2.1 Isolation and screening of inulin- degrading fungal strains
The method of isolation was performed using the surface soil of the rotten wood of the mangrove detritus situated in the estuary of Halady river, on the Herikudru island, Kundapura Taluk (13°38'28"N, 74°42'01"E), India. Here, the mangrove area is mostly comprised of mangrove trees that have died due to urbanization and deforestation. The soil collection was done in July 2017. Firstly, a serial dilution of the soil sample was carried out followed by plating 0.1ml of the sample on the Czapex Dox Agar medium (CDA). CDA medium consists of (g/l): sucrose - 30, K2HPO4 - 1, NaNO3 - 2, KCl - 0.50, MgSO4 - 0.50, FeSO4 - 0.01 and agar - 15. It is used for the isolation of fungal species. The pH of the media was kept at 7.4 and autoclaved for 45min at 121℃ and 15-20 psi. The plates were incubated for 3-4 days at 30℃ to get diverse colonies of fungal species . In the process of primary screening, individual colonies from different fungal colonies were selected and plated aseptically on inulin media. The inulin media consists of (g/l): inulin - 6.00, Na2HPO4 - 33.9, NH4Cl - 5, NaCl - 2.50, KH2PO4 - 15, and agar - 15. The plates were incubated for three days at 30℃. The pH of the media was kept at 6.7 . Lugol’s plate assay was performed to screen the microorganisms producing extracellular inulinases .
2.2 Identification of the fungal strain
The fungal strain which gave the highest activity was recognized using 18s rRNA gene sequencing (⁓571bp) at National Centre for Microbial Resource (NCMR) Pune, India. Using the Tamura-Nei model, the evolutionary history was inferred by using the Maximum Likelihood method. The tree was constructed using 1000 bootstrap. The bootstrap support is 95 for the parent node. Tree construction method was selected based on the best model fitting option in MEGA6 .
2.3 Inoculum preparation
The fungal spores were totally removed from the agar plates using 0.05% of sodium chloride solution. A haemocytometer (Improved Neubauer, Rohem, India) was used to count the number of cells. For SSF, 2×106 spores/ mL of spore suspension was used .
2.4 Pretreatment of solid substrate
Stems of S. arundinaceum collected from Chitpady colony, Udupi, India were cleaned properly couple of times in running water. Small cubes of the stems were prepared and sun-dried for a couple of days to remove moisture. Subsequently, fine powder was made by grinding the dried cubes in a mixer grinder and passed through a 1.18mm sieve. After sieving, the particles with larger sizes were removed and the fine powder was used in SSF as a solid substrate.
2.5 Solid-State Fermentation
SSF was carried out by mixing 2g of S. arundinaceum powder and 5ml of inulin media in 250ml conical flasks. Moisture on a dry basis was maintained at 77.8%. All the flasks were tightly covered with hydrophobic cotton and autoclaved. The flasks were allowed to cool followed by the addition of 2ml of the spore suspension prepared earlier. Proper mixing of the solid substrate, fermentation media and the spore suspension was done to ensure the availability of nutrients uniformly. The flasks were incubated at 30℃ for five days . During the process of preliminary screening, it was found that the maximum production of inulinase occurred at 96th h. All the subsequent experiments were thus carried out for 96 h.
2.6 Extraction of Inulinase enzyme
For the extraction of enzyme after the completion of fermentation, the fermented contents were mixed with 30ml of sodium acetate buffer (0.1molar) having a pH of 4.75. The flasks were then kept for 1 h on a rotary shaker (180rpm) to extract the enzyme. Subsequently, a clean muslin cloth was used to filter out the enzyme extract followed by centrifugation at 844 G-force (10,000 rpm in rotor 9, Tabletop Superspin Centrifuge –V/FM, Plasto Crafts., Mumbai) at 4℃ for 10 min. The determination of reducing sugars was done by the dinitrosalicylic acid (DNS) method using the cell-free supernatant .
