Evaluation of Food-Grade Additives on the Viability of Ten Shigella flexneri Phages in Food to Improve Safety in Agricultural Products

Abstract

Bacteriophages can be used as biocontrol agents in agriculture to improve food safety, provided they can remain viable in food environments. The viability of ten Shigella phages (AShi, Shi3, Shi22, Shi30, Shi33, Shi34, Shi40, Shi88, Shi93, and Shi113) was evaluated against different additives and biocides used daily in food applications. In addition, the influence of additives on phage viability in a food matrix was investigated. Treatments with lactic and citric acid were the most effective to inactivate phages. In addition, the acetic acid was the most phage-friendly treatment evaluated. Preservatives such as acetate, lactate, benzoate, sorbate, and propionate proved to be highly compatible with all the phages tested. Regarding the influence of the food matrix on phage viability, an equal or higher viability was found for most phages tested when compared with the corresponding organic acid. Finally, when phages were exposed to sodium hypochlorite, ethanol, quaternary ammonium chloride (QAC), and H₂O₂, most of them were sensitive to long incubations and high concentrations. However, when biocide concentrations employed are low, 10³-10⁴ PFU mL^-1 phage particles remains viable. Thus, the phages evaluated could be used in combination with additives and biocides as a biocontrol tool against the foodborne pathogen S. flexneri in agricultural products.

Keywords: Shigella flexneri; bacteriophage; biocides; food additives; viability.

Figure 1

Phage viability at 25 °C without (■) and with 2% (□) and 4% () of acetic acid after 5 min (filled), 15 min (filled and dashed), 30 min (filled and bricks), and 60 min (filled and arrows) of incubation. Values are the mean ± standard deviation (error bars) of three determinations (treatment: phage, phage concentration, incubation time) were compared against controls using Student’s t test at p < 0.05.

Figure 2

Phage viability at 25 °C without (■) and with 2% (□) and 4% () of lactic acid after 5 min (filled), 15 min (filled and dashed), 30 min (filled and bricks), and 60 min (filled and arrows) of incubation. Values are the mean ± standard deviation (error bars) of three determinations (treatment: phage, phage concentration, incubation time) were compared against controls using Student’s t test at p < 0.05.

Figure 3

Phage viability at 25 °C without (■) and with 2% (□) and 4% () of citric acid after 5 min (filled), 15 min (filled and dashed), and 30 min (filled and bricks) of incubation. Values are the mean ± standard deviation (error bars) of three determinations (treatment: phage, phage concentration, incubation time) were compared against controls using Student’s t test at p < 0.05.

Figure 4

Phage viability in meat at 25 °C without (■) and with 4% of acetic (□), lactic (), and citric () acid after 30 min (filled), and 60 min (filled and dashed) of incubation. Values are the mean ± standard deviation (error bars) of three determinations (treatment: phage, phage concentration, incubation time) were compared against controls using Student’s t test at p < 0.05.

Figure 5

Phage viability at 25 °C without (■) and with 50 ppm (□), 100 ppm (), and 500 ppm () residual-free chlorine (sodium hypochlorite) after 1 min (filled) and 10 min (filled and dashed) of incubation. Values are the mean ± standard deviation (error bars) of three determinations (treatment: phage, phage concentration, incubation time) were compared against controls using Student’s t test at p < 0.05.

Figure 6

Phage viability at 25 °C without (■) and with 10% (□) and 70% () of ethanol after 15 min (filled), 30 min (filled and dashed), and 60 min (filled and bricks) of incubation. Values are the mean ± standard deviation (error bars) of three determinations (treatment: phage, phage concentration, incubation time) were compared against controls using Student’s t test at p < 0.05.

Figure 7

Phage viability at 25 °C without (■) and with 2% (□) and 3% () of quaternary ammonium chloride (QAC) after 15 min (filled), 30 min (filled and dashed), and 60 min (filled and bricks) of incubation. Values are the mean ± standard deviation (error bars) of three determinations (treatment: phage, phage concentration, incubation time) were compared against controls using Student’s t test at p < 0.05.

Figure 8

Phage viability at 25 °C without (■) and with 2% (□) and 3% () of hydrogen peroxide (H₂O₂) after 15 min (filled), 30 min (filled and dashed), and 60 min (filled and bricks) of incubation. Values are the mean ± standard deviation (error bars) of three determinations (treatment: phage, phage concentration, incubation time) were compared against controls using Student’s t test at p < 0.05.