Separating And Screening Of Strains That Can Produce Pancreatic Alpha-amylase Inhibitors From Mangrove

Alpha-amylase inhibitors are a kind of glycosidase inhibitors that can inhibit the activity of alpha-amylase, which are widely used in medicine and agriculture. In medicine, alpha-amylase inhibitors are mainly used for the prevention and treatment of diseases such as diabetes, hyperlipidemia and obesity; In agriculture, alpha-amylase inhibitors can be made into biological pesticide or improve the insect resistance of plants by using transgenic technology.This paper was researched on separating and screening soil microbial strains with alpha-amylase inhibitory activity in Guangxi mangrove nature reserve. According to the morphological characteristics, physiological and biochemical characteristics and16SrDNA sequences analysis to determine the classification status. To optimize the strains’fermentation condition at the same time, in order to improve the strain fermentation products in the content of alpha-amylase inhibitors.This paper screened137strains by using dilution coating separation method from mangrove soil (in northern Haikou national mangrove nature reserve in Guangxi). Using agar plate hole method we first screened9strains (T1-T9) with pancreatic alpha-amylase inhibition effect from the strains’fermentation liquor. And then screened the strains T1-T9again by the method of Bernfeld, with acarbose as positive control, we found that strains T2from the roots of mangrove avicennia marina with the best inhibiting effect. The pancreatic alpha-amylase inhibition rate of the fermented stoste is34.88%.Identifying the morphological characteristics, culture characteristics and physiological and biochemical characteristics of the goal strains. We inoculated the goal strain T2to the Zobell-2216E culture medium, after5days’thermostatic cultivation, it formed a diameter of about4mm white and slightly transparent colony of bacteria. After10days we found, the surface of the colony is dry and how wrinkled, and the opposite of the colony is yellow, with the smell of mud. The colony combined with culture medium closely, is not easy to pick. We inoculated the goal strain T2to multiple culture medium to identify the characteristics of culture. Through observation we found that the strain can make gelatin liquefaction, make milk coagulation and peptone, can make the nitrate reduction and starch hydrolysis, does not produce H2S and hydrolysis of cellulose, can absorb and utilization of glucose, sucrose, soybean meal, mannitol and inositol these five carbon sources. Then sequence determination and analysis of the extracted16SrDNA of the goal strain, and compared the results with the sequences in GenBank library, the results showed that, the sequence homology of the16SrDNA sequences of the goal strain T2and the type species of U570682.1Streptomyces sampsonii and AB249937.1Streptomyces globisporus is very high, up to98%; The figure3-3shows that the goal strain T2and EU570682.1Streptomyces sampsonii are in the same branch. According to the observation of morphology, cultivate characteristics of the strain, physiological and biochemical characteristics and16SrDNA identification, preliminarily identified the goal strain T2belongs to Streptomyces streptomyces.By single factor test and orthogonal test, to explore the fermentation conditions of goal strain T2. The results show that the carbon source and nitrogen source that were better for goal strain T2to use were glucose and peptone, among the four conditions of fermentation (the fermentation time, culture temperature, initial pH and bottling), the fermentation time had the greatest influence on the output of alpha-amylase inhibitors of strains, primary and secondary order of the impact is fermentation time> bottle quantity> temperature> initial pH valu. The optimal level of various factors:A2(fermentation time168h), B2(temperature28℃), C3(initial pH7.5), D1(70mL bottle quantity). In the optimal combination A2B2C3D1alpha-amylase inhibiting rate of up to57.43%.Extracted the active ingredient of fermented liquid by ultrasonic extraction method (respectively using water, ethanol and petroleum ether as solvent), and made determinations of the inhibitory activity. The results showed that the alpha-amylase inhibition rate of concentrated solution of water extraction is as high as70.32%(equivalent to9.65mL of the fermentation stoste);the alpha-amylase inhibition rate of the concentrated solution of alcohol extraction is17.13%(equivalent to2.35mL of the fermentation stoste); the concentrated solution of petroleum ether extraction is6.73%(equivalent to0.92mL of the fermentation stoste). The results showed that the polarity of the objective product is bigger, choosing water for the subsequent experiments. After comparing with five kinds of resi, choosing macroporous resin D101for the first step purification, to found that water as eluent elution composition of the highest inhibition rate, at67.43%(equivalent to9.26mL of the fermentation stoste), followed by a component of the ethanol concentration of20%(equivalent to2.91mL of the fermentation stoste). Using thin-layer chromatography and silica gel column chromatography, and the elution system of chloroform:methanol (ratio of3:1), then obtained the component witch maximum inhibitory activity of alpha-amylase is81.69%(equivalent to11.22mL of the fermentation stoste). At the same time, the inhibition rate of1mg/mL of acarbose on alpha amylase is78.32%. After comparison, found that the inhibition effect of fermented liquid that after several purification on alpha-amylase is better than1mg/mL of acarbose. Obtaining extractum after decompressing and concentrating the collected component, making chemical composition prediction experiments, preliminarily determined the main ingredient of the component are indican and protein.