Biocontrol of Listeria monocytogenes in minimally processed brined refrigerated cucumbers

by (Laura del Rosario) Reina, Laura D.

REINA, LAURA D. Biocontrol of Listeria monocytogenes in minimally processed brined refrigerated cucumbers (Under the direction of Henry Fleming and Fred Breidt) The purpose of the research has been to develop a biocontrol model system for the inhibition of the food-borne bacterial pathogen Listeria monocytogenes in minimallyprocessed fruits and vegetables in order to improve the safety of these products. One application of this model was to evaluate the potential use of a bacteriocin-producer Lactococcus lactis or bacteriocin-nonproducer Lactobacillus curvatus as a biocontrol bacterium at refrigeration and at abuse temperatures (18ºC) in the food system. The research involved the characterization of the microflora growing at refrigeration temperatures in cucumbers held in air and in brine, characterization of the microflora responsible for spoilage of the product at refrigeration and at abuse temperatures in cucumbers held in brine, and the potential use of the microflora (lactic acid bacteria) responsible for acidic spoilage of the product as biocontrol agents in this type of products. Isolates were tested for bacteriocin production against other lactic acid bacteria, Gram-negative spoilage microorganisms, and 5 strains of Listeria monocytogenes. The results showed that Listeria varied in sensitivity to the bacteriocin-like substance produced by some of the isolates, being strains serotype 1/2a and 1/2b more resistant than serotype 4b. Considerations were also given to the role of the natural microflora present in the cucumber, salt concentration, and the antibacterial properties of spices and garlic that can interfere with the biocontrol bacterium. The combination of salt and garlic was more inhibitory for the biocontrol bacterium and for the pathogen than garlic or salt alone; pickling spices did not affect either the biocontrol or the pathogen. To avoid interference of the natural microflora present in cucumbers, a blanching step was included in the product preparation; sterile aquaresin garlic and irradiated pickle spices were used for seasoning. The shelf life of the product was extended with this approach, and allowed the enumeration of the biocontrol bacterium and the pathogen added in the product. The use of Lactococcus lactis in the product was limited due to its low salt tolerance, cold temperatures and low pH. However, survival in the product was enhanced by previous treatment of the strain with salt and glycine betaine at 20ºC, enabling L. lactis to inhibit Listeria at abuse temperatures. The use of nisin in the product allowed the survival of L. lactis in unblanched cucumbers due to inhibition of the natural microflora, but natural nisin-resistant LAB (lactic acid bacteria) were able to grow and inhibit the Lactococcus by reduction of the pH. Listeria was able to survive at 5ºC, but did not grow in the product. It grew at 18ºC when the biocontrol agent or the natural LAB were not present. The Listeria death rate was faster in the product containing a bacteriocinproducer biocontrol bacterium than when the nonproducer was present. The rapid decline of Listeria was likely due to a synergistic effect of the nisin produced by Lactococcus, competitive inhibition, and lowering of pH. A salt, cold tolerant biocontrol bacterium able to produce a bacteriocin would be the ideal biocontrol agent for this type of products. Results from this study showed the potential of Lactobacillus curvatus as the biocontrol bacterium for minimally processed brined refrigerated cucumbers. It was able to grow slowly at 5ºC in cucumber juice containing 4% salt, and it was able to grow in the product. However, being able to produce bacteriocin would be a plus. Results from this study demonstrated that brining fruits and vegetables with a blanching step in the process and the use of sterile garlic oil and irradiated spices, and the addition of a biocontrol bacterium will ensure the safety of this product. Refrigeration and sanitation will still be required. The use of several hurdles is advised due to the ability of Listeria to withstand the stress conditions present in the environment.