Optimisation of Culture media through Response surface methodology to improve Antioxidant activity of E. coli
Neha Sharma1, Sharmistha Banerjee1, Shuchi Kaushik2, Rajesh Singh Tomar1
1Amity Institute of Biotechnology, Amity University Madhya Pradesh, Maharajpura Dang,
Gwalior (MP) - 474005, India.
1National Institute of Technology, Raipur, Chhattisgarh.
2DNA Unit, State Forensic Science Laboratory, Sagar, Madhya Pradesh, India.
*Corresponding Author E-mail: nsharma2@gwa.amity.edu
ABSTRACT:
Antioxidants play a significant role in oxidative stress management and health protection. One variable at a time response surface methodology (RSM) was used to formulate different composition of media to increase the antioxidant property of selected microorganism (Escherichia coli MTCC no. 40). 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assays was used to measure antioxidant activity of selected microorganism. The reduction potentiality of DPPH radical was determined by the DPPH% antioxidant or scavenging activity of the solution. In the present study, we have measured antioxidant activity of both intracellular and extracellular metabolites in dry and wet extracellular metabolites in supernatant respectively. In DPPH assay, extracellular metabolites showed better antioxidant potential in comparison with the reference compound. The comparison was based on antioxidant activity in different responses (number 1-16) for both dry and wet biomass. According to the results maximum DPPH % antioxidant or scavenging activity was showed by response 9 (wet biomass) and response 6 (dry biomass) that is 24.8447 and 35.0142 respectively in comparison to standard (4.4636). Hence, response surface methodology is used in bioprocess technology to optimize the medium components which is the primary step involved to enhance the antioxidant activity of particular microorganism.
KEYWORDS: Response surface methodology, DPPH % antioxidant, Antioxidant, Escherichia coli.
INTRODUCTION:
1, 1-diphenyl-2-picrylhydrazyl or DPPH assay is an effective method used to analyzed antioxidant activity. 1, 1-diphenyl-2-picrylhydrazyl assay is based on the reduction of DPPH by molecules having reducing property such as antioxidants which have DPPH scavenging activity. Antioxidants have hydrogen releasing property.3,4 When DPPH react with any antioxidant, stable free radicals become released in the presence of free radical scavenging antioxidant or a hydrogen donor.5 Zho and Elledge evaluated the antioxidant activity of isolated E.coli by DPPH in their in-vitro studies.6 DPPH assay has been the most accepted method for measuring the free radical scavenging activity of any new molecules.7,8
Aim of the present work was to increase the antioxidant activity of selected microorganism i.e. Escherichia coli MTCC no. 40 by response surface methodology and to evaluate antioxidant activity in different wet and dry biomass.
MATERIALS AND METHODS:
Pre-optimization of media composition:
Experimental design for One-factor-analysis-at-a-time is based on standard method of media formulation.9,10 Media components were optimized through changes in one component at a time in RSM. It was used to increase the antioxidant activity and biomass of pre-selected microorganism (Escherichia coli MTCC no. 40) in different optimized media. A variety of carbon, nitrogen and mineral sources were used to formulate different composition of media. Different media formulation was done by using all variables in one medium and media components were selected on the basis of absorbance.11
DPPH scavenging activity:
In DPPH assay, we measured the ability of antioxidants to reduce 2, 2- diphenyl picrylhydrazyl (DPPH).4
Preparation of DPPH solution:
0.1 M DPPH solution was prepared in methanol and kept for 2 hours in dark room. This was done to stabilize the as after one-hour incubation, absorbance of solution was found to be decreased empirically. After 2 hours, working solution of DPPH was prepared freshly by adding 1ml of DPPH, 200μl of methanol and finally 800μl (0.1M) Tris HCL buffer and pH was adjusted to 7.4 and kept in dark bottle during the assay as suggested by Tomoko et al 2014.12
Procedure of DPPH scavenging assay:
The analytical sample for absorbance was prepared by adding 200μl sample of (dry and wet supernatant) of cultured E. coli and 800μl of Tris HCl buffer (pH 7) in a test tube with 1ml of working DPPH solution. The solution was mixed properly for few seconds and left in dark room for 30 minutes. A blank solution was prepared that contain 1.2ml of ethanol and 800μl Tris HCl. Absorbance of both sample and blank solution was recorded at 517nm.
Percentage of DPPH scavenging activity was calculated by following formula:
% DPPH scavenging activity = (AC –AT)/A C) ×100
Where, AC = Absorbance of control and AT = Absorbance of crude extracts.
