Study On The Structure And Content Of Tobacco Cell Wall Polysaccharides By 2D NMR

Plant cell wall polysaccharides consist of cellulose,hemicellulose and pectin,among which the content of cellulose and hemicellulose accounts for more than 70%of cell wall polysaccharide.In tobacco,cellulose is the basic substance of the tobacco cell tissue and skeleton,which determines the integrity of the tobacco leaf.Hemicellulose has the function of strengthening the lodging resistance and biological invasion of tobacco plants.However,during cigarette combustion,cellulose and hemicellulose are prone to produce harmful substances such as aldehydes,tar,and polycyclic aromatic hydrocarbons,which have a negative impact on the smoking quality and safety of tobacco.Therefore,this paper focuses on the structural composition and content distribution of cellulose and hemicellulose in tobacco,which is an important guide to improve the smoking quality and safety of tobacco.Two-dimensional heteronuclear single-quantum coherence NMR technique（2D HSQC NMR）has become one of the important means to characterize the structure of lignocellulosic biomass due to its excellent spectral resolution.In this work,we firstly established a method to characterize the structure units of tobacco cell wall polysaccharides and their content distribution by using 2D HSQC NMR technique,and realized the qualitative and semi-quantitative analysis of cellulose and hemicellulose structure units and their contents in different types of tobacco samples.In addition,the quantitative analysis of tobacco cellulose was achieved by selecting suitable internal standards and relaxation reagents.Finally,the established 2D NMR spectroscopy method of tobacco cell wall polysaccharides was used to analyze different types of tobacco samples in an attempt to find the correlation between tobacco "ligneous odour"and the chemical structure of polysaccharide macromolecules.Specifically,the main contents of this paper was as follows:1.One method for characterization of tobacco cell wall polysaccharide structural units and their content distribution was established.In NMR experiments,effective solubility of samples in a suitable solvent system is one of the keys to obtain high quality spectra.In this paper,tobacco samples were firstly pretreated by acetylation to obtain acetylated tobacco cell wall material,which increased the solubility of samples in NMR solvents.In order to obtain higher signal intensity and rich structural information,three solvent systems,DMSO-d₆,DMSO-d₆/pyridine-d₅（4:1 v/v）,and DMSO-d₆/HMPA-d₁₈（4:1 v/v）,were selected to explore the effect of solvents on the 2D NMR spectra of tobacco cell wall substances.Based on the richness of the spectral signal and the higher signal intensity,DMSO-d₆/pyridine-d₅（4:1 v/v）was determined as the optimal solvent.In addition,standard microcrystalline cellulose samples were selected in the optimal solvent for the precision evaluation of the method.The results revealed that the deviations of the chemical shifts of the characteristic peaks of microcrystallinc cellulose were all less than 0.1 ppm,and the relative standard deviations（RSD,n=4）were all less than 0.22%,indicating the good precision of the method and the high stability of the instrument.Six major monosaccharide structural units,including glucose,xylose,mannose,arabinose,galactose and glucuronic acid structural units,were identified by attributing the signals in the spectra according to the chemical shifts.Among them,the glucose structural unit was mainly derived from the glucan structure of cellulose,and the remaining five monosaccharides belonged to the structural unit composition of hemicellulose.The results of quantitative analysis revealed that tobacco cell wall polysaccharides contained the most glucose structural units,followed by xylose structural units,and the remaining monosaccharide structural units had slightly different distribution in different types of tobacco samples.Finally,the monosaccharide analysis of tobacco cell wall polysaccharides was performed using gas chromatography-mass spectrometry（GC-MS）,and the good agreement between the results of the two methods was verified.2.A new method for the quantitative analysis of tobacco cellulose by 2D HSQC NMR was developed.Traditional chemical methods for cellulose content analysis involve successive extraction and separation steps,which are time-consuming and environmentally unfriendly.In this paper,acetylation was used to make the conversion of cellulose in tobacco samples into acetylated cellulose,which can be dissolved in DMSO-d6/pyridinc-ds solvent for NMR analysis.In addition,the sensitivity of the analytical method was improved by adding acetylacetonate chromium relaxation reagents to reduce the T1 value of acetylated cellulose.A standard curve of cellulose with 1,3,5-trimethoxybenzene as the internal reference was established to overcome the quantitative errors of 2D NMR quantification.The results revealed that the limits of detection and limits of quantification were 0.74 mg/mL and 2.45 mg/mL,respectively,and the spiked recoveries of cellulose were between 90.66 and 103.12%with the RSD（n=3）of 2.39-3.54%.Compared with chemical methods,this method is simple,accurate and environmentally friendly,which could expand a new idea for the quantitative analysis of various plant macromolecules.3.Correlation analysis of cell wall polysaccharide composition and sensory quality in different types of tobacco samples.Using the established 2D HSQC NMR method for structural composition and semi-quantitative analysis of tobacco cell wall polysaccharides,different types of samples,such as flue-cured tobacco,tobacco stems,reconstituted tobacco were analyzed.Sensory evaluation of the "ligneous odour" of the samples was performed by professionals to obtain sensory scores.Correlation and stepwise regression analyses were performed between the data obtained on the distribution of polysaccharide structural unit content and the sensory evaluation scores by SPSS 25.0 statistical software to find the correlation between the "ligneous odour"and the chemical structure of polysaccharide macromolecules in tobacco.The results revealed that there was a significant positive correlation between the content of glucose structural units and sensory evaluation scores,while the content of xylose,mannose,arabinose and galactose structural units was significantly and negatively correlated with the sensory evaluation scores.In addition,the results of stepwise regression analysis suggested that the higher the content of glucose structural units,the higher the score of sensory evaluation of "ligneous odour",which is unfavorable to the sensory quality of tobacco,while the content of arabinose structural units has a negative effect on the score of sensory evaluation of "ligneous odour",and increasing the content of arabinose structural units in polysaccharides may be beneficial to improve the "ligneous odour"of tobacco and improve the sensory quality of samples.In summary,this paper successfully established a 2D NMR spectroscopy method for the characterization of tobacco cell wall polysaccharides,which revealed the structural composition and content distribution of tobacco cellulose and hemicellulose,and applied the established method to the correlation analysis of polysaccharide structure and content distribution of different types of tobacco samples with the"ligneous odour" of tobacco,which provides some technical support and theoretical guidance to find the intrinsic connection between tobacco cell wall polysaccharide and the smoking quality and safety of tobacco products.