IMPACT of PARTICLE SIZE of WHEAT BRAN on the PRODUCTION of POLYGALACTURONASE by SOLID STATE FERMENTATION USING Aspergillus Tubingensis Dr
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e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science Volume :02/Issue :10/October -2020 Impact Factor- 5.354 www.irjmets.com IMPACT OF PARTICLE SIZE OF WHEAT BRAN ON THE PRODUCTION OF POLYGALACTURONASE BY SOLID STATE FERMENTATION USING Aspergillus tubingensis Dr. Viral N. Patel*1, Dr. Samir C. Parikh*2 *1Department of Microbiology, Smt. L. M. Shah Science College, Radhanpur, Gujarat, India *2 Department of Microbiology, Smt. S. M. Panchal Science College, Talod, Gujarat, India ABSTRACT During the study of polygalacturonase production using various physico-chemical factors, it was observed that agro residues such as wheat bran when used with different particle sizes shows great impact on the production of the enzyme. It was observed that particle size range of 300-500µ was found most suitable for fermentative production of polygalacturonase using Aspergillus tubingensis under SSF. Small to moderate particle size of the substrate allows for the optimized production of the pectic enzyme, such as polygalacturonase. Keywords: wheat bran, pectinase, solid state fermentation, Aspergillus tubingensis I. INTRODUCTION Polygalacturonases, more broadly categorized under pectinases, have got widespread applications in the clarification of apple juices (Rombouts and Pilnik, 1986). It is also widely used in fruit juice industries for the clarification of orange juices and cloudy lemon juices. The production of this enzyme using solid state fermentation always requires a lot of physico-chemicals factors to be taken care of. One such important factor is the particle size of the SSF substrates used for the production of this enzyme. Wheat bran being widely used as the substrate of choice for SSF production of enzymes, it become highly necessary to explore this enzyme for its role at different particle sizes. In the present study, we have used different mesh sizes to separate wheat bran particles on the basis of their size and have used them in this experimental work. While simply changing the particle sizes of wheat bran in each sets, we have tries to keep all the other parameters constant so as to check the actual effect of the particle size of wheat bran on the fermentative production of polygalacturonase. II. MATERIALS AND METHODS Wheat bran was the solid substrate used for the formulation of media for solid state fermentation process. The particle size of the solid substrate used was also tested for the maximum enzyme production. The different particle sizes of the locally purchased wheat bran were prepared by sieving it through standard mesh sizes. For control, the wheat bran purchased was used as is. The different mesh sizes used were 300-425µ, 425-500µ, 500- 600µ and 600-850µ. Production of Pectinolytic Enzymes in SSF: 10 grams of the pretreated and dried wheat bran (previously sorted by different particle sizes) was taken in a 100 ml Erlenmeyer flask or even using sterile glass petri plate and it was then moistened with salt solution to keep the moisture content at a level of 70% (Acuna-Arguelles et al., 1995). Then all the flasks were autoclaved at 120°C at 10 lbs for 15 minutes in order to prevent degradation of pectin. The flasks were then cooled and inoculated with 2.0 X 107 spores / gram dry matter and incubated at 28°C for 3-5 days. Recovery of Pectic Enzyme: Polygalacturonase: After the fermentation period of 72 hours, the fermented media was extracted with 40 ml of 0.05 M Sodium acetate buffer (pH- 5.0). For the extraction process, the flasks were shaken at 150 rpm for 30 minutes at 25° C and kept for one hour before they were filtered through muslin cloth. The extract was then centrifuged at 8,000 rpm in a centrifuge (REMI Research Centrifuge, Model No.: R-24) and the supernatant was then filtered through Whatman No.1 filter paper adjusted into a glass filtration assembly to remove the fungal spores completely. The filtrate was then used for enzymatic assays. The activity of polygalacturonase enzyme is mentioned as U/gds (Units/gram dry substrate) Assay of Polygalacturonase Activity: The activity of Polygalacturonase was measured by determining the amount of reducing substances released according to the method outlined by Nelson (1944) and Somogyi (1952). www.irjmets.com @International Research Journal of Modernization in Engineering, Technology and Science [729] e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science Volume :02/Issue :10/October -2020 Impact Factor- 5.354 www.irjmets.com Definition of Unit Activity: One unit of polygalacturonase activity is defined as the amount of enzyme that converts substrate into one micro mole of galacturonic acid/ml/minute under the standard assay conditions. Reagents: 0.2 M Tris acetate buffer (pH 4.5), 0.01 M CaCl2, Polygalacturonic Acid (Sigma Aldrich) 