Peer Review #2 of "Production and purification of mannan oligosaccharide with epithelial tight junction enhancing activity (v0.1)"

Abstract

Background. Mannanan oligosaccharide (MOS) is well-known as effective supplement food for livestock to increase their nutrients absorption and health status, but the structure and identification of bioactive MOS remain unclear. In this study, MOS production was accomplished, using enzymatic hydrolysis of pretreated coconut meal substrate with recombinant mannanase. Methods. Mannanase gene was cloned from Bacillus subtilis cAE24, then expressed in BL21. Purified Mannanase exhibit stability over a wide range of pH and temperature from pH 6-8 and 4 °Celsius to 70°C elsius, respectively. SEM analysis revealed that sonication could change the surface characteristic of copra meal, which gave better MOS yield, compared to untreated substrates. The separation and purification of each MOS were achieved using Biogel-P2 column chromatography. Determination of biological active MOS species was also investigated. T84 cells were cultured and treated with each of the purified MOS species to determine their tight junction enhancing activity. Results. Scanning electron microscope imaging showed that pretreatment using sonication could disrupt the surface of copra meal better than grinding alone, which can improve the production of MOS. Pentamer of MOS (M5) significantly increased tight junction integration of T84 cells measured with TEER (p < 0.0001).