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\def\RunningAuthor{Antony V Samrot et al.}
\firstPage{7476}
\articleType{Original Article}
\receivedDate{05 Oct 2020}
\acceptedDate{20 Nov 2020}
\revisedDate{17 Nov 2020}
\journalVolume{2020, 11}
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\journalDoi{ijrps.v11i4.3949}
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\title{\textbf{Optimized production of EPS and evaluating its Antimicrobial, Antioxidant and Anticancer activity }}
\author{Prakash~P\textsuperscript{1},  
            Aishwarya~B\textsuperscript{1},  
            Poornimaa~B\textsuperscript{1},  
            Shaik~Asma~Taj\textsuperscript{1},  
            Narendrakumar~G\textsuperscript{1},  
            Antony~V~Samrot\textsuperscript{*}\textsuperscript{2},  
            Abirami~S\textsuperscript{3}, 
            Jane~Cypriyana~P~J\textsuperscript{1}~\\[5pt]{\textsuperscript{1}Department of Biotechnology, School of Bio and Chemical Engineering, Sathyabama Institute of Science and Technology, Semmancheri, Chennai-600119, Tamil Nadu, India}~\\{\textsuperscript{2}School of Bioscience, Faculty of Medicine, Bioscience and Nursing, MAHSA University, Bandar Saujana Putra, Selangor-42610, Malaysia}~\\{\textsuperscript{3}Department of Microbiology, Kamaraj College, CGE Colony, Thoothukudi- 628003, Tamil Nadu, India}}

\begin{abstract}
Recently the ability of microbes to produce exopolysaccharides attracted many researchers and most attention has been given to the prokaryotic producers. Exopolysaccharides have been known to have various bioactive potential and also known to increase the nutritional value of food consumed by human population. For last two decades fungal exopolysaccharides are considered to be the high value biological macromolecules as it has several applications in various industries including food, pharmaceutical, medicine etc. In this current study, Plackett - Burman design matrix method was used for the selection of nutrients for the EPS production from Curvularia lunata under submerged batch-culture condition. Subsequently, Response Surface Methodology (RSM) was used to determine the optimal condition. 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. In order to identify the antioxidant activity, radical scavenging activity and anticancer activity the produced EPS was subjected to DPPH assay, ABTS assay and anticancer activity was tested against HeLa cell lines. EPS was found to be having a good antioxidant activity, Free radical scavenging activity and showed effective anticancer activity against HeLa cells.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}EPS,\newline Optimization,\newline bioactivity studies,\newline Response surface methodology
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Antony~V~Samrot~\\ Phone:\ ~\\  Email:\ antonysamrot@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{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 fields 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) \unskip~\citep{950337:20927697}. 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 \unskip~\citep{950337:20927705}. These EPS are carbohydrates, they also contain proteins, DNA, and glycolipids \unskip~\citep{950337:20927711}. 

Certain extracellular polysaccharides produced from microalgae possess several bioactivities such as anti-tumour \unskip~\citep{950337:20927709}, anti-inflammatory \unskip~\citep{950337:20927702}, and antiviral activity \unskip~\citep{950337:20927660} that contributes to potential prospects for pharmaceutical applications \unskip~\citep{950337:20927663}. 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 \unskip~\citep{950337:20927710,950337:20927673}, but the reports are meagre. The fungus \textit{Curvularia lunata} is known to occur on a wide range of hosts including black gram \unskip~\citep{950337:20927670} and it was selected in this study to investigate the potential role of EPS secreted by \textit{Curvularia lunata } as a potential bioactive agent. Thus, the production of EPS by \textit{C.lunata} was optimized by RSM and the produced EPS was characterized and utilized for in-vitro bioactivity studies.
    
\section{Materials and Methods}
\textbf{Isolation, extraction and identification 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) \unskip~\citep{950337:20927706}. 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 identification by LPCB staining method, 18S rRNA sequencing and was used for further study. 

\textbf{Optimization of medium for EPS production}

\textbf{Plackett- Burman Design (PBD)}

The medium components such as date syrup, jaggery, yeast extract, KH\ensuremath{_{2}}PO\ensuremath{_{4}}, K\ensuremath{_{2}}HPO\ensuremath{_{4}}, MgSO\ensuremath{_{4}}, CaCl\ensuremath{_{2}} were screened (Table~\ref{tw-ba9e6ea30674}) using PBD. After 4 days of the incubation period, the grown culture was filtered and the filtrate 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~(\ref{dfg-34b60d2585c4})
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The influence 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~(\ref{dfg-08438ccf975e})
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The error in the experiment was determined by taking an average of the mean squares of the dummy effects of E and G (Table~\ref{tw-ba9e6ea30674}) that is expressed as Equation~(\ref{dfg-87b600fe12db}). 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. 
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\begin{array}{@{}l}\frac{2(\Sigma A(H)-\Sigma A(L))}8\end{array}
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The regression coefficient, F value and P-value of the factors were determined for biomass production using statistical design.

