Isolation of Microbes for Enzymatic Production of Short Chain Oligosaccharides and its Evaluation as Prebiotic

Abstract

Short chain fructooligosaccharides are potential prebiotics with several applications in food and newlinefeed industries. Due to its health promoting benefits and several industrial applications, there is newlineexponential market growth. Enzymatic production of SC-FOS from inulin is advantageous than newlineany other method available. Endoinulinase is the enzyme that hydrolyzes inulin to SC-FOS. The newlinepresent research was aimed to isolate and identify endoinulinase producing fungi, production and newlinepartial characterization of fungal endoinulinase, extraction of inulin from the chicory roots, newlineenzymatic hydrolysis of inulin to produce SC-FOS by optimizing various factors, followed by in- newlinevivo prebiotic evaluation of SC-FOS through high throughput technology. In the present study, newlineendoinulinase was produced and partially purified from Aspergillus fumigatus. (Genbank newlineaccession number: MK640673). Optimum endoinulinase production (1.85 IU/ml of culture newlinefiltrate), was noticed with 4% chicory root powder after 72 h of growth at pH 6.5. The enzyme was newlinepartially purified using solid ammonium sulfate at 60% level of saturation. Temperature 55and#8451;; pH newline5.5 with 0.5% substrate concentration was considered as optimum for endo activity of the enzyme. newlineThrough the in-silico approach, 17 new fungal species were identified as endoinulinase producers. newlineIn addition, the co-existence of exoinulinase and endoinulinase as a single protein complex was newlineidentified through the in-silico study. A higher yield of inulin (42.39%) was obtained from chicory newlineroot powder following precipitation with 100% ethanol with three volumes of the extract at room newlinetemperature. HPLC and TLC analysis revealed that 0.5 IU/mL of the enzyme is sufficient to newlinerecover higher yield of SC-FOS (99%) from inulin at 55and#8451; temperature, pH 5.5 after 4 h of newlinehydrolysis. The DP of oligosaccharides produced in the enzymatic hydrolysate ranged from 3- newline7 with kestose and nystose as main products. It was evident that the endoinulinase from A. newlinefumigatus exhibited higher substrate to product conversion efficiency with a lower dose of the newlineenzyme within a short period of enzymatic hydrolysis. A peculiar phenomenon of oligosaccharide newlinere-synthesis was noticed during the 4 th of hydrolysis at 50 and#8451; temperature at all pH and enzyme newlinedose. An animal trial was conducted to evaluate the prebiotic efficacy of SC-FOS derived through newlineenzymatic hydrolysis of inulin; sourced from chicory roots. From the study, it was identified that newlineSC-FOS supplementation significantly reduced blood glucose levels in mice. TRFLP and NGS newlineanalysis revealed that SC-FOS supplementation increased the growth of beneficial bacteria and newlinesuppressed the growth of pathogens in the gut. newline