Production and purification of mannan oligosaccharide and its role in tight junction assembly in epithelial cells

Abstract

Mannan Oligosaccharide (MOS) is well-known as prebiotic supplement that help increasing life quality of livestocks and animals. Many reports revealed that MOS supplement can help improve intestinal morphology, enhancing growth performance, raising body weight, and increasing metabolic and stress response. MOS feeding also affected several biological processes in peripheral blood mononuclear cells in pigs and modulates expression of many immune-related genes that helps to prevent and reduce the severity of viral infection. However, the mechanism of action of MOS is remained unclear and the production, purification, identification of bioactive MOS molecular type is needed. In this study, we developed a method for mannan oligosaccharide (MOS) production by using a recombinant mannanase. We found that the reaction can be performed in the wide range of temperature and pH, from 4 to 80 degree celcius and 2.5 to 10.0, respectively. Interestingly, low manannase concentration at below 1Unit per 1 gram of coconut meal dried weight yielded a higher amount of MOS rather than using a higher enzyme/ substrate ratio. Longer incubation time exceed 18hr also gave smaller oligosaccharides. MOS products were seperated through Biogel P2 column yielding purified MOS from 2 to 9 mers. We have also investigated the biological effects of MOS on prevention and a treatment of an epithelium inflammation models by using Transepithelial electrical resistant (TEER) assay on T84 cells. The results obtained showed that MOS enhanced cells integrity and MOS pentamer (MOS-5) showed the highest activity and the optimum dose was 10 uM with an activating time of 24 hours. The study on tight junction integration recovery with calcium ions switching shown that MOS5 have the ability to reassemble the cells tight junction. A strudy on MOS5 and Dorsomorphine, an AMPK inhibitor, also shown that MOS5 may activate cellular integration through AMPK signaling pathway. The mechanism of action of MOS5 to an integration of cellular tight junction through AMPK was confirmed with western blot analysis. A structure determination of MOS5 was performed by enzyme hydrolytic assay together with mass spectrometry and nuclear magnetic resonance. The results shown that MOS5 is a galactomannan polymer composed of mannantetrasaccharide with beta-1,4-glycosidic linkage with one galactose unit attached on the second mannose unit from non-reducing end with alpha-1,6-glycosidic linkage.