\textbf{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~\ref{tw-81a65530ad7c}.

\textbf{Characterization of EPS}

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

\textbf{Bioactivity studies }

\textbf{Antimicrobial activity}

Antibacterial activity was done against \textit{E. coli} by agar well diffusion method \unskip~\citep{950337:20927701,950337:20927698}.

\textbf{Determination of antioxidant activity}

The impact of given samples on DPPH radical was assessed according to the method described by \unskip~\cite{950337:20927707}. The percentage inhibition of the DPPH radical by the samples was estimated \unskip~\citep{950337:20927675}. ABTS method was done for the samples to find its scavenging ability \unskip~\citep{950337:20927665}. Hydroxyl radical \unskip~\citep{950337:20927699}, H\ensuremath{_{2}}O\ensuremath{_{2}}\ensuremath{^{}}\unskip~\citep{950337:20927671}\ensuremath{^{}}and Superoxide scavenging activity \unskip~\citep{950337:20927704,950337:20927676} for fungal EPS was performed. 

\textbf{Anticancer activity}

Addition of 10000 cells/ 20 \ensuremath{\mu}L cell suspension (HeLa cells / MCF cells) to a 96 well culture plate was done followed by incubation at 37 \ensuremath{^{0}}C in a humidified CO\ensuremath{_{2}} incubator with 5\% CO\ensuremath{_{2.\ }} The cells were visualized under an inverted tissue culture microscope following 48h of incubation. With 80 \% confluence of cells, cytotoxicity assay was performed along with different concentrations of EPS sample (0 to 200 \ensuremath{\mu}g/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 \ensuremath{\mu}L 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 \unskip~\citep{950337:20927668}. 
    
\section{Results and Discussion}
\textbf{Isolation, extraction and identification 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~\ref{tw-3845bc9d447c}). The black mold species were identified by staining method and 18S rRNA sequence as \textit{Curvularia lunata} (Gen Bank Accession number is MK 646037). 


