INTRODUCTION: Celiac disease is a T-cell mediated-inflammatory disorder of the small intestine precipitated by gluten ingestion. Gluten can be effectively degraded by Rothia aeria, a natural resident oral microbe. Rothia bacteria can potentially be developed as a first probiotic for celiac disease. Aims: To select the optimal culture conditions for R. aeria enzyme expression in vitro and determine the ratio of R. aeria to gluten to achieve digestion within 2 hr incubation, which is the residence time of foods in the stomach/upper intestines. MATERIALS AND METHODS: R. aeria were cultivated in Brain Heart Infusion (BHI) with different strength (4%, 20% and 100%), and chemical defined media M9 supplemented with different carbon sources (glucose, succinate, glycerol, or casein). The effect of incubation time and temperature (28°C and 37°C) on the enzyme expression of the bacteria grown in BHI was also assessed. The enzyme activities of R. aeria (standardized with OD 0.6) were measured with the para-nitroanilide-derivatized substrate. Gliadin degradation was investigated by incubating a fixed gliadin concentration (250 µg/ml) with different amount of R. aeria cell (OD620 1.0, 0.5, 0.25), and analyzed by SDS-PAGE. RESULTS: The OD620 of 24 hr R. aeria culture in 4%, 20%, and 100% BHI were 0.240, 0.932, and 2.033, respectively. No bacterial growth was observed in M9 broth +2% glucose, succinate, or glycerol, but grew well in M9 broth +2% casein with OD620 19.54. R. aeria exhibited highest activity when grown in 100% BHI (0.22AU/min) and lowest in 4% BHI (0.1AU/min). Enzyme activities in M9+2% casein were low (0.035AU/min), not in proportional to the OD of R. aeria culture. The specific enzyme activities of R. aeria in 100% BHI was high in log phase (9 to 12 hr or 12 to 18 hr), but the yield of total activity was less than that of in stationary phase (20 to 44 hr). The activity of the cells grown at 37°C was higher than at 28°C. R. aeria suspension with an OD620 of 1.0 exhibited rapid degradation of gliadins at 250ug/ml. CONCLUSIONS: Full strength BHI broth and 20 to 44 hr cultivation at 37°C are considered as optimal cultivation condition to obtain a R. aeria cell culture with high enzyme activity. Starvation condition do not enhance enzyme expression.

INTRODUCTION: Foods enter the oral cavity and mix with saliva. Some foods are not well tolerated, for instance, gluten proteins in individuals suffering from celiac disease (CD). Celiac disease is a chronic immune-mediated inflammation of the duodenum, triggered by gliadin component of gluten contained in wheat, barley and rye. In previous studies we showed that oral Rothia bacteria can degrade gliadin in vitro. The objective of this study was to gain more insights into the role of Rothia bacteria and subtilisin-A enzyme on gliadin digestion in vivo, with the ultimate goal to find new therapeutic options for CD. MATERIALS AND METHODS: Part I: Rothia bacterial proteins were analyzed for enzyme activity and subjected to LC-ESI-MS/MS. For in vivo, mice chow was prepared with and without R. aeria. Gliadin epitope abolishment was assessed in the mice stomach contents (n=9/group) by ELISA. Part II: Subtilisin-A was dissolved in various solutions, temperatures and incubation time to assess enzyme activity by using enzyme substrate (Suc-AAPF-pNA). Part III: PEGylation of subtilisin-A (Sub-A) was performed by mixing Sub-A with methoxy-polyethylene glycol (mPEG) and further encapsulated by polylactic-glycolic acid (PLGA). The activity of the modified enzyme to detoxify the immunogenic gliadin epitopes was evaluated at pH3.0. RESULTS: Part I: R. aeria gliadin-degrading enzyme was found to be a member of the subtilisin family. In vivo, gliadins immunogenic epitopes were reduced by 32.6%. Part II: Sub-A dissolved at pH1.5 showed a band of 27kDa, while it only showed bands below 10kDa when dissolved at pH7.0, suggesting auto-proteolysis. The enzyme activity was completely lost at temperatures exceeding 60°C and also reduced 4-fold after 6hr incubation at 37°C. Part III: PEGylation protected Sub-A from autolysis. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gliadin immunogenic epitopes were decreased by 60% in the stomach of the mice fed with chow containing Sub-A-mPEG-PLGA. CONCLUSION: The results provide proof for the contribution of oral Rothia bacteria to gliadin digestion and pharmaceutical modification can protect Sub-A from auto-digestion as well as from acidic insults, thus rendering the usefulness of coated subtilisins as a digestive aid for gluten degradation. / 2019-01-13T00:00:00Z