Document Type

Thesis - University Access Only

Award Date

1995

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

William R. Gibbons

Abstract

Besides lactic acid, a major fermentation product of many lactic acid bacteria are proteinaceous metabolites (bacteriocins), such as nisin. Nisin has an inhibitory effect on the vegetative cells and spores of many gram positive bacteria and can be used in foods and fermentation processes to control the growth of unwanted microbes. The objective of this research was to investigate the use of alternative methods of neutralizing and/or extracting lactic acid to maintain optimal pH and develop a method of continuous recovery/concentration of the bacteriocin nisin. Initial trials investigated the use of batch vs. fed-batch fermentation in a stillage-based medium. It was found that periodic substrate (glucose) addition increased nisin and lactic acid production parameters, while not causing catabolic repression. The effectiveness of 6N NaOH, 6N NH4OH, and 10-20% NH4HCO3 in neutralizing lactic acid to maintain high levels of nisin and lactic acid production was next investigated. It was observed that 6N NH4OH out-performed the other bases tested, and was therefore used in subsequent trials. The next phase of the investigation explored the use of an ion exchange resin to remove lactic acid during fermentation (in-situ). Prior to this, however, the effects of filtering the stillage component of the medium were explored, as it was postulated that particulates in stillage could interfere with functions of the resin column. Results demonstrated that the filtered stillage-based medium supported increased nisin production, while the nonfiltered stillage-based medium favored lactic acid production. Thus, filtered stillage-based medium was chosen for subsequent RPC and SpinTrex trials, since nisin was the primary metabolite of interest. During trials with the resin, it was observed that use of the filtered stillage-based medium did not interfere with operation of resin packed columns and permitted production of both nisin and lactic acid. Moreover, the resin packed columns permitted in-situ removal of lactic acid, but seemed to direct Lactococcus lactis metabolism more towards lactic acid production at the expense of nisin production. It was felt, however, that the advantages of resin packed columns to remove lactic acid outweighed the slight reduction in nisin production. The next phase of the investigation characterized various techniques which could be used to concentrate and recover nisin from cells. Previous investigators had demonstrated that nisin would adsorb onto cells at pH 6.5 and desorb at pH≤ 3.0. Centrifugation and pH manipulation were investigated initially for concentration and recovery of nisin, but it was observed that the centrifugation process stripped producer cells of nisin even at high pH (6.5). A milder separation process (microfiltration) was then explored to investigate if nisin would stay attached to cells during concentration (pH 6.5). Preliminary results using LTB medium showed that nisin could be at least partially concentrated with cells via microfiltration, but when the filtered stillage-based medium was used, nisin production was boosted to levels which exceeded the holding capacity of producer cells. As a result, a large quantity of nisin was released from the cells (109.2%) even at high pH (6.5). Therefore, this process for concentrating nisin did not appear feasible, however, future research may be able to use microfiltration to release and recover nisin at high pH (6.5).

Library of Congress Subject Headings

Nisin
Bacteriocins
Lactococcus lactis

Format

application/pdf

Publisher

South Dakota State University

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Rights Statement

In Copyright