Date of Award:

5-1-1987

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Department name when degree awarded

Biology

Committee Chair(s)

Gary H. Richardson

Committee

Gary H. Richardson

Committee

Frederick J. Post

Committee

Jeffery K. Kondo

Committee

Ronald V. Canfield

Abstract

Twelve proteinase positive (Prt+) and twelve proteinase negative (Prt-) variant pairs of Streptococcus cremoris and three Prt+ and three Prt- variant pairs of S. lactis were assayed for proteolysis in milk using spectrophotometric o-phthaldialdehyde (OPA) and trinitrobenzene sulfonic acid (TNBS). Both methods separated Prt+ and Prt- cultures. The TNBS data range was wider between Prt+ and Prt- strains and thus greater differentiation was possible. The correlation of determination (R2) between OPA and TNBS methods was 0.88 with a coefficient of variation (CV) within of 2.2 and 3.0%. The data were compared to a cell mass increase method. The R2 between these methods and cell mass increase in milk were respectively 0.77 and 0.80. Casein solutions were inoculated with Prt+ and Prt- variant paus of Streptococcus cremoris and S. lactis. After incubation for 12 h degradation products were examined by high performance liquid chromatography (HPLC). A 0.1% trifluoroacetic acid (TFA) in solvent A, and 0.075 % TFA in 90% acetonitrile in solvent B gave the optimum chromatographic results. Within both Prt+ and Prt- strains, significant differences (P < 0.05) were obtained for the peak eluted after 24 min. Reported yield for these strains examined correlated well to the HPLC observations of proteolysis variation between the Prt+ and Prt- variants. The HPLC results indicated that S. cremons Prt- UC 85 and 91 strains were low proteolytic strains, further cheese yield analysis is suggested to investigate their application in cheese manufacturing. The Prtmutant were more susceptible to agglutination than the Prt+ strains. S. cremoris Prt- UC 45 or 85 were low agglutinating and low proteolytic strains, thus could be used for cottage cheese manufacturing. Water soluble fractions of ammo acids and peptides generated from casein by different strains of Streptococcus lactis and Streptococcus cremoris were reacted with phenylisothiocyanate, and analyzed by HPLC on a reversed-phase ultrasphere ODS column. The accessibility of the native casein to cellular enzymatic activity gave rise to free amino acids and peptides of different elution times. Production of the different concentrations of these amino acids and peptides was strain dependant producing higher concentrations of hydrophobic than hydrophilic amino acids. Although the 15 amino acids studied dominated the chromatogram, a number of other peaks were also present. These other peaks could be due to other amino acids, not included in the standard, or peptides.

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