L-ascorbic acid (0.1%) was used as the standard for comparison. Experiments were performed in triplicate.
RESULTS AND DISCUSSION:
Antioxidants are free radical scavenging substances which are effective at low concentrations when compared to other oxidizable substances and have reducing properties. It is scavenging the free radical species and prevents the cell from adverse effects of reactive oxygen (ROS) and nitrogen species (RNS).13,14 The intake of plant derived antioxidants is reported by a number of researchers to be involved in the prevention of degenerative diseases such as Cancer, Parkinson, Alzheimir and Atherosclerosis.15-18
We have calculated DPPH activity for supernatants in both dry and wet form. The supernatant is the extract of cultured E. coli in broth obtained after pre- optimization of the media constituents. During the optimization process we got sixteen different optimized media which were used to culture E. coli.10
Free radical scavenging activity or DPPH activity of each extracts at different concentration was measured on the basis of change in color from pink to yellow and confirmed that they have free radical scavenging capacity.19 In DPPH assay we obtained that both form of supernatant showed better antioxidant potential (Wet biomass 24.8447 and Dry biomass 11.7064) in comparison to ascorbic acid (4.4636) DPPH % antioxidant or scavenging activity. The reduction capability of DPPH radical is determined by the DPPH % antioxidant or scavenging activity of solution. (Table 1)
Table 1: DPPH % antioxidant or scavenging activity of E. coli
|
S. No. |
Wet Biomass |
Dry Biomass |
|
1. |
9.3536 |
9.3062 |
|
2. |
12.2381 |
13.2119 |
|
3. |
16.8228 |
20.7074 |
|
4. |
14.7805 |
7.8324 |
|
5. |
12.9382 |
7.1481 |
|
6. |
12.4571 |
35.0142 |
|
7. |
13.2329 |
- |
|
8. |
3.1008 |
- |
|
9. |
24.8447 |
11.7064 |
|
10. |
17.3491 |
7.2218 |
|
11. |
16.3490 |
- |
|
12. |
8.8219 |
- |
|
13. |
11.7170 |
12.0644 |
|
14. |
19.1914 |
12.8750 |
|
15. |
11.8170 |
12.8750 |
|
16. |
5.4058 |
6.3269 |
Half normal plot (fig. 1) obtained through response surface methodology showed the distribution of experimental as well as experimental predicted value where obtained data points are close to diagonal line supporting the model is adequate enough to explain DPPH scavenging activity of dry biomass. The parity plots the values of given media across the standard line with different shade of color that signifies that some media gives positive result for media in which biomass production is high which relatively increase in the scavenging activity of bacteria in dry biomass whereas few media given the negative result.
Half normal plot (fig. 2) showed the distribution of experimental as well as model predicted value where obtained data points are close to diagonal line supporting the model is adequate enough to explain DPPH scavenging activity of wet biomass. The values of given media across the standard line with different shade of color that signifies that some media gives positive result for percent scavenging activity of wet biomass whereas few media given the negative result. This figure suggested that out of fifteen media, twelve media are above the standard line with maximum positive result whereas three are present below the line with positive and negative result.
Many studies reported that response RSM was a useful technique to optimize experimental parameters to enhance enzyme production and bioprocessing activities which support our results.20-22 It is reported that E. coli is a good host system which is used in industries to produce maximize the intracellular recombinant proteins.23 So in the present study, we have planned to optimised condition to increase the antioxidant activity of E. coli as few antioxidants are proteinaceous or enzymatic in nature and action such as catalase, superoxide dismutase etc.
Half normal plot in fig. 1 and fig. 2 clearly validates the application of RSM in bioprocess and pharmaceutical technology to optimize the medium components to enhance the antioxidant activity of any selected microorganism.
CONCLUSION:
Antioxidant activity of E. coli was obtained higher after optimization of media components and culture conditions using RSM. The results of this study suggested that RSM can be used to enhance production of microbial antioxidant activity.
ACKNOWLEDGEMENTS:
The authors are thankful to Amity Institute of Biotechnology, Amity University Madhya Pradesh. Gwalior (M.P.) for their support to carry out this work.
CONFLICT OF INTEREST:
The authors declare no competing or conflicts of interest.
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Received on 08.05.2020 Modified on 11.07.2020
Accepted on 15.08.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(9):4909-4912.
DOI: 10.52711/0974-360X.2021.00853