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\caption{\boldmath {EPS Yield in Response to a) MgSO\ensuremath{_{4\ }} and Jaggary b) Date syrup and KH\ensuremath{_{2}}PO\ensuremath{_{4\ }}c) Jaggery and Date syrup d) Jaggery and KH\ensuremath{_{2}}PO\ensuremath{_{4\ }}e) Date syrup and MgSO\ensuremath{_{4\ }} f) KH\ensuremath{_{2}}PO\ensuremath{_{4}} and MgSO\ensuremath{_{4}}}}
\label{f-717c7a7a326a}
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\caption{\boldmath {FTIR analysis of EPSs}}
\label{f-ccf0cd8f29f0}
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\caption{\boldmath {Thermo gravity analysis of EPSs}}
\label{f-0592f4fa767b}
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\makeatother 
\caption{\boldmath {GC-MS analysis of EPSs. Blue arrow denotes the mannitol and the red arrow denotes the glucose presence}}
\label{f-45bc50c38d4d}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/21bb8761-a16c-44a3-bd6d-5e33eb48faae-upicture5.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/21bb8761-a16c-44a3-bd6d-5e33eb48faae-upicture5.png}{\includegraphics{images/21bb8761-a16c-44a3-bd6d-5e33eb48faae-upicture5.png}}{}
\makeatother 
\caption{\boldmath {Anticancer activity of EPS}}
\label{f-38f786c1b401}
\end{figure*}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {Media components used in Plackett-Burman method} }
\label{tw-ba9e6ea30674}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2127\linewidth-2\tabcolsep}p{\dimexpr.2894\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}p{\dimexpr.2479\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Trails & \cAlignHack Components & \cAlignHack High & \cAlignHack Low\\
\tblmidrule 
\cAlignHack A &
  \cAlignHack Date Syrup &
  \cAlignHack 5 &
  \cAlignHack 2.5\\
\cAlignHack B &
  \cAlignHack Jaggery &
  \cAlignHack 5 &
  \cAlignHack 2.5\\
\cAlignHack C &
  \cAlignHack Yeast Extract &
  \cAlignHack 3 &
  \cAlignHack 1.5\\
\cAlignHack D &
  \cAlignHack KH\ensuremath{_{2}}PO\ensuremath{_{4}} &
  \cAlignHack 1 &
  \cAlignHack 0.5\\
\cAlignHack E &
  \cAlignHack K\ensuremath{_{2}}HPO\ensuremath{_{4}} &
  \cAlignHack 1 &
  \cAlignHack 0.5\\
\cAlignHack F &
  \cAlignHack MgSO\ensuremath{_{4}} &
  \cAlignHack 0.25 &
  \cAlignHack 0.125\\
\cAlignHack G &
  \cAlignHack CaCl\ensuremath{_{2}} &
  \cAlignHack 0.25 &
  \cAlignHack 0.125\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Variables used in Response Surface Methodology} }
\label{tw-81a65530ad7c}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1816\linewidth-2\tabcolsep}p{\dimexpr.2997\linewidth-2\tabcolsep}p{\dimexpr.2687\linewidth-2\tabcolsep}p{\dimexpr.25\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack S. No. & \cAlignHack Components & \cAlignHack High (ml) & \cAlignHack Low (ml)\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack Jaggery syrup &
  \cAlignHack 5 &
  \cAlignHack 2.5\\
\cAlignHack 2 &
  \cAlignHack Dates syrup &
  \cAlignHack 10 &
  \cAlignHack 5\\
\cAlignHack 3 &
  \cAlignHack KH\ensuremath{_{2}}PO\ensuremath{_{4}} &
  \cAlignHack 1 (g) &
  \cAlignHack 5 (g)\\
\cAlignHack 4 &
  \cAlignHack MgSO\ensuremath{_{4}} &
  \cAlignHack 0.25 (g) &
  \cAlignHack 0.125 (g)\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Screening of fungal species} }
\label{tw-3845bc9d447c}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2263\linewidth-2\tabcolsep}p{\dimexpr.4047\linewidth-2\tabcolsep}p{\dimexpr.369\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Morphology & \cAlignHack Exopolysaccharides (g) & \cAlignHack Concentration (\ensuremath{\mu}g)\\
\tblmidrule 
\cAlignHack White &
  \cAlignHack 0.1 &
  \cAlignHack 400\\
\cAlignHack Green &
  \cAlignHack 0.21 &
  \cAlignHack 430\\
\cAlignHack Black &
  \cAlignHack 0.2 &
  \cAlignHack 450\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Plackett{\textendash} Burman Design} }
