| Backgroud Muramic acid (Mur), C9H17NO7,Muramic acid is an amino sugarthat forms part of the peptidoglycan in prokaryotic cell walls, Since muramic acid isfound only in prokaryotes it has been used as a measure of bacterial and cyanophytebiomass， It can be used to characterise organic nitrogen of microbial origin, may beuseful for elucidating the microbial origin of organic nitrogen in differentenvironments (dust, soil, sediment, even animal tissue, etc.). It’s one of thecomposition of sleep-promoting factor, and one of the imporent composition ofmuramyl dipeptide (MDP), MDP is proposed to be the minimal essential structuralunit of bacterial cell-wall components required to stimulate a variety of biologicalactivities. Mur as one of the Leguminosae paly possible role in plant-bacteriainteractions, potential enzyme inhibitors of bacterial, anti-tumor,and other researchfield have a great prospect.Corynebacterium glutamicum can be used to ferment different products (glutamicacid, lysine, arginine, organic acids, etc.). The raw material of experiment isfermentation residue of Corynebacterium Glutamicum which fermented Glutamicacid to be used to produce monosodium glutamate (MSG). It’s annual production upto hundreds of tons, which is the main byproduct of the MSG industry..At present, several methods of measurement have been developed to detect thecontent of Mur, it can be directly analyzed by Chromatography System without otherstreatment, also can be derivatized to colored, stable, or volatile compounds, then usedColorimetric Assay, Mid-infrared spectroscopy, Gas Chromatography/GC-MS/LC-MS, HPLC, TLC and other laws. By far the most common method is the GC andHPLC methods. HPLC method has speeded analytical, short time, high sensitivity,widely application, and so on. amino sugar be detected by HPLC commonly userefractive index detector in order to keep sample, but the disadvantage is the sensitivity is low, instability, vulnerablely affected by temperature and mobile phase,for carbohydrate have wavelength absorption of nearby190nm in Near UltravioletBand (the maximum absorption wavelength of Muramic acid is199nm), thesolvent,impurities have also absorption wavelength in short-wavelength region, Itshould be use UV-visible detector after derived. Gas chromatography method has highsensitivity, minimum sample, speeded analytical, and low cost. If we choice gaschromatography to detect Mur, the problem is the Mur which has high boiling point,don’t volatility at low temperatures, so it is converted to derivatives of volatile,more stable to heat before gas chromatographic analysis.Objective on the basis of the previous studies, the purpose of this paper was designedto explore a method to extract muramic acid in rich fermentation residue ofCorynebacterium glutamicum which is hydrolysis as the raw material, To qualitativemuramic acid sample by the use of gas chromatography-mass spectrometry, massspectrometry and high performance thin layer chromatography, and quantitative Murby HPTLC scanning spots. muramic acid extraction process was optimized for factoryproduction. We also scanned purified muramic acid sample in Methanol solution ofspectral scanning, watched it’s crystallization crystal shape with microscope, we canhave a better understanding of the nature of muramic acid.Methods The extraction of muramic acid in fermentation residue of Corynebacteriumglutamicum: washed residue with distilled water, removed teichoic acid withtrichloroacetic, and contribute to lysate cell, hydrolyzed with hydrochloric acid, in theprocess of impurity in the sample, after acid hydrolysis, firstly, acid solution wasseparation with chloroform and water in liquid-liquid separation to remove lowpolarity material, then adsorpted colored substances and other impurities withactivated carbon of sugar, extracted in methanol or ethanol, adjusted PH from6.6to6.8, detected directly the crude samples in HPTLC or derived generate nitrileacetate derivatives of sugar for GC derivatives, analysised Mur sample whichseparated with column chromatography of activated carbon in Mass spectral. Forcritical process steps of Mur-hydrolysis of ermentation residue, select type of acidhydrolysis, time, concentration, temperature of hydrolysis, solid-liquid ratio, experimented these single factor to get the best experimental program. We simplifiedextraction process of muramic acid can be reduce extraction steps so that reduce thecost of industrial production. Extract the purity of muramic acid in HPTLC.Studied on the nature of muramic acid mainly scanned methanol solution ofmuramic acid’s spectral scanning, know its maximum absorption wavelength and thecharacteristic absorption peak; observated Mur’s crystal shape by microscopic.Results: The sample turn red from Colorless by Ehrlich reagent; The experiment havedetected Glucosamine link with Mur in the GC-MS analysis; The sample andmuramic acid have the same color spot in the same Rf, After scanned in the reflectionabsorption wavelength detected by high performance thin layer chromatography, wefound spots and Mur’s curve are consistent, so we can speculate that the samplecontains Mur or Mur derivatives, this spot was separated and purified by columnchromatography, the detection of sample that the relative molecular mass is251.3bymass spectrometry, this substance is identified Muramic acid. Muramic acid wasdetected by HPTLC will use mixing developer: two developing solvent-methanol/acetone/isopropanol/acetic acid/water (5:3:1:1:1, v/v) and acetic acid/n-butylalcohol/water (1:4:5, v/v), two kinds of staining methods-ninhydrin reagentethanol and Aniline-Phthalates acid reagent. TLC scanner scanned the pre-expandedHPTLC plates with492nm/365nm absorption wavelength, qualitative andquantitative of Mur sample, HPTLC scan detects bacterial，the value of Rf were0.84±0.01and0.60±0.02, the results of investigation for the experiment method:Intra-day variation was2.2396%-3.5324%（RSD）， inter-day variation was2.6545%-3.70534%（RSD）,the average recovery was97.22%, the content of Mur inthe fermentation residue is11.05192mg/g by HPTLC. The optimum extractionprocess: hydrolysis→decolorization→ethanol extraction→column chromatographyof activated carbon→purity detection (HPTLC), solid-liquid ratio is2,4mol/L ofhydrochloric acid hydrolysis6h at65℃heated, the mass activated carbon is0.1times with residue mass, decolorizated by activated carbon adsorption in water bath ofat45℃. The maximum absorption wavelength of Mur in methanol is199nm, crystalof Mur shape is divergence acicular. Conclusion The substance can be comprehensive identified by Ehrlich reagent,GC-MS, HPTLC, mass spectrometry. HPTLC was establish that it is an effective wayto measure the content of muramic acid in ermentation residue of Corynebacteriumglutamicum, It is aexperiment methods were rapide, feasible, accurate and practical.We worked out a simple and viable extraction process of Mur, and the maximumabsorption wavelength, crystal shape. |
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