

Screening of Fungal Strains
Based on the batch studies, the xylanase enzyme production by P11 and P15 strains were found to be higher on the 7 th day when compared to 5 th and 6 th day. The strain P15 showed higher xylanase production compared to P11 strain on 7 th day. Thus P15 strain was considered to be a potent producer of xylanase and was used for further studies. The results of which are represented in Fig. 1.
The screening of the isolates was performed by estimating the zone of clearance formed on xylan agar plates. The results of which are depicted in Fig. 2.

Screening by Placket Burman Design
The variables significantly affecting the response with two-factor interactions were analyzed in PB design. The design matrix with the response (Enzyme Activity) is shown in Table 2. The Pareto chart shows the effect of the media components on enzyme activity. The results indicated that except MgSO 4 , NaH 2 PO 4 , sugarcane, and (NH 4 ) 2 SO 4 all other components have a significant effect on xylanase activity, as shown in Fig. 3. Among them, wheat bran (Pvalue 0.006), yeast extract (P-value 0.011), KCl (P-value 0.008) showed a major effect on xylanase production, as shown in Table 3. Xylanase activity was found to be in the range of 15.792 U/mlto 30.66 U/mL. Similar results were obtained by other researchers [33,34].
The maximum enzyme activity observed from screening, the process parameters was found to be 30.66 U/mlwhich was the 10 th run consisting of wheat bran, yeast extract, peptone, urea, Na 2 HPO 4 , CaCl 2 , FeSO 4 , ZnSO 4 K 2 HPO 4 . In addition, the R 2 value predicted was 99.15% which indicates the model is of good quality.

Optimization by Response Surface Methodology
In order to optimize the media components, three factors were considered. 3 3 experiments were performed to check the effect on xylanase activity. The Full factorial design matrix with the response (Enzyme Activity) is shown in Table 4. After performing the full factorial design, P-Value was analyzed to check the effect of factors. The factors were considered to be significant whose P-Value is less than 0.15. ANOVA Table 5 shows the following P-Values (wheat bran: 0.096, KCl: 0.000, Yeast extract: 0.14). The highest xylanase activity of 26.15 U/ml was observed for 13 th run, which consisted of wheat bran and yeast extract at mid-levels of 8 g/L and 3 g/L respectively. While KCl is at a low level of 0.1 g/L. The results indicated that wheat bran significantly affects xylanase production at mid-level and decreases at high and low levels. KCl significantly affects xylanase production at a low level, and its effect decreases at a high level. Yeast extract significantly affects Xylanase production at midlevel, and its effect decreases at high and low levels.
The result showed an average optimum enzyme activity of 27 In the present study Pareto chart results indicate that wheat bran potentially affects the xylanase activity compared to KCl and Yeast extract. And the interaction effect between wheat bran and Yeast extract are found to be more significant than the interaction effect between Wheat bran and KCl. Similar results were obtained by other researchers [35,36].    Table 5, the following inferences were drawn. The regression model is significant (P-value < 0.15 level of significance). All the main effects and interaction effects were significant except the interaction of KCl *Yeast Extract (P-value< 0.15 level of significance). The third order interaction between Wheat bran* KCl *Yeast Extract is found to be insignificant. The adequacy of the model can be verified by the coefficient of determination. From the regression analysis, the coefficient of determination(R 2 ) was 94.96% (0.9496) which is very close to 1. This indicates 94.96% of total variability is explained by the regression model. The adjusted R 2 value is 92.96% and the predicted R 2 value is 85.49%.
Through main effect plots (Fig. 6), it was observed that as the quantity of wheat bran increases from low (2 g/L) to mid-level (8 g/L) the enzyme activity increases from 14 U/mlto 23 U/mland then decreases to 15 U/mlas wheat bran is further increased to 14 g/L. Similarly, it was observed that as KCl increases from low (0.1 g/L) to mid-level (1.1 g/L) the enzyme activity decreases from 20 U/mlto 17 U/mland then further decreases to 15 U/mlas KCl is further increased to 2.1 g/L. Similarly, it was observed that as yeast extract increases from low (0.5 g/L) to mid-level (3.0 g/L) the enzyme activity increases from 17.5 U/mlto 19.5 U/mland then decreases to 16.5 U/mlas yeast extract is further increased to 5.5 g/L. From the Interaction Plot (Fig. 7) it can be inferred that there is considerable interaction among the media parameters (wheat bran* Yeast extract). Similar results were obtained from other workers [37,38].
From contour plots, as shown in Fig. 8(a) it was observed that maximum enzyme activity was seen for a low level of KCl and mid-level of wheat bran. Similarly, in Fig. 8(b), maximum enzyme activity was observed at the mid-level of yeast extract and wheat bran. In Fig. 8 (c), the maximum enzyme activity was observed at midlevel yeast extract and low level of KCl.