\label{tw-f34a2e325113}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.121\linewidth-2\tabcolsep}p{\dimexpr.10540000000000001\linewidth-2\tabcolsep}p{\dimexpr.10359999999999998\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.11\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Trails & \cAlignHack A & \cAlignHack B & \cAlignHack C & \cAlignHack D & \cAlignHack E & \cAlignHack F & \cAlignHack G & \cAlignHack Net wt of EPS (g)\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack H &
  \cAlignHack H &
  \cAlignHack H &
  \cAlignHack L &
  \cAlignHack H &
  \cAlignHack L &
  \cAlignHack L &
  \cAlignHack 0.69\\
\cAlignHack 2 &
  \cAlignHack L &
  \cAlignHack H &
  \cAlignHack H &
  \cAlignHack H &
  \cAlignHack L &
  \cAlignHack H &
  \cAlignHack L &
  \cAlignHack 0.62\\
\cAlignHack 3 &
  \cAlignHack L &
  \cAlignHack L &
  \cAlignHack H &
  \cAlignHack H &
  \cAlignHack H &
  \cAlignHack L &
  \cAlignHack H &
  \cAlignHack 0.61\\
\cAlignHack 4 &
  \cAlignHack H &
  \cAlignHack L &
  \cAlignHack L &
  \cAlignHack H &
  \cAlignHack H &
  \cAlignHack H &
  \cAlignHack L &
  \cAlignHack 0.61\\
\cAlignHack 5 &
  \cAlignHack L &
  \cAlignHack H &
  \cAlignHack L &
  \cAlignHack L &
  \cAlignHack H &
  \cAlignHack H &
  \cAlignHack H &
  \cAlignHack 0.64\\
\cAlignHack 6 &
  \cAlignHack H &
  \cAlignHack L &
  \cAlignHack H &
  \cAlignHack L &
  \cAlignHack L &
  \cAlignHack H &
  \cAlignHack H &
  \cAlignHack 0.66\\
\cAlignHack 7 &
  \cAlignHack H &
  \cAlignHack H &
  \cAlignHack L &
  \cAlignHack H &
  \cAlignHack L &
  \cAlignHack L &
  \cAlignHack H &
  \cAlignHack 0.5\\
\cAlignHack 8 &
  \cAlignHack L &
  \cAlignHack L &
  \cAlignHack L &
  \cAlignHack L &
  \cAlignHack L &
  \cAlignHack L &
  \cAlignHack L &
  \cAlignHack 0.56\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Statistical Analysis of Plackett {\textendash} Burman Design} }
\label{tw-a907c7c86a21}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1086\linewidth-2\tabcolsep}p{\dimexpr.1241\linewidth-2\tabcolsep}p{\dimexpr.1273\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack A & \cAlignHack B & \cAlignHack C & \cAlignHack D & \cAlignHack E & \cAlignHack F & \cAlignHack G\\
\tblmidrule 
\cAlignHack EH &
  \cAlignHack 2.67 &
  \cAlignHack 2.52 &
  \cAlignHack 2.55 &
  \cAlignHack 2.52 &
  \cAlignHack 2.42 &
  \cAlignHack 2.43 &
  \cAlignHack 2.45\\
\cAlignHack EL &
  \cAlignHack 2.09 &
  \cAlignHack 2.24 &
  \cAlignHack 2.21 &
  \cAlignHack 2.24 &
  \cAlignHack 2.34 &
  \cAlignHack 2.33 &
  \cAlignHack 2.31\\
\cAlignHack D &
  \cAlignHack 1.16 &
  \cAlignHack 0.56 &
  \cAlignHack 0.68 &
  \cAlignHack 0.56 &
  \cAlignHack 0.16 &
  \cAlignHack 0.2 &
  \cAlignHack 0.28\\
\cAlignHack MS &
  \cAlignHack 0.023113 &
  \cAlignHack 0.0098 &
  \cAlignHack 0.01445 &
  \cAlignHack 0.0098 &
  \cAlignHack 0.0008 &
  \cAlignHack 0.00125 &
  \cAlignHack 0.00245\\
\cAlignHack MSE &
  \cAlignHack 0.001025 &
  \cAlignHack 0.001025 &
  \cAlignHack 0.001025 &
  \cAlignHack 0.001025 &
  \cAlignHack 0.001025 &
  \cAlignHack 0.001025 &
  \cAlignHack 0.001025\\
\cAlignHack F Test &
  \cAlignHack 22.54878 &
  \cAlignHack 9.560976 &
  \cAlignHack 14.09756 &
  \cAlignHack 9.560976 &
  \cAlignHack 0.780488 &
  \cAlignHack 1.219512 &
  \cAlignHack 2.390244\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Response Surface Methodology - Central composite design matrix for optimization} }
\label{tw-defaaa0c6f72}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.0879\linewidth-2\tabcolsep}p{\dimexpr.14350000000000001\linewidth-2\tabcolsep}p{\dimexpr.17919999999999998\linewidth-2\tabcolsep}p{\dimexpr.1494\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Run & \cAlignHack A: Jaggery & \cAlignHack B: Date syrup & \cAlignHack C: KH\ensuremath{_{2}}PO\ensuremath{_{4}} & \cAlignHack D: MgSO\ensuremath{_{4}} & \multicolumn{2}{p{\dimexpr(.30\linewidth-2\tabcolsep)}}{\cAlignHack Response}\\
\rowcolor{kwdboxcolor}\cAlignHack  & \cAlignHack G & \cAlignHack Ml & \cAlignHack G & \cAlignHack g & \cAlignHack Actual & \cAlignHack Predicted\\
