Study On Quantitative Analysis Method Of Pectin In Tobacco

Pectin is an important component of plant cell walls.It not only has an important impact on the physiological and quality traits of plants,but is also widely used as a food additive in human production and life.Pectin is a hydrophilic colloidal substance,which can maintain the moisturizing properties of tobacco by controlling the absorption of water,and has a binding and supporting effect on the cells of the tobacco plant.However,higher pectin content will lead to the production of acetic acid during cigarette processing and fermentation and the release of aldehydes and ketones harmful substances during the combustion process,which is extremely detrimental to the quality and safety of smoking.Therefore,the development of a quantitative analysis method is very necessary to accurately and quickly determine the content of pectin in tobacco for its effective control.This paper employed solid-state 13C CP/MAS NMR spectroscopy,near-infrared spectroscopy and meta-hydroxybiphenyl method to establish three quantitative analysis methods for pectin.Through the evaluation of their applicability and accuracy,it was found that they can be applied to the determination of pectin content in tobacco.The main work carried out in this paper was as follows:First,an analysis method was developed for quantification of pectin content in tobacco samples by solid state 13C CP/MAS NMR.A 5.5 mm outer diameter of dimethyl silicone rubber tube was designed and utilized as a reference of intensity,which was packed into a 5.5 mm inner diameter zirconia NMR rotor to construct a NMR sample tube.The powder samples filled into the sample tubes were detected to obtain 13C CP/MAS NMR spectra.The peak of C-6 at 171 ppm was processed with spectral deconvolution to eliminate interference from overlapping peaks.The calibration curve was established with the area ratios of assigned C-6 peak to intensity reference peak and the mass of the polygalacturonic acid(PGA).This method was used to determine the pectin content in six different tobacco samples.The relative error was ranged from-4.94%to 3.84%compared with the results measured by the standard method.The recovery of PGA from spiked tobacco samples was ranged from 94.33%to 102.77%,the RSD(n=5)was less than 2.32%.It shows that the 13C CP/MAS NMR method with a novel intensity reference is fast,accurate and simple,which is suitable for the quantitative analysis of pectin content in tobacco.Second,an analysis method that can quickly determine the content of pectin in tobacco was developed by using Fourier transform near-infrared spectroscopy.First,the near-infrared spectra of 203 tobacco samples were collected,and SNV+Second derivative+Savitzky-Golay smoothing filter(9-point)were applied to preprocess the NIR spectrum.Then,partial least squares method(PLS)was selected to establish a calibration model of pectin in tobacco among 4000?9000cm-1 of spectrum.At the same time,the optimal calibration model established by principal component regression(PCR)was utilized to comparison with that of PLS.The precision and external verification experiments were applied to investigate stability and applicability of the established calibration model.The results showed that the correlation coefficient(R2)of calibration model by PLS method was 0.9875,the root mean square error of cross validation(RMSECV)was 0.298,RPD(the ratio of standard deviation of validation set to square error of prediction)was greater than 5.80,and the ratio of the standard deviation of prediction(SEP)and the standard deviation of correction(SEC)was less than 1.2.Compared with the calibration model established by PCR method,the robustness and predictive ability of calibration model established by PLS method was better.Then,five tobacco samples were randomly selected and repeatedly determined for 10 times,it was found that the relative standard deviation RSD of the predicted values were all less than 1.80%,indicating that the precision and stability of the established calibration model were good.The absolute value of relative deviation of the chemical reference value of 45 externally verified samples and the pectin content predicted by the near-infrared calibration model was between 0.00%and 5.46%,and the average prediction relative deviation was 1.94%.Therefore,the calibration model established by FT-NIR spectroscopy and PLS method has good robustness and high prediction accuracy,and can be rapidly and accurately applied to the determination of pectin in tobacco samples.Third,a new method for the rapid and accurate determination of tobacco pectin content was established by the m-hydroxybiphenyl in this study.The ethanol solution of pH 2.0 and 80%volume fraction was utilized to quickly remove soluble sugars and pigments,and pectin was specifically extracted by enzymatic hydrolysis.Then the extracted pectin was completely hydrolyzed in a bath of concentrated sulfuric acid and boiling water.After cooling,adding meta-hydroxybiphenyl to react and measure its absorbance at 525 nm wavelength.For the measurement of 7 different tobacco samples,it can found that the relative error of this method was ranged from-2.93%to 3.45%compared with the standard method.The recovery rate ranged from 90.99%to 103.92%,the average recovery rate reached 96.97%,and the RSD(n=3)was less than 0.45%,the accuracy of this method was higher.In addition,the developed analysis method was evaluated and found that the color stability(n=6,RSD was 0.76%),reproducibility(n=5,RSD was 0.93%),and precision(n=5,RSD was 0.30%)were all good.When the glucose content was one times the pectin content,the absorbance only increased by 0.005,and the relative error of the absorbance was 3.79%,which had a certain anti-interference effect on neutral sugars.Therefore,the enzymatic hydrolysis-m-hydroxybiphenyl method established in this study is fast,accurate and simple,and is suitable for the determination of pectin content in all tobacco samples.