2.7 Inulinase assay
For the assay of inulinase enzyme, 2g inulin was added to 10ml of 0.1M sodium acetate buffer to make a substrate solution. Then the reaction mixture which consisted of 0.9 ml of substrate solution and 0.1 ml of the cell-free supernatant was incubated for 10 min, followed by the addition of 1ml of DNS reagent. The mixture was heated for 10 min in a water bath. For the blank, the enzyme was deactivated by preheating the reaction mixture. The quantity of enzyme which produces 1μmol of fructose per min is defined as one unit of inulinase activity.
Similar to inulinase assay, the invertase assay was determined using sucrose in the reaction mixture and the activity of invertase was determined with respect to the sucrose activity .
2.8 Inulinase to Sucrose activity (I/S ratio)
Usually, the inulinase activity is characterized from the activity of invertase using the I/S ratio. An I/S ratio higher than 10-2 proves to have a high production of inulinase whereas an I/S ratio lower than 10-4 proves to have a high production of invertase enzyme in the culture media .
2.9 Optimization of Inulinase Production
Proper understanding and former knowledge of bioprocess are necessary to obtain a model with accuracy. The data obtained from the experiments were studied using ‘‘Minitab 188.8.131.52”, a design software. Using Central composite design, a quadratic model was obtained. A fractional CCD 2(5-1) was used here and the significance level was kept at 95%. CCD consists of factorial trials and star points which assist the defining of quadratic effects. The centre points assess the pure variance with inulinase production as the output variable . The optimization of the five significant media constituents which increased the inulinase production was done using RSM. A set of thirty-two experiments were performed by studying the five components at 5 different levels, keeping zero as the central coded value. The experimental results of all the 32 experiments – the inulinase activity (Y) were recorded and submitted to ANOVA to carry out the analysis of data using multiple regression . Lastly, an empirical model showing the relationship between the independent components and the measured response was achieved.
2.10 Analysis of end products
The pattern in which inulinase enzyme works on inulin was figured out by thin-layer chromatography (TLC) analysis. To perform the reaction of hydrolysis, 1.0 ml of the cell-free supernatant was mixed with 0.01g of inulin. The reaction mixture was incubated at 56℃ for six h. Then, the reaction was stopped by boiling the reaction mixture for 10 min. Along with the sample test (T), four standards namely, deactivated inulinase (I), fructose (F), glucose (G), sucrose (S), were spotted on the TLC plates. It was then air-dried and dipped in a developer solution composed of butanol:ethanol:water (50:30:20). The plate was again air-dried and sprayed with diphenylamine reagent (1% diphenylamine, 10% phosphoric acid and 1% aniline). This reagent develops colors on reaction with sugars. Finally, the plate was kept in a hot air oven for 10 min at 120℃ for drying .
2.11 Seliwanoff’s test
To distinguish between aldoses and ketoses, Seliwanoff’s color test is carried out. Ketose sugar can be differentiated from aldose sugar by the formation of a burgundy or cherry red color with resorcinol on condensation, whereas aldose sugar doesn’t give any color . Heating for a long time is avoided as aldose sugars will also give a red color . To perform this test, three controls namely fructose, glucose and deactivated inulinase with inulin was used and a test sample containing the supernatant along with inulinase and inulin was used. 0.5 ml of the test sample was mixed with 1ml of Seliwanoff’s reagent and heated for 2 min. To 1ml of Seliwanoff’s reagent, 1ml each of the following, namely, standard fructose, standard glucose, and a pre-heated mixture of deactivated inulinase and inulin were also mixed and heated for 2 min.
2.12 Osazone formation test
Osazone test is performed to determine and identify the sugars qualitatively. It is a simple test in which sugars form distinctive crystals of osazone on reaction with phenylhydrazine. Distinctive broom and needle- shaped osazones are formed by monosachharides like glucose and fructose. The following test was carried out using the optimized cell-free liquid of inulinase and inulin mixture along with the fructose control. 2 ml of the control fructose and 2 ml of the supernatant were mixed with phenylhydrazine hydrochloride solution followed by heating for 30 min in a water bath . After cooling down of the solutions, the distinctive images of the osazone crystals were visualized by a light microscope (Euromex iscope, Holland) at 40x magnification.