Masters Theses

Date of Award

8-2005

Degree Type

Thesis

Degree Name

Master of Science

Major

Food Science and Technology

Major Professor

David A. Golden

Committee Members

Svetlana Zivanovic, F. Ann Draughon

Abstract

A study was conducted to determine the importance of cellular surface charge on susceptibility of yeasts to the natural biopolymer chitosan. The test organisms utilized were Saccharomyces cerevisiae, Candida krusei, and Zygosaccharomyces bailii. Surface charge was determined at various culture ages and under selected environmental conditions. Bovine serum albumin (BSA) was used as a protein standard to ensure an accurate method to measure microbial surface charge. Yeasts cells were grown to the early stationary phase, washed and suspended in potassium chloride with absorbance value (A600nm) of 0.1 to 0.2, and charge was measured using a phase analysis light scattering (Zeta PALS) apparatus. The chosen absorbance was predetermined using BSA, which had minimal standard deviation within surface charge measurements.

Surface charge of S. cerevisiae cells was measured after growth in yeast-mold (YM) broth for 12, 18, 24, 36, 48, and 72 hr to determine changes in charge as a function of growth phase. The effect of short term exposure to various pH on surface charge was determined by suspending S. cerevisiae cells in acetate buffer adjusted to pH 3-11 using 0.1 N NaOH or 0.1 N HCl. Additionally, S. cerevisiae cells were adapted over time to pH 3, 4, and 8 to evaluate prolonged effects of growth pH on yeast surface charge.

Flocculation and viability of the three yeasts were also evaluated. Cells were washed in sodium chloride and resuspended in acetate buffer (pH 4.0) to achieve an absorbance (600 nm) of 3.0. Chitosan was added to the yeast suspensions to achieve concentrations of 0.00001-0.001%. The test concentrations were relatively low due to the increase in viscosity of suspensions with higher chitosan concentrations. Cells were observed using a phase contrast microscope to detect morphological differences between species, at selected pH, and when chitosan was added.

Surface charge data for bovine serum albumin corresponded with previously published literature. Surface charge of yeasts cells was shown to be influenced by growth phase, species, environmental pH, and adaptation to non-optimal pH. After 48 hr, the surface charge of S. cerevisiae cells showed a significant increase or decrease (p< 0.05), and there were overall surface charge differences observed among the various pH values. However, pH adapted cells developed resistance to non-optimal pH due to adaptation, and only showed differences in pH between pH 3 and 8 and pH 4 and 8. This study showed that there were significant differences (pS. cerevisiae cells was -19.6 mV, -12.07 mV for C. krusei and -25.82 mV for Z. bailii. Candida krusei had the least negative surface charge. This yeast was least affected by the antimicrobial affects of chitosan. Z. bailii had the most negative charge, which may be the reason chitosan was more effective against Z. bailii. This study demonstrated that chitosan, which is positively charged, may be more effective as an antimicrobial agent against microbial cells with more negative charges.

Flocculation patterns of yeasts cells were altered by chitosan and lower log counts of some yeasts were observed when chitosan was added to suspensions. Chitosan was shown to inhibit growth of yeast species differently which may be partially explained by the surface charge differences of the cells. Yeasts cells were observed microscopically to identify changes in overall appearance and morphology when cells were exposed to chitosan. When chitosan was added to S. cerevisiae cell suspensions, cells appeared less dense, and more rounded, as compared to S. cerevisiae control cells. Clustering, or clumping, of cells was also noticed when chitosan was present.

The surface charge of yeasts was shown to be affected by environmental pH, age, and species. These influential factors are important when determining the most desirable conditions for chitosan to serve as a natural food antimicrobial. Chitosan is currently approved as a dietary supplement by the Food and Drug Administration, and it has the potential to be used as an antimicrobial agent and inhibit microbial growth in foods.

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