\tblmidrule 
\cAlignHack 1 &
  \cAlignHack 2.5 &
  \cAlignHack 10 &
  \cAlignHack 0.5 &
  \cAlignHack 0.13 &
  \cAlignHack 0.31 &
  \cAlignHack 0.32\\
\cAlignHack 2 &
  \cAlignHack 5 &
  \cAlignHack 5 &
  \cAlignHack 1 &
  \cAlignHack 0.13 &
  \cAlignHack 0.48 &
  \cAlignHack 0.46\\
\cAlignHack 3 &
  \cAlignHack 2.5 &
  \cAlignHack 5 &
  \cAlignHack 1 &
  \cAlignHack 0.13 &
  \cAlignHack 0.51 &
  \cAlignHack 0.51\\
\cAlignHack 4 &
  \cAlignHack 5 &
  \cAlignHack 10 &
  \cAlignHack 1 &
  \cAlignHack 0.25 &
  \cAlignHack 0.33 &
  \cAlignHack 0.32\\
\cAlignHack 5 &
  \cAlignHack 3.75 &
  \cAlignHack 7.5 &
  \cAlignHack 0.75 &
  \cAlignHack 0.19 &
  \cAlignHack 0.79 &
  \cAlignHack 0.78\\
\cAlignHack 6 &
  \cAlignHack 1.25 &
  \cAlignHack 7.5 &
  \cAlignHack 0.75 &
  \cAlignHack 0.19 &
  \cAlignHack 0.53 &
  \cAlignHack 0.49\\
\cAlignHack 7 &
  \cAlignHack 5 &
  \cAlignHack 5 &
  \cAlignHack 0.5 &
  \cAlignHack 0.13 &
  \cAlignHack 0.33 &
  \cAlignHack 0.32\\
\cAlignHack 8 &
  \cAlignHack 3.75 &
  \cAlignHack 2.5 &
  \cAlignHack 0.75 &
  \cAlignHack 0.19 &
  \cAlignHack 0.42 &
  \cAlignHack 0.44\\
\cAlignHack 9 &
  \cAlignHack 2.5 &
  \cAlignHack 5 &
  \cAlignHack 1 &
  \cAlignHack 0.25 &
  \cAlignHack 0.44 &
  \cAlignHack 0.46\\
\cAlignHack 10 &
  \cAlignHack 3.75 &
  \cAlignHack 7.5 &
  \cAlignHack 0.75 &
  \cAlignHack 0.19 &
  \cAlignHack 0.79 &
  \cAlignHack 0.78\\
\cAlignHack 11 &
  \cAlignHack 5 &
  \cAlignHack 5 &
  \cAlignHack 0.5 &
  \cAlignHack 0.25 &
  \cAlignHack 0.4 &
  \cAlignHack 0.37\\
\cAlignHack 12 &
  \cAlignHack 5 &
  \cAlignHack 10 &
  \cAlignHack 1 &
  \cAlignHack 0.13 &
  \cAlignHack 0.4 &
  \cAlignHack 0.4\\
\cAlignHack 13 &
  \cAlignHack 3.75 &
  \cAlignHack 7.5 &
  \cAlignHack 0.75 &
  \cAlignHack 0.19 &
  \cAlignHack 0.81 &
  \cAlignHack 0.78\\
\cAlignHack 14 &
  \cAlignHack 6.25 &
  \cAlignHack 7.5 &
  \cAlignHack 0.75 &
  \cAlignHack 0.19 &
  \cAlignHack 0.37 &
  \cAlignHack 0.42\\
\cAlignHack 15 &
  \cAlignHack 2.5 &
  \cAlignHack 10 &
  \cAlignHack 0.5 &
  \cAlignHack 0.25 &
  \cAlignHack 0.33 &
  \cAlignHack 0.34\\
\cAlignHack 16 &
  \cAlignHack 3.75 &
  \cAlignHack 7.5 &
  \cAlignHack 0.75 &
  \cAlignHack 0.19 &
  \cAlignHack 0.79 &
  \cAlignHack 0.78\\
\cAlignHack 17 &
  \cAlignHack 3.75 &
  \cAlignHack 12.5 &
  \cAlignHack 0.75 &
  \cAlignHack 0.19 &
  \cAlignHack 0.4 &
  \cAlignHack 0.39\\
\cAlignHack 18 &
  \cAlignHack 3.75 &
  \cAlignHack 7.5 &
  \cAlignHack 0.75 &
  \cAlignHack 0.31 &
  \cAlignHack 0.24 &
  \cAlignHack 0.24\\
\cAlignHack 19 &
  \cAlignHack 3.75 &
  \cAlignHack 7.5 &
  \cAlignHack 0.75 &
  \cAlignHack 0.19 &
  \cAlignHack 0.77 &
  \cAlignHack 0.78\\
\cAlignHack 20 &
  \cAlignHack 3.75 &
  \cAlignHack 7.5 &
  \cAlignHack 0.25 &
  \cAlignHack 0.19 &
  \cAlignHack 0.26 &
  \cAlignHack 0.28\\
\cAlignHack 21 &
  \cAlignHack 3.75 &
  \cAlignHack 7.5 &
  \cAlignHack 0.75 &
  \cAlignHack 0.06 &
  \cAlignHack 0.24 &
  \cAlignHack 0.26\\
\cAlignHack 22 &
  \cAlignHack 5 &
  \cAlignHack 10 &
  \cAlignHack 0.5 &
  \cAlignHack 0.13 &
  \cAlignHack 0.33 &
  \cAlignHack 0.3\\
\cAlignHack 23 &
  \cAlignHack 2.5 &
  \cAlignHack 5 &
  \cAlignHack 0.5 &
  \cAlignHack 0.13 &
  \cAlignHack 0.29 &
  \cAlignHack 0.28\\
\cAlignHack 24 &
  \cAlignHack 2.5 &
  \cAlignHack 10 &
  \cAlignHack 1 &
  \cAlignHack 0.25 &
  \cAlignHack 0.42 &
  \cAlignHack 0.43\\
\cAlignHack 25 &
  \cAlignHack 2.5 &
  \cAlignHack 10 &
  \cAlignHack 1 &
  \cAlignHack 0.13 &
  \cAlignHack 0.51 &
  \cAlignHack 0.51\\
\cAlignHack 26 &
  \cAlignHack 3.75 &
  \cAlignHack 7.5 &
  \cAlignHack 0.75 &
  \cAlignHack 0.19 &
  \cAlignHack 0.75 &
  \cAlignHack 0.78\\
\cAlignHack 27 &
  \cAlignHack 5 &
  \cAlignHack 10 &
  \cAlignHack 0.5 &
  \cAlignHack 0.25 &
  \cAlignHack 0.33 &
  \cAlignHack 0.33\\
\cAlignHack 28 &
  \cAlignHack 3.75 &
  \cAlignHack 7.5 &
  \cAlignHack 1.25 &
  \cAlignHack 0.19 &
  \cAlignHack 0.51 &
  \cAlignHack 0.51\\
\cAlignHack 29 &
  \cAlignHack 5 &
  \cAlignHack 5 &
  \cAlignHack 1 &
