Puri ication, Characterization, Optimization and Evaluation of Bioactivity Potential of Exopolysaccharides of Curvularia Lunata

Prakash P1, Aishwarya B1, Poornimaa B1, Shaik Asma Taj1, Narendrakumar G1, Antony V Samrot*2, Abirami S3, Jane Cypriyana P J1 1Department of Biotechnology, School of Bio and Chemical Engineering, Sathyabama Institute of Science and Technology, Semmancheri, Chennai-600119, Tamil Nadu, India 2School of Bioscience, Faculty of Medicine, Bioscience and Nursing, MAHSA University, Bandar Saujana Putra, Selangor-42610, Malaysia 3Department of Microbiology, Kamaraj College, CGE Colony, Thoothukudi628003, Tamil Nadu, India

rides secreted by microorganisms into the surrounding environment. These polysaccharides have tremendous application in pharmaceutical industries. For the past two decades, exopolysaccharides of fungus have been known to have high-value biomacromolecules. In most of the studies, sugar compositions of fungus Exopolysaccharaide and variation in molecular weight depend upon culture medium composition and different physical conditions that take place during the process of fermentation. In this current study, the selection of nutrients for the EPS production from Curvularia lunata under submerged batch-culture conditions was made using the Plackett -Burman design matrix method. Subsequently, the optimal condition was determined using Response Surface Methodology (RSM). Thus, produced EPS was characterized by GC-MS and thermogravimetric analysis. It was found to have glucose and mannitol and also showed good thermal stability. EPS was subjected to DPPH assay for analyzing antioxidant activity. Free radical scavenging activity was observed by subjecting the sample to the ABTS method and anticancer activities were tested against HeLa cells. EPS was found to be having a good antioxidant activity, Free radical scavenging activity and showed effective anticancer activity against HeLa cells.

INTRODUCTION
Extracellular polymeric substances are bounteous in our environment are produced by a variety of prokaryotes (bacteria) and eukaryotes (fungi, algae, and plants). The exact knowledge on the application value of this compound in various ields was established only in recent decades. Exopolysaccharides (EPS) are excreted by numerous microbes upon fermenting number of diverse carbon sources. Most EPS are obtained from seaweeds (agar, alginates and carrageenan) (Shukla et al., 2019). A substantial number of Gram-positive and Gram-negative bacteria, yeasts and many fungi produce EPS fermenting glucose, sucrose, molasses, hydrolyzed starch, methanol and different hydrocarbons (Verma et al., 2020). These EPS are carbohydrates, they also contain proteins, DNA, and glycolipids .
Certain extracellular polysaccharides produced from microalgae possess several bioactivities such as anti-tumour (Xie et al., 2020), antiin lammatory (Sun et al., 2020), and antiviral activity (Barbalace et al., 2019) that contributes to potential prospects for pharmaceutical applications (Philippis et al., 2011). Microalgae are known to yield a different type of exopolysaccharide. These EPS reported in yeasts and fungi are very less, though they contribute to interesting pharmacological properties. There are reports on EPS produced by several fungi and its potential bioactivity like antibacterial, anticancer and antioxidant activity (Cui et al., 2019;Rajoka et al., 2020), but the reports are meagre. The fungus Curvularia lunata is known to occur on a wide range of hosts including black gram (Lal et al., 2013) and it was selected in this study to investigate the potential role of EPS secreted by Curvularia lunata as a potential bioactive agent. Thus, the production of EPS by C.lunata was optimized by RSM and the produced EPS was characterized and utilized for in-vitro bioactivity studies.

Isolation, extraction and identi ication of fungal species producing EPS
10 g of soil was collected from different area and were enriched with fruit wastes (especially banana waste), vegetable wastes etc. From these sources, fungal organisms were isolated by pour plate method on PDA (potato dextrose agar) (Hertwig et al., 2020). The pure culture was inoculated to PDB (potato dextrose broth) and incubated for 72 h. The medium further subjected to centrifugation at 3000 rpm for 15 min and the acquired pellet was discarded. EPS was extracted as precipitate by adding an equal amount of ice-cold ethanol, and obtained solution was subjected for Anthrone test for carbohydrate presence. The fungal organism that produced more EPS was subjected for identi ication by LPCB staining method, 18S rRNA sequencing and was used for further study.

