**5. Conclusion**

418 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

treatment with lipolytic or glycolytic enzymes did not affect the activity of antimicrobial compound produced by strain, suggesting that produced bacteriocins do not belong to the controversial group IV of the bacteriocins, which contain carbohydrates or lipids in the active molecule structure [45-47]. It is important to note that, their activities were not due to hydrogen peroxide or acidity, as antimicrobial activity was not lost after treatment with catalase. Both of the presumptive were considered to be heat stable. Although heat stability of antibacterial substances produced by *Lactobacillus* spp. has been well established [39,48,49,50-53] heat stability of *L. casei* AP8 121ºC for 10 min is novel. The result of pH stability were not coherent with previous report that had indicated the tolerance of bacteriocins to acidic pH rather than alkaline [36,54]. The loss of antimicrobial activity of AP8 bacteriocin at pH > 10 might be ascribed to proteolytic degradation, protein aggregation or instability of proteins at this extreme pH [39,48,55]. *L. casei* AP8 bacteriocin showed an increase in the inhibitory activity after treatments with SDS and EDTA, may be due to the ability of these compounds to break down the proteinaceous complex from its large form into smaller more active unite [21]. Similar to Lactocin RN78 and Plantaricin LC74, both bacteriocin *L. casei* AP8 and *L. plantarum* H5 were found to be stable after treatment with organic solvents like butanol, ethanol and methanol confirming their proteinaceous and soluble nature [18,21,56]. Pronounced inhibitory potential against various species of Grampositive bacteria were shown, including pathogenic and spoilage microorganisms such as *A. hydrophila, A. salmonicida, C. perfringens*, *B. cereus* and *L. monocytogenes*. Observed effects were consistent with reports about bacteriocins produced by other strains of LAB [1,3,17,19,20,25,41,49,55,57 59,60]. Although bacteriocins from LAB usually are ineffective against Gram-negative bacteria and rather relate to a narrow antimicrobial spectrum [9,51,53], both presumptive bacteriocins AP8 and H5 showed broad antimicrobial activity against several genera of Gram-positive and Gram-negative bacteria. Even representatives of *Pseudomonas, Salmonella, E. coli, A. hydrophila*, *A. salmonicida and V. anguillarum* could be inhibited. Moreover a high level of inhibitory activity against *Listeria monocytogenes* was observed. Earlier studied have shown that several marine bacteria may produce inhibitory substances against bacterial pathogens in aquaculture systems [1,16,19]. Hence the use of such bacteria releasing antimicrobial substances in now gaining importance in fish farming as a natural alternative to administration of antibiotics [1,61-63]. In kinetic studies, both crude bacteriocins were continuously produced during logarithmic phase followed by optimal production during stationary growth phase, suggesting that these peptides may be secondary metabolites. Similar results were reported for some bacteriocins produced by some LAB isolates [5,64,65] and is contrary for other *Lactobacillus* species bacteriocins [1,16,25,55,60,66]. Bacteriocin H5 showed a decrease in activity towards the end of stationary growth may be due to proteolytic degration, protein aggregation, and feedback regulation as has been observed for Lactacin ST13BR, Lactacin B, Helveticin J and Enterocin1146 [53,55,67]. *L. casei* AP8 crude bacteriocin demonstrated a bactericidal mode of action, as the immediate decrease in the optical density of *L. monocytogenes* was observed in mix culture. In the case of H5 bacteriocin a bacteriostatic mode of action was observed. Crud H5 bacteriocin showed a growth inhibition, followed by decrease activity for remained time,

Bacteriocins AP8 and H5 showed a wide spectrum of antibacterial activity against seafood borne pathogens like *Listeria*, *Clostridium*, *Bacillus* spp, *S. aureus* and even Gram-negative pathogens like *Pseudomonas, Salmonella* and *E. coli*. Some of these foodborne pathogens can produce toxins resulting in human illness. In addition to the broad inhibition spectrum, theirs technological properties and especially cold, heat and storage stability, indicate that bacteriocins AP8 and H5 have potential for application not only as biopreservative agents to control pathogens in food products that are pasteurized and cook-chilled but also as bioprotect compounds at aquaculture. Accordingly *L. casei* may be of great interest as probiotics strains because of their ability to adhere to intestinal epithelial cells and being of human origin. Several authors have reported the production of bacteriocins by *L. casei* and *L. plantarum* strains from plant, dairy or meat origin. However, very few bacteriocins from *L. plantarum* have been reported to be isolated from fish and also based on our knowledge this is the first report of a *L. casei* bacteriocin isolated from fish.
