In vitro Evidence

Antibacterial Activity of Subtilosin Alone and Combined with Curcumin, Poly-Lysine and Zinc Lactate Against Listeria monocytogenes Strains
Probiotics & Antimicro. Prot.  2010;2(4):250–57
Listeria monocytogenes, a Gram-positive bacterium, commonly occurring in the environment as well as in a variety of commodities, such as uncooked meats and vegetables, and processed foods like soft cheeses. It has been known to cause listeriosis, a serious public health problem, mainly affecting pregnant women, newborns and adults who are low on immunity. Often, various approaches are being adapted to control this pathogen in foods, including the use of food preservatives. In the last couple of decades, particular attention has been focused on the use of nature-derived antimicrobial substances like bacteriocins from lactic acid bacteria.

However, due to limited spectrum of activity and efficacy only at very high concentrations has made these natural antimicrobials less attractive. In addition, high concentration has been found to alter the organoleptic properties of treated foods. As a result, combination of antimicrobials with different mechanism of action at low concentrations has become a promising approach for the food industry. This way, higher rates of microbial killing and better spectrum of activity can be achieved along with lower resistant variants and reduced antimicrobial concentrations in food preservation.

Subtilosin A is bacteriocin produced by the Gram-positive, spore-forming bacterium Bacillus subtilis, which is known to demonstrated broad-spectrum antimicrobial activity. However, to-date, no study has been conducted to evaluate the effects of subtilosin on L. monocytogenes, a food-borne pathogen, alone or in combination with other antimicrobials, such as Curcumin, poly-lysine, a peptide, and zinc lactate.

To assess the susceptibility of L. monocytogenes against subtilosin combination with Curcumin (plant extract-derived compound) versus encapsulated Curcumin, poly-lysine and zinc lactate.

Study Design:

1. Determination of Minimum Inhibitory Concentration:

  • All the substances were evaluated for minimum inhibitory concentration (MIC) (i.e. antimicrobial activity) using the broth microdilution method
  • Here, different concentrations of all the antimicrobial agents were inoculated separately with culture of two strains of L. monocytogenes (i.e. ScottA and NR30)
  • The absorbance was recorded at 630 nm every 30 min. over a 24 h incubation period at 30 ⁰C

2. Checkboard Assay:

  • The assay was used to determine interaction between antimicrobials against L. monocytogenes strains
  • Here, interaction of two antimicrobial substances was studied at different concentrations at the same time
  • Fractional inhibitory concentration (FIC) index of paired combinations of antimicrobials was calculated
  • Two-dimensional array of serial concentrations of test compounds was adapted to demonstrate synergistic or additive effects
Results and Discussion:

1. Determination of MIC:

  • All antimicrobial agents were found to inhibit the growth of both bacterial strains, particularly subtilosin was more active at lower pH
  • At pH 5, MICs of subtilosin were 12-fold (L. monocytogenes ScottA) and 8-fold (L. monocytogenes NR30)
  • L. monocytogenes ScottA was found to be more sensitive than L. monocytogenes NR 30 to subtilosin and pure curcumin
  • However, L. monocytogenes NR 30 was very sensitive to encapsulated curcumin and zinc lactate, while both bacterial strains exhibited the same sensitivity to poly-lysine

2. Checkboard Assay:

  • Bacterail growth curve data showed partial inhibition caused by subtilosin alone and encapsulated curcumin alone, whereas full inhibition was induced by the combination of subtilosin with encapsulated Curcumin
  • The interaction between subtilosin and encapsulated curcumin (15 and 60 µg/mL, respectively) was found to be synergistic
  • Similarly, synergistic effects were observed with subtilosin-zinc lactate combination against both the strains, while an additive effect was witnessed when subtilosin was combined with pure curcumin or poly-lysine

Overall, combining subtilosin with other antimicrobial agents like curcumin, poly-lysine or zinc lactate could be effective in controlling L. monocytogenes infection.