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.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/31317 |
| Date | 08 August 2018 |
| Creators | Yang, Chin-Hua |
| Contributors | Wei, Guoxian, Trackman, Philip |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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