The Extraction,Identification And Preparation Of Nano-liposomes Of Carotenoid From Arctic Ocean Rhodococcus(Rhodococcus Sp.) B7740

Rhodococcus sp.B7740 was separated from the Arctic seawater scholars,its lyophilized powder was cultured by Ningbo Key Laboratory of Microbial and Environmental Engineering.In present,Few studies had reported about the growth environment,metabolism,reproduction and secretion of Rhodococcus sp.B7740.This paper had been combined with microbial production of carotenoids literature reports,we used enzyme hydrolysis to break the wall,and combined with organic solvent extraction to extract carotenoids,Then experimented by saponification,HPLC to separate preliminary,used the LC/MS to identify the structure of carotenoids;Taking into account the physical and chemical properties and bioavailability of carotenoids,we used the thin-film dispersion method to prepare Nanoliposomes of carotenoids,and analyzed its storage stability and thermal stability,oxidation resistance.The main conclusions were as follows:(1)The optimization of carotenoids extracts from Rhodococcus sp.B7740Based on UV-visible wavelength scan figure of carotenoid,we found acetone/methanol(7:3,v/v)is the optimum extraction solvents of carotenoid.After single element and orthogonal experiment of enzyme hydrolysis and organic solvent extraction to extract carotenoids,we got the optimum conditions of extracting carotenoids:First,we used 1mg/m L egg white lysozyme(p H6.2 PBS)enzyme hydrolysis to break the wall,In accordance with lyophilized buffer with lysozyme material 1/150(m/v)to enzymatic90min at 40?,Centrifugaed separation and got the lower precipitation,In accordance with freeze-dried powder and solvent 1/250(m/v)ratio to extract 90min at 40?,Centrifuge supernatant were the carotenoids extracts.The extraction carotenoid content was about 1000?g/g,and its primary extraction rate was 88.15%.(2)Isolation and characterization of carotenoids extract from Rhodococcus sp.B7740Saponification could make the content of carotenoid from Rhodococcus sp.B7740increased,this was because the carotenoids esters or carotenoids acid are degradated in the system,while free carotenoid degradated less,which would lead free carotenoid content increaseing;then we used methanol+MTBE(1:1)as the solvent by high performance liquid chromatography(HPLC)to isolate 38 pesks,and preliminary identificated that it contains?-carotene.By LC/MS analysising,we found the extracted carotenoids belong menaquinone class,specific conclusions were as follows:MK₈(H₂)and MK₈(H₄),MK₇(H₂)and MK₇,C₄₀H₅₆O₂.These substances were composed of eight Isoamyl Diene and a methyl-naphthoquinone composition,their structure was different from common carotenoids.(3)Preparation and characterization of nano-liposomes of carotenoids extract from Rhodococcus sp.B7740Through the optimization of ultrasound,ultrasonic power,loads of carotenoids and buffer solution p H,We got the optimum conditions of preparation of nano-liposome as follows:Weighing the load 1%the carotenoid and L-?-lecithin,polysorbate 80(polysorbate 80 and L-?-lecithin to 1:0.72 quality)and completely dissolved in 7m L chloroform;used thin-film dispersion method and vacuum evaporation 60min to remove chloroform,then transfered it to a beaker to hydration mixing 60min;used the power of240W to ultrasonic 30min,standing for some time,then we can get the carotenoids nano-liposomes,The average particle size of liposomes was 50nm,dispersion coefficient(PDI)was less than 0.3,the embedding of carotenoids was 96.5%.Make the liposomes be storaged at 4?,37?and 80?,blank liposomes as controls,Liposomes of carotenoids had a certain degree of thermal stability and storage stability,it could be stored under at37?for 3days,80?for 2days and remain stability for 18days at 4?.We still found that oxidation resistance of carotenoids liposome was better than carotenoid extract and?-carotene in solution by DPPH free radical scavenging capacity and determination of reduced iron.