Plackett-Burman Design (PBD)
The medium components such as date syrup, jaggery, yeast extract, KH 2 PO 4 , K 2 HPO 4 , MgSO 4 , CaCl 2 were screened (Table 1) using PBD. After 4 days of the incubation period, the grown culture was iltered and the iltrate was subjected for solvent precipitation method for extracting EPS. The obtained EPS was estimated by the Anthrone method. The basic design was utilized for the current experimental studies (8 runs) in which three replicates and randomized experiments were used.
Determination of the main effect of the experiment was observed by the difference between the mean of H (high) and L (low) responses for each independent and dummy variable which was expressed as, Equation (1) The in luence of an independent variable on the response is determined by the difference between the mean response of four experiments at the high level and the mean value for four experiments at the low level and it is expressed as, Equation (2 The error in the experiment was determined by taking an average of the mean squares of the dummy effects of E and G ( Table 1) that is expressed as Equation (3). The last step is to detect the factors displaying large effects. F-test (Factor mean square/error mean square) was applied for the above experiment.
The regression coef icient, F value and P-value of the factors were determined for biomass production using statistical design.

Medium Optimization by Response surface methodology
The selected variables using PBD were further characterized using RSM. The high and low actual values are displayed in Table 2.

Characterization of EPS
The produced EPS was subjected to FTIR analysis using Shimadzu Af inity 1s. Thermal stability was analyzed by Thermogravimetry analysis, GC -MS Analysis was performed using Shimadzu GC-2010 Plus gas chromatography.

Determination of antioxidant activity
The impact of given samples on DPPH radical was assessed according to the method described by Gadow et al. (1997). The percentage inhibition of the DPPH radical by the samples was estimated . ABTS method was done for the samples to ind its scavenging ability (Khammuang and Sarnthima, 2008). Hydroxyl radical (Selvarani et al., 2019), H 2 O 2 (Ngonda, 2013)and Superoxide scavenging activity (Tang et al., 2017;Qi et al., 2006) for fungal EPS was performed.

Anticancer activity
Addition of 10000 cells/ 20 µL cell suspension (HeLa cells / MCF cells) to a 96 well culture plate was done followed by incubation at 37 0 C in a humidi ied CO 2 incubator with 5% CO 2. The cells were visualized under an inverted tissue culture microscope following 48h of incubation. With 80 % conluence of cells, cytotoxicity assay was performed along with different concentrations of EPS sample (0 to 200 µg/mL). Following 48h of incubation, MTT was added to the wells and left undisturbed in room temperature for 3h. The substances present in the well were removed using a pipette and 100 µL 10% SDS in DMSO was included to disperse the formazan crystals. The absorbances were studied using the Read Well Touch microplate reader at 570 nm (Mosmann, 1983).

Isolation, extraction and identi ication of fungal species producing EPS
Various fungal species were isolated and screened; amongst all, a black colour mold fungus species was producing more amount of EPS (Table 3). The black mold species were identi ied by staining method and 18S rRNA sequence as Curvularia lunata (Gen Bank Accession number is MK 646037).
EPS are believed to be involved in enhancing the production of enzymes by acting as their inducing substrates. Extracting the EPS from the medium involved many procedures such as heating at high temperature (D' Abzac et al., 2010), centrifugation and iltration process etc (Nielson and Jahn, 1999). All the processes are costly or require special equipment. Choosing a proper solvent system to extract would reduce the cost and time; thus, the extraction system in this study was ethanol. Table 4 illustrates the output of 8 runs. Thus, the optimal medium condition was obtained through the Plackett Burman model.

Response Surface Methodology
The current study emphasizes on optimizing the compositions present in the medium, especially the carbon source, nitrogen source and mineral salts for the secretion of exopolysaccharides. Box-Behnken design of RSM was used to acquire the data that suits a quadratic polynomial model. Regression analysis was carried out on the data acquired, Equation (4) EPS production attained by optimizing the media components were observed to be signi icantly affected by sugar composition and nitrogen source composition in the designed medium. Steytr stated that the low nitrogen content in the growth environment in luences the microbial synthesis of extracellular biopolymers extensively. Several earlier reports on EPS synthesis states that it was commonly favoured by the occurrence of excess carbon source, in association with another limited nutrient like Nitrogen or Oxygen as reported by Chawla et al. (2009). The calculation of regression analysis gives the value of the determination coef icient R 2 =0.988728 for Curvalaria lunanta respectively and the P-value Prob F less than 0.0500 indicates that the model terms are signi icant ( Table 5). The models used for estimating the response of signi icant variables for EPS production is based on the suggestions of the statistical tool ( Table 6).
The effect of four variables on EPS production was calculated using Response Surface Methodology. Table 6 represents the Central Composite design matrix that comprises of experimental and predicted values for both the responses like EPS production. The regression equation projects EPS production as an empirical function in terms of coded.
The signi icance for response surface polynomial model for EPS production was implied using ANOVA analysis that showed P values of the model as P-0.0001. The coef icient of variation of the model was (CV = 5.836205%). The validity for a it of the model was assessed using determination coef icient (R 2 = 0.988728), and it was inferred that the variation of the sample more than 97.1% were ascribed to the variables. The modi ied determination coef icient (AdjR 2 = 0.978208) was also found to be suitable for      Response surface graphs (three-dimensional graphs) and contours plots aids in interpreting the form of interaction between variables and also to acquire the optimum conditions. Six response surfaces (AB, AC, AD, BD, BC and CD) were revealed by considering all the feasible permutations. A circular contour plot displays secondary interface amongst the independent variables, while absolute interaction designates elliptical contours. The maximum predicted value is characterized by the surface restricted in the least ellipse in the contour illustration (Figure 1). The optimum value of each variable was recognized based on the dome in the 3D plot, or from the focal point of the conforming contour plot. Each contour curve signi ies a vast number of combinations between the two tested variables, while the other two is maintained at zero levels. Each diagram represents the interaction between the variables and the effective response can be predicted.