. Counter plots representing interaction effect of (a) KCl and Wheat bran, (b) Yeast extract and Wheat bran, (c) Yeast extract and KCl
3D surface plots as shown in Fig. 6, were plotted to study the interaction effects of media components on enzyme production. In surface plots, the vertical axis represents Enzyme Activity (EA), and two horizontal axes represent the levels of two independent variables, keeping other variables at their control level. From the plots, it can be inferred that there is a nonlinear effect of the factors on enzyme activity.

Model Validation
Experimental validation of the regression model was performed by carrying out experiments at the optimum settings predicted by the RSM optimizer as shown in Fig. 10. The experiments were performed in triplicates as per the optimum settings as shown in Table 6 and enzyme activity was found to be 26.15 U/mL, which is in good agreement with the model predicted value of 27.43 U/mL. Hence Model is validated as model output is in line with observed data.

Ammonium sulfate precipitation
The enzyme produced in solid-state fermentation was purified by ammonium sulfate fractionation followed by dialysis. The results indicated that maximum enzyme activity after dialysis was 3.231 U/mlwith specific activity of 0.0237 U/μg.

Gel filtration chromatography
Purification of xylanase was performed by gel filtration chromatography on Sephadex G-50. Atthe 6 th minute the maximum xylanase activity was obtained. The activity was high for volume fraction 2 to 7 as shown in Fig. 11. Hence it was concluded that the elution time of 6 min is ideal for the purification of the xylanase enzyme.

Application of xylanase in fruit juice clarification
The orange juice clarification carried out at 60°C liberated high reducing sugar for pure enzyme (10.477 µM/mL) and higher %clarity was achieved at 60°C for pure enzyme (85%). The results indicated that the samples treated with pure enzyme showed maximum % clarity and high reducing sugar were liberated. The results of which are indicated in Table 8.

CONCLUSIONS
In the present work, optimization of xylanase production was carried out using solid state fermentation by PB Design and RSM-FFD. The applied Statistical tools proved to be efficient for optimizing xylanase enzyme production by locally isolated Aspergillus species. Plackett-Burman design was used to test the relative importance of medium components on xylanase production. Among the several variables, Wheat bran, Rice bran, Sugar cane bagasse, Corn cob, Orange peel, Yeast extract, Peptone, Na 2 NO 3 , Urea, KCl, Na 2 HPO 4 , CaCl 2 , FeSO 4 , ZnSO 4 , Na 2 CO 3 and KH 2 PO 4 were found to be significant. Response surface methodology using Full Factorial design was proved to be a powerful statistical tool for optimization of media components for the enhanced production of xylanase. Maximum production of enzyme was obtained with the media composition of wheat bran (8.4 g/L), KCl (0.1 g/L) and yeast extract (2.5 g/L). Under optimal conditions of independent variables, the experimental responses showed close agreement with predicted responses, confirming the validity of regression model. Partial purification of xylanase produced was carried out using ammonium sulfate precipitation and dialysis. Application of xylanase in orange juice clarification was studied and optimum temperature was found to be 60ºC.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

University,
Hubballi for their constant encouragement and support in carrying out this research work.