  \cAlignHack 0.25 &
  \cAlignHack 0.42 &
  \cAlignHack 0.41\\
\cAlignHack 30 &
  \cAlignHack 2.5 &
  \cAlignHack 5 &
  \cAlignHack 0.5 &
  \cAlignHack 0.25 &
  \cAlignHack 0.33 &
  \cAlignHack 0.33\\
\multicolumn{2}{p{\dimexpr(.2314\linewidth-2\tabcolsep)}}{\cAlignHack Std. Dev.} &
  0.026874 &
  \multicolumn{2}{p{\dimexpr(.28939999999999998\linewidth-2\tabcolsep)}}{\cAlignHack R\ensuremath{^{2}}} &
  \multicolumn{2}{p{\dimexpr(.30\linewidth-2\tabcolsep)}}{0.988728}\\
\multicolumn{2}{p{\dimexpr(.2314\linewidth-2\tabcolsep)}}{\cAlignHack Mean} &
  0.460467 &
  \multicolumn{2}{p{\dimexpr(.28939999999999998\linewidth-2\tabcolsep)}}{\cAlignHack Adj R\ensuremath{^{2}}} &
  \multicolumn{2}{p{\dimexpr(.30\linewidth-2\tabcolsep)}}{0.978208}\\
\multicolumn{2}{p{\dimexpr(.2314\linewidth-2\tabcolsep)}}{\cAlignHack C.V. \%} &
  5.836205 &
  \multicolumn{2}{p{\dimexpr(.28939999999999998\linewidth-2\tabcolsep)}}{\cAlignHack Pred R\ensuremath{^{2}}} &
  \multicolumn{2}{p{\dimexpr(.30\linewidth-2\tabcolsep)}}{0.946677}\\
\multicolumn{2}{p{\dimexpr(.2314\linewidth-2\tabcolsep)}}{\cAlignHack Press} &
  0.051247 &
  \multicolumn{2}{p{\dimexpr(.28939999999999998\linewidth-2\tabcolsep)}}{\cAlignHack Adeq Precision} &
  \multicolumn{2}{p{\dimexpr(.30\linewidth-2\tabcolsep)}}{28.86004}\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{The "Pred R\ensuremath{^{2}}" of 0.9467 is inreasonable agreement with the "Adj R\ensuremath{^{2}}" of 0.9782}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Anti oxidantactivity of EPSs} }
\label{tw-fb4bcc7a9f15}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.06299999999999999\linewidth-2\tabcolsep}p{\dimexpr.1032\linewidth-2\tabcolsep}p{\dimexpr.14940000000000005\linewidth-2\tabcolsep}p{\dimexpr.1613\linewidth-2\tabcolsep}p{\dimexpr.17569999999999997\linewidth-2\tabcolsep}p{\dimexpr.15890000000000002\linewidth-2\tabcolsep}p{\dimexpr.1885\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\cAlignHack Sl. No. & \cAlignHack Conc. (uL)  & \cAlignHack \% DPPH Scavenging activity & \cAlignHack \% ABTS Inhibition & \cAlignHack   \% H\ensuremath{_{2}}O\ensuremath{_{2}} scavenging activity & \cAlignHack \% Hydroxy radical scavenging & \cAlignHack \% superoxide scavenging activity\\
\tblmidrule 
\cAlignHack  1 &
  \cAlignHack 50 &
  \cAlignHack 0 &
  \cAlignHack 40.7 \ensuremath{\pm}0.25 &
  \cAlignHack 16.5 \ensuremath{\pm} 0.44 &
  \cAlignHack 23.63 \ensuremath{\pm} 0.35 &
  \cAlignHack 27.46 \ensuremath{\pm} 0.39\\
\cAlignHack 2 &
  \cAlignHack 100 &
  \cAlignHack 87.48 \ensuremath{\pm} 0.29 &
  \cAlignHack 66.36 \ensuremath{\pm}0.59 &
  \cAlignHack 27.6 \ensuremath{\pm}0.42 &
  \cAlignHack 39.13 \ensuremath{\pm}0.49 &
  \cAlignHack 40.9 \ensuremath{\pm} 0.64\\
\cAlignHack 3 &
  \cAlignHack 300 &
  \cAlignHack 88.09 \ensuremath{\pm}0.52 &
  \cAlignHack 69.53 \ensuremath{\pm} 075 &
  \cAlignHack 31.27 \ensuremath{\pm}0.64 &
  \cAlignHack 46.6 \ensuremath{\pm}0.7 &
  \cAlignHack 41.43 \ensuremath{\pm} 0.45\\
\cAlignHack 4 &
  \cAlignHack 500 &
  \cAlignHack 90.29 \ensuremath{\pm} 0.66 &
  \cAlignHack 73.36 \ensuremath{\pm} 0.59 &
  \cAlignHack 40.93 \ensuremath{\pm}0.41 &
  \cAlignHack 48.37 \ensuremath{\pm}0.52 &
  \cAlignHack 54.28 \ensuremath{\pm}0.24\\
\cAlignHack 5 &
  \cAlignHack 800 &
  \cAlignHack 91.08 \ensuremath{\pm} 0.61 &
  \cAlignHack 80.07 \ensuremath{\pm} 1.12 &
  \cAlignHack 49.73 \ensuremath{\pm}0.24 &
  \cAlignHack 60.63 \ensuremath{\pm}0.35 &
  \cAlignHack 63.41\ensuremath{\pm} 0.59\\
\cAlignHack  6 &
  \cAlignHack 1000 &
  \cAlignHack 91.49 \ensuremath{\pm} 0.29 &
  \cAlignHack 83.26 \ensuremath{\pm} 0.46 &
  \cAlignHack 52.6 \ensuremath{\pm}0.26 &
  \cAlignHack 70.3 \ensuremath{\pm}0.55 &
  \cAlignHack 72.31 \ensuremath{\pm} 0.56\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
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 \unskip~\citep{950337:20927657}, centrifugation and filtration process etc \unskip~\citep{950337:20927677}. 

