Date of Award:

1999

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Nutrition, Dietetics, and Food Sciences

Advisor/Chair:

Bart C. Weimer

Abstract

Lactococci are widely used in the cheese industry as a starter culture. Starter cultures face carbohydrate starvation due to the absence of a fermentable carbohydrate in the cheese curd after pressing. Starvation leads to a decreased ability to synthesize ATP, generate a proton motive force, and accumulate nutrients necessary to maintain viability. The aim of this work was to investigate the culturability of lactococci grown with and without lactose in a chemically defined medium, and to define the metabolic changes that occur during carbohydrate starvation.

Lactose metabolism provided energy for logarithmic phase growth and greater cell density in L. lactis ssp. lactis ML3 and L. lactis ssp. cremoris S2. However, the rate of lactose metabolism was strain dependent in that L. lactis ssp. lactis 11454 did not metabolize lactose as rapidly as did ML3 and S2. In the absence of lactose the cells became nonculturable on agar.

In addition to becoming nonculturable, the aminopeptidase and lipase/ esterase activity became nonmeasurable after 21 d, and cellular metabolism was altered because of carbohydrate starvation. Nevertheless, the cells remained viable for up to 42 d in spent media as measured by fluorescent viability stains and intracellular ATP content. Fluorescent viability staining demonstrated that the cells maintained an intact cell membrane to contain their DNA, as well as to contain enzymes and ATP necessary to maintain viability and metabolic activity.

With the addition of arginine to the basal medium, the survival time, cell number, and ATP concentration increased. Amino acids, including arginine, provided energy after carbohydrate exhaustion. At the onset of lactose exhaustion, the extracellular concentrations of arginine, glycine/valine, glutamate, and glutamine decreased in the media when energy was present for their transport. There was a significant increase in serine and methionine concentrations in the spent media over the same time period.

These data indicated lactococci remained viable and metabolically active, but were nonculturable in response to carbohydrate starvation. Additionally, amino acids are in a dynamic state during carbohydrate starvation, and utilization of amino acids, such as arginine and serine, could facilitate lactococcal cells in maintaining viability in harsh environments such as ripening cheese.

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