New insight into the antibacterial activity of emulsifying salts used in processed cheese: from in vitro to in situ application

Field: Pharmacology, Toxicology and Pharmaceutics (Life Sciences)

Processed cheese (PC) is a dairy product with multiple end-use applications, where emulsifying salts play a fundamental role in physicochemical changes during production. Moreover, some of these salts may be a strategy to control spoilage and pathogenic microorganisms, contributing to safety and shelf-life extension. In the construction of this thesis, four steps were employed to assess the antimicrobial activity of different emulsifying salts. (i) In the first step, the inhibitory activity of a commercial polyphosphate (JOHA ® HBS, at different concentrations, from 0.2% to 3.0%) was tested in vitro against 21 bacterial strains by streak assay, agar-spot method, spot-on-the-lawn and well-diffusion method. Different culture medium (Brain Heart Infusion Broth, Nutrient, Plate Count, Trypticase Soy), either in liquid or solid form, were used. In all the protocols performed, the solid culture medium (Nutrient agar) was more effective for the inhibition of the target bacteria; results of the streak assay showed that 11 out the 21 tested strains were highly inhibited at 0.5% of JOHA ® HBS. (ii) In this step, we selected Bacillus thuringiensis CFBP 3476 and Clostridium perfringens ATCC 13124 to access the in situ inhibitory activity of two emulsifying salts (ESSP = short polyP and BSLP = long polyP). PC samples were produced through laboratory-scale and pilot-scale, inoculated and analyzed until 45 days of storage at 6 ± 2 ºC. C. perfringens ATCC 13124 growth was not affected (p > 0.05), but both of the treatments reduced B. thuringiensis CFBP 4376 counts in the tested conditions (p < 0.05); a higher and faster reduction in samples produced by the laboratory-scale method was obtained. (iii) In the third step, a total of 14 treatments (T1-T14) composed by mixtures of emulsifying salts (ESSP, BSLP and trisodium citrate) and antimicrobial additives (nisin and potassium sorbate) were evaluated against eight target strains. PC samples were produced through laboratory- scale, inoculated and analyzed until 90 days of storage at 6 ± 2 ºC. Most treatments resulted in some level of bactericidal or bacteriostatic effect against the target microorganisms. Bactericidal activity was evident against Bacillus spp., and bacteriostatic effect was clear against C. perfringens ATCC 13124, Enterococcus faecalisFAIR-E 179, Listeria monocytogenes Scott A and Staphylococcus aureus ATCC 6538 (p < 0.05) during the storage period. The treatments in which BSLP was applied alone showed greater inhibitory activity; that is, equal concentrations of BSLP result in less bacterial inhibition when in the presence of ESSP. (iv) In the last step, the bacteriostatic effect of six emulsifying salts, containing different concentrations of phosphorus pentoxide (P 2 O 5 ), was investigated in PC samples deliberately contaminated with B. cereus spores. Also, the influence of PC processing methods (pasteurization and creaming), storage temperature (6 and 30 ºC) and storage time (until 120 days) were evaluated. Results showed that lower storage temperature and emulsifying salts significantly influenced bacterial growth. In addition, the creaming process and the P 2 O 5 content enhanced the inhibitory effect of emulsifying salts; PC treated with the highest concentration of P 2 O 5 , the lowest average value of both vegetative cells and spore was detected. Finally, the four steps of this study reveals that: (i) Nutrient medium and agar- based tests are required to properly test the inhibitory activity of polyphosphates; (ii) the inhibitory effect of emulsifying salts against B. thuringiensis in PC obtained by two different methods was confirmed; (iii) the composition of emulsifying salts significantly influences bacterial inhibition and the phosphate interactions may infer such activity and; (iv) the creaming process and the P 2 O 5 content can improve the inhibitory effect of emulsifying salts on B. cereus vegetative and spore growth in PC. Taken together, the results contribute to expanding the understanding of emulsifying salt application in PC, with a focus on microbiological safety and shelf-life. However, more detailed studies are needed to determine the specific emulsifying salt composition and the relation of P 2 O 5 content responsible for bacterial inhibition. Keywords: Dairy products. Emulsifiers. Microbiological stability. Pathogenic bacteria. Polyphosphates. Shelf-life.