Production of nanoliposomes carrying nisin with chitosan coating and evaluation of physical and antibacterial properties of the product against Bacillus cereus and Staphylococcus aureus

Nisin is one of the antimicrobial substances that is used today as a preservative in various foodstuffs. It is a bacteriocin comprised of 34 amino acids and a molecular weight of 3.5 Da. With all the benefits of nisin, there are barriers to its use in dairy and protein rich products. One of these barriers is the combination of nisin with fats, proteins and sugars and the consequent reduction of its antibacterial activity. In the food science and industry, the use of the technique of encapsulation and production of liposome is the best possible solution in such cases. Also, by adding an antimicrobial agent such as chitosan to the coating of nanoliposomes, the antibacterial activity of the product may be increased. The aim of the present research was to produce nanoliposomes carrying nisin with (and without) chitosan coating and to evaluate the physical and antibacterial properties against two gram-positive bacteria, Bacillus cereus and Staphylococcus aureus. 

In this study, four treatments of nanoliposomes carrying nisin (NN), nanoliposomes carrying nisin coated with chitosan 0.05% ((NN-CH (0.05)), nanoliposomes carrying nisin coated with chitosan 0.1% (NN-CH (0.1)) and nanoliposomes carrying nisin coated with chitosan 0.5% (NN-CH (0.5)) were prepared and examined in terms of physical properties (average particle size, particle dispersity index, zeta potential and encapsulation efficiency) and antibacterial activity (against two gram-positive bacteria, Bacillus cereus and Staphylococcus aureus with two diffusion methods in agar medium and microdilution test). This research was conducted in a completely randomized design and SPSS and EXCEL softwares were used for statistical analysis and drawing of diagram, respectively. Data were analyzed by one-way analysis of variance and the difference between the means was evaluated by Duncan's test at 95% confidence level. 

The results showed that the average particle sizein different treatments with each other are significantly different (P<0.05) and vary from about 110 to 327nm; Also as the amount of chitosan in the coating increased, the particle size increased (P<0.05). This indicates the successful binding of chitosan to the surface of the nanoliposome, which results in the formation of a layer around the nanoliposome and an increase in particle size. Particle dispersity index was recorded less than 0.3 in all treatments and was not related to the amount of chitosan in the coating. With increasing the amount of chitosan in the coating of nanoliposomes, zeta potential increased significantly (P<0.05). This index changed from -55.34 in NN treatment to 53.14 mV in NN-CH (0.5) treatment. In fact, chitosan as a cationic polysaccharide changes the potential to positive values. As the amount of chitosan in coating of nanoliposomes increased, the encapsulation efficiency increased significantly in the treatments (P<0.05); this index increased from 32.19% in NN treatment to 75.14% in NN-CH (0.5) treatment. The results of the antibacterial activity of nisin in two methods of diffusion in agar medium and microdilution test showed that its antibacterial activity increased with nanoencapsulation of nisin with (and without) chitosan coating (p<0.05). Also, with the increase in chitosan concentration, the antibacterial activity of carrier nanoliposomes increased and the highest antibacterial activity was recorded in NN-CH (0.5) treatment (p<0.05). The diameter of the non-growth halo of Bacillus cereus against the research treatments (with five concentrations of 2.5 to 25 μg/ml) varied from about 4.5 to 17.5 mm. This amount for Staphylococcus aureus was recorded from 2.1 to 26.5 mm. By increasing the concentration of nisin and carrier nanoliposomes, the diameter of the halo of non-growth of both bacteria increased significantly (p<0.05). But an exception was recorded in this case; The diameter of the non-growth halo for Staphylococcus aureus in two concentrations of 2.5 and 5 μg/ml of treatments was the same and had no significant difference (p>0.05). The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the examined treatments for Bacillus cereus were in the range of 100 to 400 and 200 to 500 μg/ml, respectively. These two concentrations for Staphylococcus aureus were recorded as 50 to 200 and 100 to 400 μg/ml respectively. Based on the values of diameter of non-growth halo, MIC and MBC it can be claimed that Bacillus cereus is more resistant to the examined treatments than Staphylococcus aureus.Nanoencapsulation of nisin in the form of carrier nanoliposomes with chitosan coating is a suitable solution to improve its physical and antibacterial properties. In such a way that by increasing the concentration of chitosan in the coating, both of the aforementioned properties improved significantly. Nanoliposomes carrying nisin with (and without) chitosan coating have the ability to inhibit the growth and killing Bacillus cereus and Staphylococcus aureus bacteria. The antibacterial activity increases with the increase in nisin and carrier nanoliposomes concentrations. The value of non-growth halo, minimum inhibitory concentration and minimum bactericidal concentration confirm that Bacillus cereus is more resistant to nisin and its carrier nanoliposomes than Staphylococcus aureus.