Method for measurement of antibacterial activity of nisin.

Abstract

Nisin. this highly effective bacteriocin has not yet deserved its adequate position among preservatives in food industry. but in the last decades the situation slowly improves. Combinations of chemicals and mild processes looks like having advantages in minimal processes food. One such „natural” chemical is nisin. In the presented experiments we aimed to develop a rapid method for screening the effect of preservative combinations on Gram-positive and Gram-negative microorganisms. A micro-method has been worked out and tested with some combined preservation treatment. 96 well flat bottom ELISA plates were used. Wells were filled with different broth compositions which contained preservatives (nisin+lactic- and propionic acid. etc.) in a basal medium in different concentrations and inoculated by bacterial suspensions at three dilutions. Absorbance changes of the wells were recorded during incubation (24 hours) by a Labsystems MULTISCAN photometer at 405. 450 and 600 nm wavelength. The basal medium was supplemented with resazurine redox indicator. The colour changes of resazurine indicator were measured reporting the reducing capacity of the bacteria during growth. The curves follow the growth of bacteria and the metabolic activity of them similarly. The inhibition effect of nisin and different combinations of treatments were compared and studied based on the changes of lag periods. the slopes of growing curves and maximal growth values. It has been concluded from the data. that an effective screening tool has been developed to test the effect of most different nisin combinations on selected Gram-positive and Gram-negative bacteria. Naturally promising nisin combination treatments selected in such tests have to be comfirmed in model experiments. especialy because high nisin concentrations were used to easily detect the effectivity of nisin in experiments. Staphylococcus aureus as a Gram-positive microorganism was readily inhibited at all nisin concentrations. on contrary the growth of Salmonella Derby was not influenced in the nisin concentrations applied; nisin seems to be ineffective with Gram-negatives according to the relevant literature too. Pseudomonas aeruginosa however well tolerated pH=5.3 made by lactic- and propionic acid but not in the presence of nisin. Listeria monocytogenes was inhibited in growth. the lag phase was elongated in the function of nisin concentration. but the slope of the log phase remained unchanged. The results were similar in the presence of SO42- anions (pH=7). but the elongation was much higher and nisin reduced the maximal growth values. To reduce microbial contamination of whole and cut vegetables combinations of nisin and tween 20. or nisin and citrate seems to be effective with Listeria monocytogenes and Xanthobacter flavus. This screening method is a very rapid technique (data are available within 24 hours). productive to give enough data. applicable for mathematical statistical evaluation. to confirm the effects. The accuracy and reproducibility of the method proved to be excellent