Atividade antibacteriana das enzimas papaína e bromelina livres e encapsuladas contra Alicyclobacillus spp

Abstract

Alicyclobacillus spp. are Gram-positive, thermophilic, acidophilic, non-pathogenic micro-organisms that have the ability to form spores. They are present in soil and are often related to the deterioration of acidic products among them citrus juices and beverages, such as orange juice. Heat treatments, such as pasteurization, are used in the orange juice concentrate industry to inactivate pathogenic and spoilage micro-organisms. However, the spore of Alicyclobacillus acidoterrestris can survive such heat treatment, germinate, grow and thus cause the juice to deteriorate after reconstitution. Research is currently focused on the application of natural antimicrobial agents to foods as they provide a potential pathway for inhibiting a wide variety of microorganisms without risk to consumer health. Papain and Bromelain are two enzymes extracted from papaya (Carica papaya) and pineapple (Ananas comosus) respectively, where several studies characterize its antimicrobial effects, however these enzymes have not been evaluated against the genus Alicyclobacillus to date. Because they are two proteolytic enzymes, such compounds may undergo denaturation when subjected to the heat treatment employed, for example, in the pasteurization of orange juice. Microencapsulation is a technology that allows the fine coating of solid particles in order to overcome limitations in the use of ingredients, such as attenuating undesirable flavors, reducing volatility and reactivity as well as increasing the stability of these solid particles under adverse conditions. Spray drying is a viable method for the preparation of microparticles and the use of natural biopolymers as an encapsulating material makes their application possible in food products. To evaluate the antibacterial activity of the enzymes papain and bromelain against Alicyclobacillus spp., focusing on the A. acidoterrestris species and to microencapsulate these enzymes with alginate and chitosan to verify its effectiveness in the protection of the antibacterial activity after being submitted to the heat treatment.The determination of minimum inhibitory and bactericidal concentration (MIC and MBC) for papain and bromelain against Alicyclobacillus strains was performed. The time course of death of the microorganism was determined in the presence and absence of the enzymes and investigated the enzymatic activity as a mechanism of action on the microorganism through enzyme inhibitor and denaturation assays. The antibacterial activity of the enzymes was also evaluated in combination with nisin. The microencapsulation of the enzymes was performed by spray drying using alginate and chitosan as encapsulating material, after which the compounds were submitted to thermal treatment and after their inhibitory and bactericidal activity was evaluated against five different species of Alicyclobacillus. Microcapsules were characterized by Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry (DSC) and Infrared Spectroscopy (FT-IR). The results showed that for the A. acidoterrestris strain, the MIC of papain was 0.98 μg/mL and the MBC was 3.91μg/mL, while the MIC of bromelain was 62.5 μg/mL and the MBC was 250 μg/mL. The concentration of 4XMIC for both the enzymes was sufficient to eliminate 4 logs of the micro-organism after 24 hours 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, but may be related to other activities, such as amidase and esterase. The synergistic activity of the enzymes revealed an 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. Microencapsulation of the particles with alginate and chitosan was evidenced through the physical analysis. The results showed that papain microencapsulated with chitosan or alginate maintained low MIC values after submission to heat treatment, demonstrating its effectiveness and potential application as a biopreservative. The application of the enzymes in reconstituted orange juice contaminated with A. acidoterrestris proved to be effective, since after 48 h of incubation at three different temperatures, the initial microbial population was eliminated. Therefore, papain and bromelain are enzymes with antibacterial action in A. acidoterrestris. The microencapsulation technique was effective in protecting the antibacterial activity of papain, characterizing this compound with great potential of application as a bioconservant in acidic products, being necessary more studies on stability and sensorial characteristics when applied to the food.