Screening Of Producing Alpha-Glucosidase Inhibitor Strain And Research On The Fermentation Technology

α-Glucosidase inhibitors（AGI）can inhibit specifically the activity ofα-glucosidase hydrolyzing glycosides in the intestine and regulate postprandial blood sugar,being mainstream drugs for type 2 diabetes.In this study,a novel AGI-producing strain was isolated and characterized as Bacillus amyloliquefaciens FSY-3 by high throughput screening model in vitro.The effects of optimization of fermentation technology on DNJ production by FSY-3 were then investigated.Batch fermentation of raw inulin extract from Jerusalem artichoke tubers was performed in a 5-L fermenter to produce DNJ.To the best of our knowledge,this is the first report of the use of inulin as raw material to produce DNJ by consolidated bioprocessing（CBP）.Assisted by high-throughput screening model in vitro,a strain with stable GI synthesis was isolated from the natural environment.After morphological observation,16S rDNA sequence analysis,and physiological and biochemical experiments,it was identified as Bacillus amyloliquefaciens and named B.amyloliquefaciens FSY-3.The crude product of FSY-3 was determined as DNJ by HPLC after pretreated the fermentation broth.The fermentation medium and fermentation conditions of DNJ production by FSY-3 were optimized with single factor and orthogonal experiments in shake-flask.The results showed that a seed culture of B.amyloliquefaciens FSY-3 was carried out in a 250-m L shake flask at 30℃and 180 rpm for 24 h.The seed culture（1.0 m L;2%,v/v）was inoculated into 50 m L of fermentation medium and cultivated at 35℃and 140 rpm for 72 h.The optimized medium comprised 27.5 g/L inulin,8.0 g/L corn steep liquor,6.5 g/LMg Cl₂,15.5 g/LCa CO₃（pH 6.0）.The highest titer of DNJ could reach 55.48±2.14 mg/L in shake-flask.The aim of the comprehensive utilization of inulin,nitrogen source CSL was used to degrade inulin,reducing the discharge of sulfur-containing wastewater.The titer of DNJ was increased by nearly 15%with train FSY-3 capable of hydrolysing the inulin by production inulinases and also synthesizing the target product from the saccharified sugars.Further optimize the parameters of fermentation tank,the results show that as the inulin was consumed,DNJ production increased to 103.68±2.67 mg/L in 5.0-L fermenter with 220 rpm and 0.9 vvm at 40℃.The Jerusalem artichoke mainly compose of polyfructose inulin,used as a low-cost carbon source in the fermentation process.The raw inulin extract was obtained from the Jerusalem artichoke powder using different methods including hot water extraction,ultrasound-assisted extraction and CSL hydrolyzing in shake-flask.According to the elucidated DNJ biosynthetic pathway,DNJ synthesis was regulated by adding precursors,intermediate analogs and glycolytic pathway inhibitors.The results suggested that the strategy facilitated strain FSY-3 to synthesize DNJ from raw inulin extract under the conditions of supplementing sorbitol（1 g/L）,sodium citrate（7 g/L）and iodoacetic acid（75 mg/L）at 0 h and 12 h,respectively after hot water extraction pretreatment.The fermentation period is 1 day shorter by utilizing CBP strategy,FSY-3 produced 189.72±2.13 mg/L of DNJ with thermophilic condition of 40℃,simultaneously saccharifing Jerusalem artichoke and efficiently synthesizing the product in 5.0-L fermenter at 48 h,suggesting that FSY-3 has the potential for industrial application.