FTIR analysis
The FTIR spectra characteristic band at 3780 cm−1 due to the -OH group. This indicates the occurrence of an Alcoholic (OH) group in the EPS, which is a prime reason behind that EPS precipitated by cold ethanol. The band at 3390 cm−1 were because of secondary amine. The peak at 1623 cm−1, 880.4 assigned to the Keto group and CH stretching vibrations of EPS. The newly emerged peak observed in the 1061 cm−1 region corresponds to the Sulfoxide groups of the EPS (Figure 2). Initially, EPS displayed weight loss between 207 and 402 • C, which may be due to the loss of moisture (Figure 3). EPS of Paenibacillus polymyxa was found to be stable till 305 0 C (Liu et al., 2012). Several reports propose that the presence of high level of carboxyl groups that are bound to water molecules may be the reason for the initial loss of moisture (Wang et al., 2015;Singh et al., 2019). An essential characteristic taken into consideration for industrial application of EPS is thermal stability, particularly in the food industry, a majority of the manufacturing and processing The "Pred R 2 " of 0.9467 is inreasonable agreement with the "Adj R 2 " of 0.9782 of the food products are usually performed at high temperature (Sajna et al., 2013). Due to the thermostability and rheological properties of EPS, it can be regarded as an ideal candidate for food application.

Analysis of compounds from EPS By GC-MS
EPS was found to have mannitol as major sugar, which is also known as sugar alcohol and used as a medication (Bartholdson et al., 2008;Felz et al., 2019). Glucose was also detected in the EPS (Figure 4). Mannitol is helpful for the organism from stress and adverse conditions (Patel and Williamson, 2016).

Antimicrobial activity
EPS was not having any antimicrobial activity against the strain used in this study. Even the polysaccharide derived from Terminalia catappa and Araucaria heterophylla did not show any antibacterial activity (Samrot et al., , 2019.

Antioxidant activity
EPS have shown to contain antioxidant and free radical scavenging properties in this study (Table 7). The IC 50 value for hydrogen peroxide scavenging activity was 802 µg / ml. The antioxidant capacity of EPS against the total hydroxyl radical was found to be 370 µg/ml. Maximum superoxide scavenging activity of 70.4% was found at 1000 mg, showing that the radical scavenging activity occurred in a dose-dependent manner ( Table 7). EPS of Rhizobium meliloti were reported to have antioxidant and free radical scavenging activity (Kishk and Al-Sayed, 2007) where EPS of a Cyanobacterium Arthrospira platensis was also having an antioxidant activity (Challouf et al., 2011). Ganoderma lucidum, a medicinal mushroom derived polysaccharide, is reported to have a good antioxidant capacity (Ghalem, 2017).

Anticancer Activity
The IC 50 was calculated as 139.346 µg/ml against HeLa cell line of EPS sample, where the EPS were not effective against MCF-7 cell line ( Figure 5). Liu et al. (2012) have reported the antitumor activity of EPS of Paenibacillus polymyxa.

CONCLUSION
EPS from Curvularia lunata was optimized using RSM. Thus, produced EPS was subjected to bioactivity studies. EPS was found to be an effective antioxidant and also an anticancer agent against HeLa cells. EPS was characterized by FTIR, thermogravimetric analysis and GC-MS and found to have mannitol and glucose. They were found to have better antioxidant and radical scavenging activity.

Funding Support
The authors declare that they have no funding support for this study.