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Updated: Jun 27, 2026
Production and Testing of Antimicrobial Peptides and Their Mimics
Published on: April 10, 2026
Effect of modified MRS medium on production and purification of antimicrobial peptide ST4SA produced by Enterococcus
Svetoslav D Todorov1, Leon M T Dicks
1Department of Microbiology, Stellenbosch University, Private Bag X1, 7602 Matieland, Stellenbosch, South Africa.
Abstract:
Highest antimicrobial activity of peptide ST4SA (51,200 AU/mL) was recorded after 14 h of growth in MRS broth with optimal production at pH 6.0 or 6.5. Growth of strain ST4SA in the presence of tryptone, yeast extract, or a combination of the two, yielded 102,400 AU/mL. An increase in production of peptide ST4SA to 102,400 AU/mL was recorded in the presence of 20.0 g/L fructose, but decreased to 25,600 AU/mL in the presence of lactose (20.0 g/L) or mannose (20.0 g/L) as sole carbon source. Lower activity (25,600 AU/mL) was recorded when 2.0 g/L K(2)HPO(4) was replaced by 2.0 g/L KH(2)PO(4) in MRS broth. An increase of K(2)HPO(4) to 10.0 g/L and 20.0g/L resulted in higher activity (102,400 AU/mL). Addition of glycerol to MRS broth had a negative effect on peptide ST4SA production. Production of peptide ST4SA required the presence of magnesium sulphate, manganese sulphate and 5.0 g/L sodium acetate. Exclusion of tri-ammonium citrate from the medium resulted in reduction of activity to 3,200 AU/mL. Maximum activity (102,400 AU/mL) was recorded in MRS supplemented with 1.0 ppm Vit. C, DL-6,8-thioctic acid or thiamine, respectively. Growth of Listeria ivanovii susbp. ivanovii ATCC 19119 in the presence of peptide ST4SA (12,800 AU/mL) resulted in 99% cell lysis after 18 h. Improved production of peptide ST4SA was recorded in MRS broth (Biolab) pre-treated with Amberlite XAD-1180. Precipitation with ammonium sulphate, followed by gel filtration chromatography, yielded the highest level of peptide ST4SA. This paper describes the partially deproteination of growth medium to facilitate peptide ST4SA purification.
Insights
Antimicrobial peptide ST4SA production is optimized by specific nutrients like tryptone and yeast extract, and conditions such as pH 6.0-6.5. This peptide effectively lyses Listeria ivanovii cells.
Area of Science:
- Microbiology
- Biochemistry
- Food Science
Background:
- Antimicrobial peptides (AMPs) are crucial in host defense and have therapeutic potential.
- Optimizing the production of specific AMPs, like ST4SA, is essential for their practical application.
- Listeria ivanovii is a pathogen that requires effective antimicrobial agents for control.
Purpose of the Study:
- To determine the optimal conditions for the production of antimicrobial peptide ST4SA.
- To investigate the effect of various medium components and supplements on ST4SA yield.
- To evaluate the antimicrobial activity of ST4SA against Listeria ivanovii.
Main Methods:
- Cultivation of strain ST4SA in de Man, Rogosa and Sharpe (MRS) broth with varied nutritional and environmental conditions.
- Analysis of peptide ST4SA activity using antimicrobial units (AU/mL).
- Testing the efficacy of ST4SA against Listeria ivanovii ATCC 19119.
- Purification of ST4SA using ammonium sulphate precipitation and gel filtration chromatography.
Main Results:
- Optimal ST4SA production occurred at pH 6.0-6.5, with tryptone and yeast extract significantly increasing yield (102,400 AU/mL).
- Fructose as a carbon source enhanced production, while lactose and mannose reduced it.
- Potassium phosphate (K(2)HPO(4)) at 10.0-20.0 g/L and supplements like magnesium sulphate, manganese sulphate, sodium acetate, Vitamin C, thioctic acid, and thiamine were crucial for high activity.
- ST4SA demonstrated potent antimicrobial activity, causing 99% lysis of Listeria ivanovii within 18 hours at 12,800 AU/mL.
Conclusions:
- The study successfully identified key factors for maximizing ST4SA production, including specific nutrients, pH, and mineral supplements.
- Partially deproteinating the growth medium, as achieved with Amberlite XAD-1180 pre-treatment, aids in ST4SA purification.
- ST4SA exhibits significant potential as an antimicrobial agent against Listeria ivanovii, warranting further investigation for therapeutic and food preservation applications.
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