Degree

Doctor of Philosophy (PhD)

Department

School of Nutrition and Food Sciences

Document Type

Dissertation

Abstract

Probiotic Lactobacillus species are widely used in functional foods and fermented dairy products because of their health benefits. Protoplast fusion provides a non-recombinant approach for combining desirable characteristics from different bacterial strains without introducing foreign DNA. This dissertation developed a reproducible, non-recombinant system for improving probiotic strains through protoplast formation, PEG-mediated fusion, cell wall regeneration, and functional characterization. In Study 1, species-specific protoplast formation was optimized using lysozyme (0, 90, and 125 µg/mL) and mutanolysin (0, 10, and 20 µg/mL). Lactobacillus acidophilus produced the highest protoplast yield (90%) using 125 µg/mL lysozyme without mutanolysin, whereas L. plantarum, L. delbrueckii subsp. bulgaricus, and L. helveticus required mutanolysin for optimal yields, demonstrating species-specific enzymatic requirements. In Study 2, PEG 4000 concentration (10, 30, and 60%) and exposure time (1, 2, and 5 min) significantly affected fusion frequency (P < 0.05). The highest frequency of single cell-to-cell fusion (25.7 ± 5.5 fusion events per microscopic field) occurred at 30% PEG after 5 min. In Study 3, regeneration media containing 10% sucrose, lactose, or glucose were evaluated using three plating techniques. Sucrose with spread plating produced the greatest recovery (3.49 log₁₀ CFU/mL), followed by sucrose after cooling (3.11 log₁₀ CFU/mL) and lactose spread plating (2.97 log₁₀ CFU/mL), whereas glucose did not support regeneration. In Study 4, regenerated fusion cultures remained metabolically active in milk. Although pH reduction and coagulation were not significantly different (P > 0.05), LH–LH and LA–LH cultures produced greater milk thickening after 6 h. LA–LA exhibited the highest viable counts (6.32 log₁₀ CFU/mL), whereas LH–LH and LA–LH had significantly lower counts (5.37 and 5.19 log₁₀ CFU/mL, respectively). In Study 5, LP–LA exhibited greater acid tolerance (6.27 log₁₀ CFU/mL) than LP–LP (5.96 log₁₀ CFU/mL) and LP–LH (5.27 log₁₀ CFU/mL). Under 0.3% bile, LP–LA (7.01 log₁₀ CFU/mL) and LP–LH (6.94 log₁₀ CFU/mL) maintained greater viability than LP–LP (6.10 log₁₀ CFU/mL). This research established a reproducible non-recombinant system for species-specific protoplast formation, PEG-mediated fusion, cell wall regeneration, and functional evaluation of probiotic Lactobacillus species with potential applications in food, dairy, and biotechnology.

Date

6-29-2026

Committee Chair

Aryana, Kayanush

LSU Acknowledgement

1

LSU Accessibility Acknowledgment

1

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