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.

\textbf{Optimization of medium for EPS production}

\textbf{Plackett - Burman Model}

Table~\ref{tw-f34a2e325113} illustrates the output of 8 runs. Thus, the optimal medium condition was obtained through the Plackett Burman model. 

\textbf{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~(\ref{dfg-df89cda77d15})
\let\saveeqnno\theequation
\let\savefrac\frac
\def\dispfrac{\displaystyle\savefrac}
\begin{eqnarray}
\let\frac\dispfrac
\gdef\theequation{4}
\let\theHequation\theequation
\label{dfg-df89cda77d15}
\begin{array}{@{}l}\begin{array}{l}Response=0.7867\;-0.0174A\;-\;0.011917B\;\\+\;0.05592C\;-0.00658D\;-0.0151AB\;\\-0.023375\;AC\;+\;0.0014AD\;-\;0.009BC\;\\+\;0.00687\;BD\;-0.026125\;CD\;-0.0813125\;A^{2}\\-\;0.0923B^{2}\;-0.0978\;C^{2}\;-0.1338\;D^{2}\;\end{array}\end{array}
\end{eqnarray}
\global\let\theequation\saveeqnno
\addtocounter{equation}{-1}\ignorespaces 
EPS production attained by optimizing the media components were observed to be significantly affected by sugar composition and nitrogen source composition in the designed medium. Steytr stated that the low nitrogen content in the growth environment influences 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 \unskip~\cite{950337:20927658}. The calculation of regression analysis gives the value of the determination coefficient R\ensuremath{^{2}}=0.988728 for \textit{Curvalaria lunanta } respectively and the \textit{P}-value Prob F less than 0.0500 indicates that the model terms are significant (Table~\ref{tw-a907c7c86a21}). The models used for estimating the response of significant variables for EPS production is based on the suggestions of the statistical tool (Table~\ref{tw-defaaa0c6f72}).

