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The effect of addition of purified nisin Z in liposomes to cheese milk and of in situ production of nisin Z by Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in the mixed starter on the inhibition of Listeria innocua in cheddar cheese was evaluated during 6 months of ripening. A cheese mixed starter culture containing Lactococcus lactis subsp. lactis biovar diacetylactis UL719 was selected for high-level nisin Z and acid production. Experimental cheddar cheeses were produced on a pilot scale, using the selected starter culture, from milk with added L. innocua (10 5 to 10 6 CFU/ml). Liposomes with purified nisin Z were prepared from proliposome H and added to cheese milk prior to renneting to give a final concentration of 300 IU/g of cheese.The nisin Z-producing strain and nisin Z-containing liposomes did not significantly affect cheese production and gross chemical composition of the cheeses. Immediately after cheese production, 3-and 1.5-log-unit reductions in viable counts of L. innocua were obtained in cheeses with encapsulated nisin and the nisinogenic starter, respectively. After 6 months, cheeses made with encapsulated nisin contained less than 10 CFU of L. innocua per g and 90% of the initial nisin activity, compared with 10 4 CFU/g and only 12% of initial activity in cheeses made with the nisinogenic starter. This study showed that encapsulation of nisin Z in liposomes can provide a powerful tool to improve nisin stability and inhibitory action in the cheese matrix while protecting the cheese starter from the detrimental action of nisin during cheese production.Nisin is a cationic polypeptide of 34 amino acids produced by Lactococcus lactis strains (18, 47). Two natural variants of nisin (nisin A and nisin Z) are known and are equally distributed among nisin-producing strains (34). These variants differ by a single substitution, at position 27, with histidine (nisin A) and asparagine (nisin Z) (34). This structural modification gives nisin Z higher solubility and diffusion characteristics which are important for food applications (12).Nisin has an inhibitory effect against a wide variety of grampositive food-borne pathogens and spoilage microorganisms (40) and can also act on several gram-negative bacteria when the integrity of their outer membranes is disrupted (23,43). The use of nisin as a food preservative dates back to 1956, when nisin was proposed to control growth and spore formation of Clostridium botulinum and Clostridium sporogenes in cheese (31). Nisin is the only bacteriocin that has been approved by the World Health Organization as a preservative in food (46), and Nisalpin, the commercial product containing 2.5% pure nisin A, is being legally used in more than 50 countries for specific food applications (10). However, the loss of nisin activity from the commercial form has been reported for several food products during storage (8,9). Moreover, the use of nisin in its free form in cheese can be expensive and results in inhibitory effects against the suitable acidifying or aroma-p...

This study aimed to evaluate the effects of incorporating liposome-encapsulated nisin Z, nisin Z producing Lactococcus lactis ssp. lactis biovar. diacetylactis UL719, or Lactobacillus casei-casei L2A adjunct culture into cheese milk on textural, physicochemical and sensory attributes during ripening of Cheddar cheese. For this purpose, cheeses were made using a selected nisin tolerant cheese starter culture. Proteolysis, free fatty acid production, rheological parameters and hydrophilic/hydrophobic peptides evolution were monitored over 6 mo ripening. Sensory quality of cheeses was evaluated after 6 mo. Incorporating the nisin-producing strain into cheese starter culture increased proteolysis and lipolysis but did not significantly affect cheese rheology. Liposome-encapsulated nisin did not appear to affect cheese proteolysis, rheology and sensory characteristics. The nisinogenic strain increased the formation of both hydrophilic and hydrophobic peptides present in the cheese water extract. Sensory assessment indicated that acidic and bitter tastes were enhanced in the nisinogenic strain-containing cheese compared to control cheese. Incorporating Lb. casei and the nisinogenic culture into cheese produced a debittering effect and improved cheese flavor quality. Cheeses with added Lb. casei and liposome-encapsulated nisin Z exhibited the highest flavor intensity and were ranked first for sensory characteristics.

This study investigated both the activity of nisin Z, either encapsulated in liposomes or produced in situ by a mixed starter, against Listeria innocua, Lactococcus spp., and Lactobacillus casei subsp. casei and the distribution of nisin Z in a Cheddar cheese matrix. Nisin Z molecules were visualized using gold-labeled anti-nisin Z monoclonal antibodies and transmission electron microscopy (immune-TEM). Experimental Cheddar cheeses were made using a nisinogenic mixed starter culture, containing Lactococcus lactis subsp. lactis biovar diacetylactis UL 719 as the nisin producer and two nisin-tolerant lactococcal strains and L. casei subsp. casei as secondary flora, and ripened at 7°C for 6 months. In some trials, L. innocua was added to cheese milk at 10 5 to 10 6 CFU/ml. In 6-month-old cheeses, 90% of the initial activity of encapsulated nisin (280 ؎ 14 IU/g) was recovered, in contrast to only 12% for initial nisin activity produced in situ by the nisinogenic starter (300 ؎ 15 IU/g). During ripening, immune-TEM observations showed that encapsulated nisin was located mainly at the fat/casein interface and/or embedded in whey pockets while nisin produced by biovar diacetylactis UL 719 was uniformly distributed in the fresh cheese matrix but concentrated in the fat area as the cheeses aged. Cell membrane in lactococci appeared to be the main nisin target, while in L. casei subsp. casei and L. innocua, nisin was more commonly observed in the cytoplasm. Cell wall disruption and digestion and lysis vesicle formation were common observations among strains exposed to nisin. Immune-TEM observations suggest several modes of action for nisin Z, which may be genus and/or species specific and may include intracellular target-specific activity. It was concluded that nisin-containing liposomes can provide a powerful tool to improve nisin stability and availability in the cheese matrix.

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