Date of Award:

1992

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Nutrition, Dietetics, and Food Sciences

Department name when degree awarded

Nutrition and Food Sciences

Advisor/Chair:

Jeffery K. Kondo

Abstract

Chapter I reviews current literature on gene transfer systems in lactic acid bacteria, how genetically altered microorganisms for food are presently regulated, and how nisin is used as a food preservative.

Chapter II investigates previous reports which linked genes for nisin biosynthesis and sucrose utilization (Nip+Suc+) to plasmid DNA in two well characterized L· lactis subsp. lactis strains. Plasmid curing studies, conjugations, and DNA-DNA hybridizations indicated that these genes were encoded by chromosomal loci in all Nip+Suc+ strains examined. Similar results were noted in nisin-sucrose transconjugants of L. lactis subsp. cremoris and S. salivarius subsp. thermophilus in Chapters III and IV.

Chapter III describes the use of conjugation to construct nisin-producing Lactococcus lactis subsp. cremoris strains. The direct-plate conjugation method was developed to facilitate transfer of Nip+Suc+ to L. lactis subsp. cremoris recipients. DNA-DNA hybridizations to transconjugant DNAs with an oligonucleotide that detected the nisin structural gene, nisA, demonstrated that this gene was transferred during conjugation. Lactococcus lactis subsp. cremoris Nip+Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. These results indicated that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for mixed and multiple starter systems.

Chapter IV investigates features of Nip+Suc+ transfer using a Lactococcus lactis subsp. lactis model system. Intergeneric transfer of nisin-sucrose genes was also achieved between lactococcal Nip+Suc+ donors and Streptococcus salivarius subsp. thermophilus recipients. Streptococcal transconjugants acquired Suc+ and nisin immunity but did not produce nisin. DNA-DNA hybridizations, however, demonstrated that nisA was present in these transconjugants. To investigate whether nisA was involved in nisin immunity, this gene was cloned and electro-transformed into Lactococcus lactis subsp. lactis LM0230. Electro-transformants did not express nisin immunity or any other trait linked to nisin production in lactococci.

Results presented in Chapter V indicate that nisin may have application for control or prevention of bovine mastitis. Gram-positive pathogens which cause bovine mastitis were examined for their susceptibility to nisin. Disc diffusion assays indicated that minimum inhibitory concentrations of nisin ranged from 10 to 250 ug per ml. In addition, 50 ug of nisin per ml in milk inhibited all gram-positive pathogens tested.

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