1%, Alkaline copper tartarate reagent (Solution A: Dissolved 2.5 gm of anhydrous Sodium carbonate, 2 gm of Sodium bicarbonate, 2.5 gm of Potassium sodium tartarate and 20 gm of anhydrous Sodium sulphate in 80 ml of water and made up the volume to 100 ml.; Solution B: Dissolved 15 gm Copper sulphate in a small volume of distilled water. Added one drop of sulfuric acid and made up the volume to 100 ml; Prepare fresh and mixed 4 ml of solution B and 96 ml of solution A before use.), Arsenomolybdate solution (Dissolve 2.5 gm Ammonium molybdate in 45 ml water. Added 2.5 ml Sulfuric acid and mixed well. Then add 0.3 gm of Sodium hydrogen arsenate dissolved in 25 ml water. Mixed well and incubated at 37 C for 24-48 hrs.), Polygalacturonate Standard Solution: (Galacturonic acid monohydrate (Fluka) 1.0%) Procedure: 1. The assay mixture was prepared with the following components: • 0.2 ml enzyme • 0.2 ml 0.2 M tris-acetate buffer (pH 4.5) • 0.1 ml 0.01 M CaCl2 • 0.5 ml of 1.0% solution of polygalacturonic acid (PGA) 2. Prepared a blank for each sample by boiling the reaction mixture before the addition of the substrate. 3. Incubated at 37C for 1 hour. 4. Stopped the reaction by heating at 100C for 3 minutes. 5. 0.5 ml of the solution mixture was taken and analyzed for reducing sugars by Nelson-Somogyi Method. 6. Made up the volume in both sample and standard tubes to 2 ml with distilled water. 7. Pipetted out 2.0 ml of distilled water in a separate tube to set up a blank. 8. Added 1.0 ml of Alkaline copper tartarate reagent and kept for 10 minutes. 9. Cooled the tubes and added 1.0 ml of arsenomolybdate reagent to each of the tubes. 10. Made up the volume in each tube to 10 ml with distilled water. 11. Read the absorbance of blue colour at 620 nm after 10 minutes in a Spectrophotometer. (Ramchandran, Sandhya 2005) All the experiments were conducted in triplicate and the mean values of all the sets of observations were taken for evaluation of the experimental results. III. RESULTS AND DISCUSSION Results of the effect of particle size on the production of polygalacturonase by Aspergillus tubingensis are shown in Table-1 and Graph-1 below. Maximum polygalacturonase production by Aspergillus tubingensis was observed at the smallest, 300-425µ particle size of wheat bran. Enzyme activity in control was found greater than that of 500-600µ and 600-850µ particles sizes in the experiments. Table – 1: Effect of particle size of wheat bran on polygalacturonase production by Aspergillus tubingensis Particle Size of Wheat bran Polygalacturonase Activity (U/gds) Mean ± S.D. (Micron) Control 15.647 0.655 300-425 31.993 0.685 425-500 18.007 0.378 500-600 8.600 0.561 600-850 3.847 0.365 During the study of various physico-chemical factors, particle size of the solid substrate was found to be an important critical factor for the growth and product formation by the fungi. Smaller substrate particles provide larger surface area for the fungal attack and may have a desirable effect on the production of enzyme (Pandey et al., 1999). www.irjmets.com @International Research Journal of Modernization in Engineering, Technology and Science [730] e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science Volume :02/Issue :10/October -2020 Impact Factor- 5.354 www.irjmets.com Polygalacturonase Activity (U/gds) 35 30 25 20 15 Enzyme activity (U/gds) 10 5 0 Control 300-425 425-500 500-600 600-850 Particle Size (Micron) Graph-1 Effect of different particle sizes of wheat bran on polygalacturonase production by Aspergillus tubingensis As per our observation, smaller particle size of the wheat bran may prove to be one of the reasons for better polygalacturonase activity. However, too small size of the substrate particles may lead the substrate to clump with each other that may further interfere with the microbial respiration and aeration during the fermentation process and therefore result in poor microbial growth. On the contrary, larger wheat bran particles facilitate for better and efficient microbial respiration and aeration, but provide limited surface area for microbial attack. This in turn necessitates a compromised particle size for a particular process (Pandey et al., 1999) IV. CONCLUSION For solid state production of polygalacturonase using Aspergillus tubingensis, it was observed that the smaller to moderate the particle size (300-500 Micron), the better the production of polygalacturonase as opposed to very small or very large particle size of the wheat bran. Small to moderate particle size of the wheat bran allows better utilization and bioconversion of the substrate for the production of the polygalacturonase enzyme. On the other hand, too much small or too much large particle sizes of the wheat bran may also be affected by various other experimental parameters such as ability to retain the moisture content, that may further affect the growth and colonization of the fungus and in turn impair the production of enzyme.