Nisin Resistance of Bacillus Cerus: Preparation of Nisin

Nisin is a peptide that is made by the bacterium Lactococcus lactis. Nisin is a small molecule that kills gram positive bacteria by binding to their membrane and by disrupting the proton motive force. When food is processed it is heated to kill bacteria, but some bacteria still survive. Adding nisin to the food provides a second barrier for the growth of the bacteria. Purified nisin has become quite expensive in the current marketplace. Therefore this study is directed at producing nisin and purifying it. L. lactis was grown in five different media (BHI, BHI + 1% glucose, BHI + 1% sucrose, BHI + 3% yeast extract, and skim milk medium). Samples of nisin from these media were filter-sterilized and tested for their ability to inhibit the growth of Lactobacillus viridescens, which is susceptible to nisin. It was found that nisin from the media BHI + 3% yeast extract after 8 hours of growth has a similar concentration as the standard, which was 1000 μg/ml. The nisin was then purified. An extraction process using ammonium sulfate was used to precipitate nisin out of the media. It was found that a 50% ammonium sulfate concentration precipitated out all of the nisin. The nisin was dialyzed against distilled water to remove the salts. The Minimal Inhibitory Concentration (MIC) of the extracted nisin, using Bacillus cereus and endospores was equivalent to a commercial nisin preparation at 1000μg/ml. 1 Weber: Nisin Resistance of Bacillus Cerus: Preparation of Nisin Published by Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato, 2004 Introduction Nisin is a peptide that is made by the bacterium Lactococcus lactis. It is a small molecule that kills gram positive bacteria by binding to their membranes and by disrupting the proton motive force (1). It appears to be active against the endospores of the food-borne, pathogenic gram positive bacteria, Clostridium botulinum and Bacillus cereus, and inhibits their outgrowth. In recent years, nisin obtained a GRAS (generally recognized as safe) status for use as a biopreservative in the food industry. When food is processed it normally goes through a heat treatment. This kills bacteria that are found in food. While vegetative cells of B. cereus are killed by heat, the endospores are more resistant. The endospores that survive turn into vegetative cells and start producing toxins. These toxins cause food poisoning. Adding nisin to the food provides a second barrier for the growth of the bacteria. However, there is concern that bacteria will develop resistance to nisin, just as they did to antibiotics like penicillin. Listeria monocytogenes, another gram positive food borne pathogen, develops a resistance to nisin (2). L. monocytogenes does not form endospores. Whether endospores can become resistant is not known. However, C. botulinum can become nisin resistant and their spore outgrowth inhibited (3). In order to produce endospores of B. cereus it is helpful to grow them on solid agar cultures. Addition of nisin to the culture medium requires large concentrations of nisin. Purified nisin has become quite expensive in the current marketplace. Therefore this study is directed at producing nisin and purifying it. Then it can be used with B. cereus cultures for the development of resistance in spores and vegetative cells. Methods Medium and stage of growth: L. lactis was grown in BHI (brain heart infusion) overnight at 30 C. Then a 0.5 ml inoculum was inoculated into 50 ml of five different media (BHI, BHI + 1% glucose, BHI + 1% sucrose, BHI + 3% yeast extract, and Skim Milk medium). The cultures were incubated in a shaker water bath set at 30 C. A 5 ml sample was taken at 4, 6, 8, and 10 hours. The last 5 ml sample was taken at 24 hrs. The samples were centrifuged and filter-sterilized and were tested for their ability to inhibit the growth of Lactobacillus viridescens, a very sensitive gram positive bacterium. Measurement of nisin activity The test organism, L. viridescens, was prepared from an overnight culture and 0.1 ml of the culture was added to 5 ml of Nutrient Agar (NA) and then poured over a layer of 15 ml of NA in a Petri dish. A 5 μl sample of nisin from the five cultures and a sample of a known nisin concentration were dotted onto the top agar. The plates were incubated for 24 hours at 37 C and examined for zones of inhibition. The size of the zone is proportional to the amount of nisin in it. This dot method allowed me to compare all 5 cultures rapidly and has been used by others (4). The time of maximum production and the medium were used to produce more nisin and then purify it. Minimum inhibitory concentrations (MIC) were also determined by a dilution assay. The nisin was serially diluted in Tryptic Soy Broth (TSB) for the test organism (Bacillus cereus and L. viridescens) and then the test organism is added to all the 2 Journal of Undergraduate Research at Minnesota State University, Mankato, Vol. 4 [2004], Art. 16 http://cornerstone.lib. mnsu.edu/jur/vol4/iss1/16 dilutions. The lowest concentration that does not show growth of the test organism is the MIC (5) Ammonium sulfate was added to the culture supernatant to yield 40, 50, 05 60% saturation and mixed for 24 hours at 15 C. The precipitated material was separated from the supernatant following centrifugation at 10,000 g for 20 minutes. The partially purified nisin was then fed into an ultrafiltration unit (Amicron) with a 10,000 Dalton molecular weight cutoff. The retentate and the filtrate were tested for nisin. Results Lactococcus lactis was grown in five different media (BHI, BHI + 1% glucose, BHI + 1%sucrose, BHI + 3% yeast extract, and skim milk medium) to determine the media that would best produce nisin the fastest and at a concentration similar to that of a standard concentration (1000 μg/ml). It was found that nisin from the media BHI + 3% yeast extract after 8 hours of growth has a similar concentration as the standard, which was 1000 μg/ml. Nisin production from the media BHI + sucrose was determined to be just below that of nisin from the media BHI + yeast extract. Nisin from BHI and BHI + glucose were similar to each but was lower than the standard concentration. The production of nisin from the media Skim Milk started out very slowly and then increased quickly. It was still increasing at the termination of the experiment. (Figure 1.)