The effect of four variables on EPS production was calculated using Response Surface Methodology. Table~\ref{tw-defaaa0c6f72} 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 significance 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 coefficient of variation of the model was (CV = 5.836205\%). The validity for a fit of the model was assessed using determination coefficient (R\ensuremath{^{2}} = 0.988728), and it was inferred that the variation of the sample more than 97.1\% were ascribed to the variables. The modified determination coefficient (AdjR\ensuremath{^{2}} = 0.978208) was also found to be suitable for the significance of the model. 

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~\ref{f-717c7a7a326a}). 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 signifies 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. 

\textbf{Characterization of EPS}

\textbf{FTIR analysis }

The FTIR spectra characteristic band at 3780 cm\ensuremath{-}1 due to the {\textendash} 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\ensuremath{-}1 were because of secondary amine. The peak at 1623 cm\ensuremath{-}1, 880.4 assigned to the Keto group and CH stretching vibrations of EPS. The newly emerged peak observed in the 1061 cm\ensuremath{-}1 region corresponds to the Sulfoxide groups of the EPS (Figure~\ref{f-ccf0cd8f29f0}). Initially, EPS displayed weight loss between 207 and 402\ensuremath{^\circ}C, which may be due to the loss of moisture (Figure~\ref{f-0592f4fa767b}). EPS of \textit{Paenibacillus polymyxa} was found to be stable till 305 \ensuremath{^{0}}C\ensuremath{^{}}\unskip~\citep{950337:20927669}. 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 \unskip~\citep{950337:20927708,950337:20927703}. 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 of the food products are usually performed at high temperature \unskip~\citep{950337:20927672}. Due to the thermostability and rheological properties of EPS, it can be regarded as an ideal candidate for food application.

\textbf{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 \unskip~\citep{950337:20927661,950337:20927664}. Glucose was also detected in the EPS (Figure~\ref{f-45bc50c38d4d}). Mannitol is helpful for the organism from stress and adverse conditions \unskip~\citep{950337:20927674}.

\textbf{Bioactivity studies}

\textbf{Antimicrobial activity}

EPS was not having any antimicrobial activity against the strain used in this study. Even the polysaccharide derived from \textit{Terminalia catappa} and \textit{Araucaria} \textit{heterophylla} did not show any antibacterial activity \unskip~\citep{950337:20927675,950337:20927700}.

\textbf{Antioxidant activity}

EPS have shown to contain antioxidant and free radical scavenging properties in this study (Table~\ref{tw-fb4bcc7a9f15}). The IC\ensuremath{_{50}} value for hydrogen peroxide scavenging activity was 802 \ensuremath{\mu}g / ml. 

The antioxidant capacity of EPS against the total hydroxyl radical was found to be 370 \ensuremath{\mu}g/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~\ref{tw-fb4bcc7a9f15}). 

EPS of \textit{Rhizobium meliloti} were reported to have antioxidant and free radical scavenging activity \unskip~\citep{950337:20927666} where EPS of a Cyanobacterium \textit{Arthrospira platensis} was also having an antioxidant activity \unskip~\citep{950337:20927659}. \textit{Ganoderma lucidum}, a medicinal mushroom derived polysaccharide, is reported to have a good antioxidant capacity \unskip~\citep{950337:20927667}.

\textbf{Anticancer Activity }

The IC\ensuremath{_{50}} was calculated as 139.346 \ensuremath{\mu}g/ml against HeLa cell line of EPS sample, where the EPS were not effective against MCF-7 cell line (Figure~\ref{f-38f786c1b401}). \unskip~\cite{950337:20927669} have reported the antitumor activity of EPS of \textit{Paenibacillus polymyxa}.
    
\section{Conclusion }
EPS from \textit{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.

\textbf{Funding Support}

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

\textbf{Conflict of Interest }

The authors declare that they have no conflict of interest for this study.
    

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