Antibacterial activity of papain and bromelain on Alicyclobacillus spp.

Alicyclobacillus spp. are spore forming bacteria that are often related to the deterioration of acidic products such as beverages and citrus juices. After the process of industrial pasteurization, the spore produced by the bacteria can germinate and the microorganism can grow, causing sensory abnormalities in the product. Alternative biopreservatives, such as the antimicrobial compounds, are of considerable importance to the food industry. Papain and bromelain are proteolytic enzymes derived frompapaya and pineapple, respectively. These enzymes are widely used in medicine and in the pharmaceutical and food industries, but while some studies have described their antibacterial action, no studies of the Alicyclobacillus spp. exist. The aimof this studywas to analyze the antibacterial effect of papain and bromelain on Alicyclobacillus spp. through 1) determining minimum inhibitory and bactericidal concentration (MIC and MBC); 2) determining the death time curve of the micro-organism in the presence and absence of enzymes; and 3) investigating the enzymatic mechanism on the microorganism. The antibacterial activity of enzymes in combination with nisin was also evaluated. The results showed that for the Alicyclobacillus acidoterrestris strain, the MIC of papain was 0.98 µg/mL and the MBC was 3.91 µg/mL, while theMIC of bromelain was 62.5 µg/mL and the MBCwas 250 µg/mL. The concentration of 4 ×MIC for both the enzymes was sufficient to eliminate 4 logs of the micro-organism after 24 h of incubation. Through the use of enzyme inhibitors specific for cysteine proteases, it was found that the antibacterial activity of papain and bromelain is not related to its proteolytic activity, butmay be related to other activities, such as amidse and esterase. The synergistic activity of the enzymes revealed a fractional inhibitory concentration (FIC) level of 0.16. Combination with nisin revealed an FIC of 0.25 for papain and 0.19 for bromelain, indicating synergism between both compounds. The application of enzymes in reconstituted orange juice contaminated with A. acidoterrestris was found to be effective, as after 48 h of incubation, at three different temperatures, the initial microbial population was eliminated. This study showed that the enzymes papain and bromelain have an antibacterial effect on A. acidoterrestris.

Resistance of Alicyclobacillus acidoterrestris spores and biofilm to industrial sanitizers.

This study evaluated the adhesion and biofilm formation of Alicyclobacillus acidoterrestris on industrial orange juice processing equipment and the bactericidal efficacy of peracetic acid, sodium hypochlorite, and quaternary ammonia after biofilm formation. The efficacy of these sanitizers against the spores of this microorganism was also evaluated. Stainless steel and nylon surfaces exhibited higher cell adhesion levels than did polyvinyl chloride surfaces. Peracetic acid was the most effective in removing biofilms from all surfaces (P < 0.05) and also reduced bacterial counts by 3 log CFU/cm² on the surface of polyvinyl chloride, but the other sanitizers also reduced the bacterial counts by 2 log CFU/cm². Quaternary ammonia exhibited the optimal minimum sporicidal concentration, preventing spore germination after only 15 s of contact at a concentration of 82 ppm. The flow cytometry results indicated that the spores and cells had low incidences of plasma membrane lysis after treatment with sanitizer, suggesting that lysis is not the principal mode of action for these sanitizers on A. acidoterrestris.