The antibacterial activity of rhamnolipid biosurfactant is pH dependent

Highlights

• Antimicrobial activity of rhamnolipid is pH dependent.

• Rhamnolipid action is favored at more acidic conditions.

• B. cereus is very sensitive to rhamnolipids.

• Rhamnolipid changes cell surface hydrophobicity and composition.

Abstract

Rhamnolipid (RL) biosurfactants have been studied as agents to control the growth of food pathogens however, to be successful applied as antimicrobial agent in food, is important to determine the effect of environmental conditions on RL activity. Once pH is a determinant factor to the development of microorganisms in food, we investigated the antimicrobial activity of RL under different pH values. The antimicrobial activity of RL against the Gram-positive pathogens L. monocytogenes, B. cereus and S. aureus was pH dependent and favored at more acidic conditions while the Gram-negative Salmonella enterica and E. coli (EHEC) showed resistance at all pH levels studied. Bacillus cereus was the most sensitive bacteria showing MIC of 19.5 μg/mL and eradication of the population was observed after 30 min with 39.1 μg/mL of RL. The sensitivity to RL was associated with reduction on cell surface hydrophobicity and cytoplasmic membrane damage. Scanning Electron Microscopy images evidenced the cell damage promoted by RL on sensitive strains. The pH is an important factor to be considered on developing RL-based strategies to the control of food pathogens.

Introduction

According to the World Health Organization, one in ten people fall ill every year from eating contaminated food, causing around 420,000 deaths (WHO. World Health Organization 2015). The most relevant bacterial pathogens involved in foodborne diseases (FBD) are Salmonella spp., Campylobacter spp., Listeria monocytogenes, Clostridium perfringens, Clostridium botulinum, Vibrio spp., Escherichia coli O157 and Staphylococcus aureus (CDC. Center for Disease Control and Prevention 2017). In Brazil, 96% of FBD outbreaks reported from 2007 to 2017 were caused by Escherichia coli, Salmonella spp., Staphylococcus aureus, Clostridium perfringens and Bacillus cereus (SVS 2017).

FBD are of great concern to food industries that must warrant safe products to consumers. Considering the emergence of bacterial resistance to preservatives, the search for new alternatives to control the growth of food pathogens represents a challenge to industry. Additionally, consumers' preference for natural additives (Carocho, Morales, & Ferreira 2015) combined to health and environmental awareness creating a demand for new “green” antimicrobials.

Microbial surfactants or biosurfactants (BS) are a natural class of surface- active compounds produced by microorganisms. The production of BS from renewable feedstocks or agricultural wastes, their biodegradability and low toxicity are in agreement with green chemistry and represent an important tool for innovation and sustainability, fulfilling the current market needs (Mulligan, Sharma, Mudhoo, & Makhijani 2014).

The rhamnolipids produced by Pseudomonas aeruginosa are glycolipids biosurfactants composed of one or two rhamnose molecules linked to one or two fatty acids alkyl chains, being naturally synthesized as a mixture of homologs molecules namely the di-rhamnolipids and mono-rhamnolipids (Magalhães & Nitschke 2013). The RL surfactants demonstrate useful characteristics such as surface, emulsifying and biological activities; and owing to this versatility, they have been considered as multipurpose ingredients in food processing (Nitschke & Silva 2018). Some studies shown the effectiveness of the RL on controlling growth of food important microorganisms as the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, Clostridium perfringes; the Gram-negative Salmonella Typhimurium, Escherichia coli, Enterobacter aerogenes and the molds Phytophthora infestans, Botrytis cinerea, Fusarium graminearum and Mucor sp. (Benincasa, Abalos, Oliveira, & Manresa 2004; Haba et al. 2003; Sha, Jiang, Menq, Zhang, & Song 2012).

In a previous work, conducted with 32 Listeria monocytogenes isolates, we have demonstrated that over 90% were susceptible to RL showing predominantly a bacteriostatic activity. The study also demonstrated that RL have a synergistic effect when combined with nisin (Magalhães & Nitschke 2013). The RL have also been explored to control sessile cells, displaying anti-adhesive, antimicrobial activity (Araujo et al. 2016) and as agents to disrupt/remove biofilms of foodborne pathogenic bacteria (Gomes & Nitschke 2012; Silva, Carvalho, Aires, & Nitschke 2017). Considering the importance of the control of pathogens to the quality and safety of food, the use of the RL represents a highly promising alternative.

To be successful applied as antimicrobial agent in food, is important to determine the effect of environmental conditions on RL activity. Considering that pH is determinant for the growth of food microbiota, in this work we investigate the antimicrobial activity of RL against Gram-negative and Gram-positive food pathogens under different pH values. The changes promoted by RL treatment in sensitive and resistant food bacteria were also compared.