Assessment of salivary enzymatic activities towards casein and gluten substrates in a large cohort of subjects
Alharkan, Daniah S.
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OBJECTIVES: To quantitate and analyze casein and gluten-degrading protease activities produced by natural inhabitant oral microorganisms in human saliva, and to isolate new gluten-degrading oral species. METHODS: Whole saliva samples were collected from visitors to the Museum of Science in Boston, MA. At total of 769 samples were collected and sample number, subject age, race, gender and dietary habits with regard to gluten consumption were reported. At least 1 ml of saliva was collected from each subject and the samples were aliquoted into several 150 µl aliquots. To one of the aliquots 150 µl 80% BHI/20% glycerol was added to preserve the microbes. Enzyme activity analysis was conducted on pre-cast casein and zymogram gels. After electrophoresis, gels were renatured and developed, and enzyme activities were visualized and analyzed. Protease activity in each sample was evident from well-defined bands in the gels resulting from casein digestion. Enzyme activities were quantitated within the 75-150 kDa region and compared among subjects. Based on this value, subjects were grouped into displaying high, medium, low or negligible enzyme activity. Lastly, saliva aliquots from four selected subjects were plated for bacteria isolation and characterization of enzyme activities on a gliadin zymogram gel. RESULTS: The average overall salivary enzymatic activity among the various race groups was in the order of other/mixed race > Caucasian > Asian > African American > Hispanics, where the Other/mixed race group showed statistically significantly higher values than any of the other race groups. With respect to age, the overall enzymatic activity was in the order of 7-12 > 31-54 > 55-80 > 13-18 > 19-30 age group. As for the gender groups, no statistically significant differences were found in activities between males and females. A good consistency was observed in banding patterns between saliva samples analyzed on a casein zymogram and a gliadin zymogram gel, indicating that the same enzymes act on both substrates. From four selected donors showing unusual and/or high salivary enzyme activities, 12-15 individual salivary bacterial strains were cultured to purity, and analyzed individually on a zymogram gel. Among these, some showed proteolytic activity ~75 kDa, and were suspected to be Rothia bacteria based on previous studies. Others showed activity in high or low MW regions indicating different and potentially novel species of oral gluten degrading bacteria. CONCLUSION: This large scale study has provided insights into the extent and inter-subject variation of salivary enzyme activities, and the apparent molecular weights of the major enzymes involved. It furthermore showed conclusively that gliadin-degrading enzyme activity is present in saliva, and that casein is a good substitute substrate for investigating such activities. Novel gluten-degrading oral species were isolated. Their enzymes could potentially be further explored for aiding in the digestion of gluten in vivo as a means to treat patients suffering from gluten-intolerance disorders