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Document Type

Thesis - University Access Only

Award Date

1992

Degree Name

Master of Science (MS)

Department

Biology and Microbiology

First Advisor

William R. Gibbons

Abstract

Nisin, a polypeptide bacteriocin produced by Lactococcus lactis subsp. lactis, is used in some types of food preservation due to its inhibitory action on Gram positive bacteria and their spores. Unfortunately, the high cost of producing nisin limits its commercial use. Therefore, the main objective of this project was to develop a means of producing nisin more economically. This involved developing both a system to maintain proper pH and a low-cost medium for nisin production. Prior to initiating this research it was first necessary to improve the sensitivity and accuracy of the nisin bioassay. This bioassay is based on horizontal diffusion of nisin through agar preinoculated with nisin-sensitive bacteria. This results in zones of inhibition with diameters that are proportional to nisin concentration. To improve the bioassay this investigation evaluated several variables. Results showed Micrococcus luteus as the most sensitive organism tested. Results also demonstrated that a lower agar concentration (0.75%) increased the sensitivity of the assay (21% improvement over standard 1.5% agar). Different buffers were also examined for their potential to alleviate the inhibitory action of low pH samples 2 on the test organism. In nonbuffered bioassay agar, the low pH of samples enlarged inhibitory zones beyond the size expected based on nisin concentration. By incorporating 1 % Na2HPO4 buffer into the bioassay agar it was possible to prevent these false inhibitory zones from developing. This resulted in a 57% improvement in accuracy and a 12% improvement in precision compared to the standard method. The second part of this investigation included development of methods for controlling the inhibitory effect of lactic acid produced during fermentation and development of a low cost medium for nisin production. Of the nine buffer systems tested, NaHC03 proved the most effective buffer for increasing nisin production (66% nisin yield increase for buffered versus nonbuffered LTB). Different complex media were compared for nisin and cell mass yields. Thin stillage, a fuel ethanol production byproduct, was shown to be the most efficient, low-cost medium for high nisin titres (723 IU/ml) and yields (20,466 IU/g cell mass).

Library of Congress Subject Headings

Nisin
Nisin -- Economic aspects
Lactococcus lactis

Format

application/pdf

Publisher

South Dakota State University

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