Integrity of proteins in human saliva after sterilization by gamma irradiation

Abstract

Microbial contamination of whole human saliva is unwanted for certain in vitro applications, e.g., when utilizing it as a growth substratum for biofilm experiments. The aim of this investigation was to test gamma irradiation for its suitability to sterilize saliva and to investigate the treatment's influence on the composition and integrity of salivary proteins in comparison to filter sterilization. For inhibition of bacterial growth by gamma irradiation, a sterility assurance level of 10(-6) was determined to be reached at a dose of 3.5 kGy. At this dose, the integrity of proteins, as measured by fluorescence, circular dichroism, and gel electrophoretic banding pattern, and the enzymatic activities of salivary amylase and lysozyme were virtually unchanged. Filtration reduced the total protein concentration to about half of its original value and decreased lysozyme activity to about 10%. It can be concluded that irradiation is suitable for sterilizing whole saliva in its native form.

FIG. 1.

Effects of irradiation or filtration on bacterial viability. (A) Suspensions of Escherichia coli, Geobacillus stearothermophilus, and Deinococcus radiodurans were irradiated with doses up to 10 kGy. Irradiation was performed in three independent experiments on pure bacterial suspensions in saline solution (saline data for D. radiodurans) or on salivary samples from three individuals spiked with D. radiodurans (saliva data). (B) Samples of whole saliva were irradiated with doses up to 5.1 kGy. Irradiation was performed in three independent experiments on samples from three individuals (data points are shown as •, ▴, and ▪). Each data point represents the result from one donor in one experiment. (C) Samples of whole saliva were filtrated in successive steps with membrane filters of decreasing pore size. Filtration was performed in two independent experiments on samples from two individuals (• and ▴). Each data point represents the result from one donor in one experiment. Aliquots of the samples were plated on agar, and bacterial colonies grown after 48 h were counted and calculated as CFU per ml of saliva. The detection limit was determined to be 20 CFU per ml of suspension and is indicated as a dashed horizontal line. Data below the limit of sensitivity were not considered for calculation of the D₁₀ value or the SAL (insert in panel B).

FIG. 2.

Effects of irradiation or filtration of whole saliva on the profile and integrity of protein bands after SDS-PAGE. Salivary samples were irradiated with doses up to 11.5 kGy (A) or filtrated in successive steps with membrane filters of decreasing pore size (B). Gels (two different donors) were stained with silver. Molecular masses of protein subunits were estimated by comparison to standard proteins (S).

FIG. 3.

Effects of irradiation or filtration of whole saliva on the concentration of proteins (A and B) and integrity of fluorophores (C and D), as well as enzymatic activities of amylase (E and F) or lysozyme (G and H). Salivary samples were irradiated (A, C, E, and G) with doses up to 10 kGy or filtrated (B, D, F, and H) with membrane filters of decreasing pore size. Irradiation was performed in three independent experiments on samples from three individuals (•, ▴, and ▪). Each data point represents the result from one donor in one experiment. Filtration was performed in five (B) or two (D, F, and H) independent experiments on samples from two individuals (• and ▴). The dashed horizontal line shows the 50% margin.