Preparation Of Heparin Sodium By Interation Of Immobilized Enzymatic Hydrolysis With Membrane Separation And Effect Of Lmwh On Intestinal Flora

Enzymatic hydrolysis with membrane separation is an important technology in modern industry,which is efficient and energy saving compared with traditional method.In this paper,preparation of heparin by interation of immobilized enzymatic hydrolysis with membrane separation（IEM）,application of iron exchange resin for heparin purification and the effects of low molecular weight heparin（LMWH）on gut microbiota were studied.The main contents as follows:1)Membrane separation was employed to recover immobilized enzyme after extracting heparin.The factors that affect the processes of separation,including operating pressure,temperature,pH,pore size of membrane and flow rate were studied.One-factor-at-a-time experiments follow by orthogonal analysis was carried out to achieve the optimal separation condition as below:at a operating pressure of 0.3MPa,temperature of 40℃,membrane pore size of 800nm,flow rate 2m/s and pH11.Under the optimal conditions,heparin transmittance is up to 92.5%,immobilized enzyme can’t through membrane,the average volum of solution through membrane was 656.13 L·m^-2·h^-1.Using 2%NaOH,the membrane flux recovery was 97.9%.Repeating heparin extraction and separation with IEM,the heparin titer was reach the 80.6%and 61.2%before recycling when repeat twice and five times.2)In this manuscript,iron exchange resin was employed to heparin purification.The factors that affect the processes of heparin purification,including kinds of resins,resins dosage,temperature,adsorption time,pH,concerntration of elution,elution volum,elute time,sampling amount,sampling flow rate and elution flow rate were studied.One-factor-at-a-time xperiments followed by response surface analysis was carried out to achieve the optimal static purification conditions as below:using FPA98Cl resin at a resin dosage of 0.30%,pH8,temperature of 44℃,adsorption time5h,4mol/L NaCl was applied as elution,the ratio of resin and elution was1:3（w/v）,elute time was 3h.Under the optimal conditions,the rate of heparin adsorption was 97.75%and the rate of desorptin was 99.1%.One-factor-at-a-time experiments was carried out to achieve the optimal dynamic purification conditions as below:the sampling amount was156BV and 4mol/L NaCl was applied as elution with flow rate 1.5BV/h.Compared with commercially available heparin,the heparin preparated by IEM have certain qualitatively competitive advantage.3)In the present study we evaluated the effects of low molecular weight heparin（LMWH）on gut microbiota.Changes in gut microbiota in BALB/C mice after subcutaneous injection of LMWH for 30 days was characterized using the high-throughput sequencing and bioinformation analysis techniques of Metagenomics.The results show that LMWH of treatment resulted in a increase of Clostridium XlVa,Alistipes and decrease of Gammaproteobacteria.