Screening Of Lactic Acid Bacteria Producing ?-amylase Inhibitor And Purification Of Active Components

?-amylase inhibitor is a kind of glycosidase inhibitor,which can inhibit the activity of ?-amylase,hinder the hydrolysis of macromolecular carbohydrates such as starch by?-amylase,and thus reduce the indexes of blood glucose and blood lipid.Therefore,?amylase inhibitors play an important role in the prevention and treatment of hyperglycemia,diabetes,hyperlipidemia and other diseases.Lactic acid bacteria is a kind of edible bacteria which is recognized to be very beneficial to human body.It has a lot of prebiotic functions.At present,more and more new functions are still being explored and studied.The development of hypoglycemic function and products of lactic acid bacteria is gradually becoming a new hotspot.In this paper,the existing lab lactic acid bacteria were screened and identified,and the fermentation conditions of producing ?-amylase inhibitors were optimized,and the inhibitors were separated,purified and characterized.The main research contents are as follows:(1)Screening and identification of Lactobacillus producing ?-amylase inhibitor168 strains of lactic acid bacteria were screened from the preserved strains in the laboratory,17 strains suspected of ?-amylase(porcine pancreas)inhibitor were screened,and a strain y9-3 with the strongest inhibition activity was screened through re screening,and the inhibition rate of ?-amylase reached 32.54%.The inhibitory effect of 1 ml y9-3 fermentation broth was similar to that of 1.565 mg acarbose.Through the morphological,physiological and biochemical characteristics of y9-3,16S r DNA sequence comparison and phylogenetic tree analysis,it is concluded that y9-3 can form clear and better colony after incubation at 37? for 48 hours.The diameter of colony is 0.2 mm-1.0 mm,the color is milky white,the surface is smooth and no other pigments are produced.Gram staining was positive,and the cells were rod-shaped without spore formation.The strain can utilize glucose,fructose,arabinose,lactose,maltose,can not hydrolyze starch,and can not liquefy gelatin.Methyl red test results show positive,hydrogen sulfide and catalase test results are negative.After homology comparison on NCBI website and phylogenetic tree analysis,the Lactobacillus y9-3 was identified as Lactobacillus plantarum.(2)Optimization of fermentation conditions for ?-amylase inhibitor producingLactobacillusThrough the selection of carbon source and nitrogen source,the inhibition activity of?-amylase was the highest when the carbon source was glucose,which was 32.04%,and the inhibition rate of ?-amylase was the highest when the nitrogen source was yeast extract,which was 32.41%.The best inoculum quantity,fermentation time,fermentation temperature and initial pH were 4%,48 hours,36?,110 ml and 6.5 respectively.Under the above conditions,the inhibition rate of ?-amylase was the highest.The optimal combination of fermentation conditions was determined by orthogonal test:The initial pH was 6.5,the bottling volume was 110 ml,the fermentation temperature was 38?,and the fermentation time was 48 h.At this condition,the inhibitory activity of bacteria fermentation supernatant on ?-amylase was the highest,reaching 47.25%.than the enzyme inhibition rate before optimization increased by 42.13%.(3)A preliminary study on the separation and purification of ?-amylase inhibitorsThe highest inhibition rate of ?-amylase in n-butanol organic phase was 84.42%.When sephadexlh-20 chromatography was carried out,the active components of the inhibitor began to be eluted from the 12th tube,and the inhibition rate reached the highest in the 23rd tube.The inhibition rate was 89.36%by combining and concentrating the obtained stream with inhibition activity.The peak with the highest inhibition activity was determined by ?-amylase inhibition rate,LC-MS/Ms.after analyzing the chemical composition and combining with the mass spectrum of the inhibitor,it was speculated that the composition of the inhibitor might be protein and carbohydrate compounds Mixture,and is a small molecular weight material.The study of its physical and chemical properties showed that the temperature had little effect on ?-amylase inhibitors.When the temperature was 45?,the inhibition activity of ?-amylase was the highest,and the inhibition rate reached 88.23%,while the pH had a great influence on ?-amylase inhibition rate,and the inhibition rate of ?-amylase reached the highest,88.69%,when the pH was 5.