Production and Characterization of Microbial Amylase and Pullulanase for Synthesis of Resistant Starch

Abstract

Starch hydrolyzing enzymes, specifically amylases and pullulanase, play vital role in food processing in the manufacturing of resistant starch. Moreover, Resistant starch (RS) is recently documented as a source of fibre and has acquired much attention for both its potential health benefits and functional properties. In current research work, resistant starch type III from maize flour was prepared using amylase and pullulanase obtained from indigenously isolated Bacillus licheniformis. The amylase and pullulanase gene from Bacillus licheniformis were amplified and cloned into thepET-24(a) and pGEX-2T vector respectively, expressed in E.coli BL21 (DE3) cells and purified by affinity chromatography. The purified amylase, pullulanase, and combination of both enzymes were used for the formulation of resistant starch which enhanced the yield of resistant starch by 16, 20 and 24-fold respectively in maize flour as compared to native maize flour. Besides, Scanning electron microscopy unveiled that the granular structure of maize flour was disordered into an opaque network with asymmetrical structure, and X-ray diffractograms sustantiated the transformation from an amorphous to a crystalline structure upon enzymatic treatment. In addition, thermogravimetric analysis disclosed increased amylose content while 1H NMR and 13C NMR indicated debranching pattern, of pullulanase treated starch compared to untreated starch. Enzymatically prepared resistant starch was analyzed for antioxidant potential by DPPH radical scavenging assay and reducing power illustrated considerable increase in antioxidant activity of maize flour after enzymatic treatment. Prebiotic effect of EM-RSIII was successfully examined during in-vitro experiment and analyzed as fermentation media for promoting growth of probiotic bacteria e.g. Lactobacillus reuteri. While, in case of in vivo study EM-RSIII fed to the 12 female Sprague delay rats for a period of 21 days. After 21 days of experiment EM-RSIII fed rats showed significant reduction in body weight gain, fecal pH, glycemic response, serum lipid profile, insulin level and modulation in gut microbiota and their metabolites. Genomic analysis of bacteria from fecal sample of rats revealed that, the numbers of starch-utilizing and butyrate producing bacteria, such as Lactobacillus, Enterococcus and Pediococcus genus, were increased after the administration of medium and high doses of EM-RSIII, while those of E.coli completely suppressed compared to control fed diet rat’s from fecal and cecum samples. The microbial metabolites including short chain fatty acids importantly butyrate precisely increased in feces of EM-RS III fed rats. Moreover, enzymatically treated maize flour caused significant enhancement of the desired properties of maize flour, such as resistant starch content, amylose, milk absorption capacity, iodine and fatty acid complexing ability, and a reduction in swelling power, water binding, oil absorption capacity, and in-vitro digestibility compared to untreated maize flour. The low swelling power, water absorption and oil absorption capacity of EM-RSIII was responsible for good handling in processing imparting crispiness and enhanced texture to the end food product like, biscuits, wafers, cookies etc. Significant difference was observed on supplementation of cookies with EM-RSIII for palatability and color parameters when compared to native flour cookies. The sensory analysis was performed by distributing questioners among individual and results were evaluated based on five-point hedonic scale. Result exhibited that consumers portrayed highest acceptability of EM-RSIII augmented cookies specifically for its appearance, sharp aroma, texture, and nutritionally important food item for consumers. EM-RSIII derived drug loaded microspheres were also synthesized by single emulsion evaporation method with average particle size of 964 nm and drug encapsulation efficiency of 86.73 %. Release studies confirmed, stability of drug loaded microspheres at acidic pH while sustained release was observed at pH 7 allowing site specific delivery of drug to colon. For validation of encapsulation efficiency, antimicrobial potential of drug loaded microspheres was analyzed through agar well diffusion method. This study concluded that amalgamation of amylase and pullulanase works efficiently for the preparation of resistant starch. The prepared resistant starch with unique physicochemical characteristics most importantly its prebiotic nature with enhanced antioxidant potential served as an important ingredient for cookies preparation and coating material for targeted and site-specific delivery of drugs. Therefore, the prepared EM-RS III is an important product to be used in different food applications with fortification of nutritionally important ingredient along with targeted delivery of vitamins, flavonoids, and probiotics