| As a kind of probiotics,lactic acid bacteria due to its excellent physical properties and biological activities have been generally researched by many workers.Peptidoglycan(PGN)as the main ingredient in the cell walls of lactic acid bacteria,its basic structure is composed of alternating N-acetylglucosamine and N-acetylmuramic acid by ?-1,4 linked key connection,and between the repeat unit bridge connecting tetrapeptide side chain by peptide interbirdge composed of polymer network structure.According to the report,as a kind of immune,PGN has made great progress on biological effects research which induce on the release of immune regulatory factors to stimulate the immune system of its anti-infection,anti-tumor and so on,but its features in live function remains to be further exploration,so this article chooses in vitro intestinal anaerobic fermentation method to study its good characteristics.In this paper,the research object is PGN which derived from Lactobacilus plantarum D1501 and L.helveticus MB2-1.Our objectives are to study the isolate and purify,structure characteristics and physiological function.1.This study extraction process of peptidoglycan is:the preciSItation of bacteria?breakage of bacteria cell wall? remove of teichoic acid ? removal of fat?removal of protein ?dialysis and purification ? centrifuge and freeze-dried.The extraction rate of PGN derived from L.plantarum D1501 is about 41.43%,The extraction rate of PGN derived from L.helveticus MB2-1 is about 16.67%.2.Using HPLC method for extraction of purity identification of PGN,and the results show that the peak area of PGN is 100%and the purity of soluble PGN is 100%.Respectively,using the method of Bradford,sulfuric acid-phenol coloration,Morgan-Elson,vanillin and ammonium molybdate to mearsure the protein,neutral sugar,amino sugar,fat,and phosphorous content of PGN,the results show that the PGN derived from L.plantarum D1501 and L.helveticus MB2-1 protein content were 35.56%and 38.46%,respectively;The neutral sugar content were 40.01%and 30.77%,respectively;Amino sugar content was 19.33%and 19.23%,respectively;Fat content were 3.56%and 9.74%,respectively;phosphorous content were 0.08%and 0.11%,respectively.The structural characterizations of PGNs were detemined by various methods.UV-wavelength spectra scanning showed D1501,MB2-1,D1501 lysozyme and lysozyme MB2-1 had protein of characteristic absorption peak at 280 nm,respectively.And there was no maximum absorbance in the range of 190?500 nm due to the PGN of no other impurities.The results of FT-IR spectra of PGN derived from L.plantarum D1501 and L.helveticus MB2-1 showed that they all had the obvioys absorption of PGN,and the absorption peak in 1070cm-1 represent the special structure of PGN samples:NAG-(?-1,4)-NAM.Amino acid analysis results of PGNs shows,the amino acid content of PGN from L.plantarum D1501 and MB2-1 is almost the same,the composition of main amino acid were Ala,Lys,Glu,Asp.Ala,Lys,Glu are the main amino acid composition of peptide chain,and Asp,Gly are as peptide bridge link components,which can be concluded that PGN from L.plantarum D1501 and L.helveticus MB2-1 are the size of Lys-1.Combining the results of 1H NMR,MALDI-TOF/TOF and above methods,we could speculate the proposed structure of PGN from L.plantarum D1501 and L.helveticus MB2-1.Observing the results of SEM,we could find that extraction of peptidoglycan structure are complete and its shape is similar to the shape of the bacteria itself which looks like a rod.Under the electron microscope,we can clearly see that there are many complete producing particles which is composed of many whole peptidoglycan mesh pocket structure of accumulation.We can get peptidoglycan molecular weight is about 68 kDa by SDS-PAGE analysis.4.The antioxidant activities of PGN in vitro were evaluated and the results showed that they have strong superoxide radical scavenging activity,moderate DPPH radical scavenging activity,hydroxide radical scavenging activity and ABTS radical scavenging activity,weaker ferrous ion chelating activity.Their antioxidant activities enhanced in the order of D1501 Lysozyme>MB2-1 Lysozyme>D1501>MB2-1,excepte the order of DPPH radical scavenging activity was D1501 Lysozyme>D1501>MB2-1 Lysozyme>MB2-1,and the order of ABTs radical scavenging activity was D1501>MB2-1>D1501 Lysozyme>MB2-1 Lysozyme.MTT assay and Tuluylene red pinocyfic test revealed that the pure PGN at the dose of 25-300 ?g/mL had no significant toxic effects on RAW264.7 cells,and can promote to the phagocytic ability of RAW264.7.Observed LPS induced macrophage RAW264.7 mice inflammation model in the activity of peptidoglycan by inverted microscope,we could find that PGN derived from L.plantarum D1501 and L.helveticus MB2-1 have certain repair effect on LPS induced inflammation.5.Total bacterial population,beneficial bacterium and harmful bacterium were enumerated by fluorescent in situ hybridization(FISH).And,the production of SCFA and lactic acid were determinated by HPLC.We could find that PGN have a certain value-added effect of beneficial bacteria,has certain inhibitory effect of harmful bacteria. |
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