Degradation Of Lignin In Tobacco Stems And Its Mechanism

Lignin, one of the three main components of cell wall in plants, accounts for 4%-8% in tobacco, which results in the quality defects of reconstituted sheets on some sensory attributes like off-taste, irradiation, cough. Moreover, lignin pyrolysis produces catechol, alkyl catechol and other substances, which can cause astringent taste and cancer risks, and hence the quality and safety of cigarette. Therefore, it is beneficial to improve the quality by lignin degradation and removal during the preparation of tobacco sheet. Laccase can selectively catalyzed lignin degradation and do not produce toxic substances under mild conditions of low temperature and atmospheric pressure, which is easy to realize industrialization. Therefore, it is of significance to study on lignin removal in tobacoo stem with laccase for the preparation of high-quality tobacco sheet, and further to explore the mechanism of the enzymic degradation of lignin in tobacco stem with laccse by using lignin model compounds.XSLacc, SUKALacc, YCLacc, DENYKEM PAP-5, DENYKEM INDICLEAN 6G+ and GrenLacc, six different laccases were used to the degrade lignin in tobacco stems. The results showed that DENYKEM PAP-5 contributed to a better effect of lignin removal. The smaller the solid-liquid ratio, the better the enzymatic hydrolysis effect is. Considering to enzymatic hydrolysis rate and the cost, the solid-liquid ratio was chosed to 3.0:100. It was found that lignin degradation rate of tobacco stem was 15.92% under the optimal reaction conditions of laccase dosage of 1.3%, the reaction temperature of 50 ℃ at pH 5.0 for 4h, and shaker speed of 150r/min.Vanillic acid, syringic aldehyde, hydroxy acetophenone etc. nine kinds of natural mediators were used into the laccase enzymatic hydrolysis reaction. The results showed that syringic acid had a positive effect in promoting the degradation of tobacco stem to a certain degree, and lignin degradation rate reached 16.64%.Tobacco sheet were prepared in the factory according to the optimal process variables of the laccase enzymatic degradation of lignin of tobacco stem. Smoking assessment by evaluating experts indicated that the properties of irritation and smoothness were improved and offensive odor was decreased significantly.The phenolic dimer lignin model compound(Ⅰ) containing β-O-4 bond was synthesized with isoeugenol and 2-bromo-4-hydroxyacetophenone as the raw material. The effects of reaction temperature and reaction time on the yield were explored. The result showed that the yield was up to 75.8% under the optimal conditions of 50℃ for 60 min. The non-phenolic dimer lignin model compound(Ⅱ) containing β-O-4 linkage was synthesized using 4-methoxy-2-bromoacetophenone and 2-methoxy phenol as raw material. A lignin model compound composed of α-O-4 and β-5 linkages(Ⅲ) was synthesized by the radical polymerization with isoeugenol as the raw material and horseradish peroxidase(HRP)/H2O2 as the catalyst. Then a phenolic tetrameric lignin model compound(Ⅳ) composed of α-O-4, β-5 and 5-5 linkages was synthesized using compound(Ⅲ) as the raw material and potassium persulfate and ferrous sulfate as redox initiator. The process variables of the synthesis were investigated. It was found that a 33.8% of yield was achieved under the optimal reaction conditions of 2:1 molar ratio of acetone to water, the total volume 30 mL, 80℃ of the reaction temperature and 10 min reaction time. The molecular weight and molecular formula of the lignin model compounds were measured by GC-MS, HRMS and EA. The structures of the products were characterized by 1HNMR and 13C-NMR.The optimal enzymatic hydrolysis conditions was applied to the phenolic dimer lignin model compound(Ⅰ) containing β-O-4 bond, the non-phenolic dimer lignin model compound( Ⅱ) containing β-O-4 linkage and the phenolic tetrameric lignin model compound( Ⅳ) composed of α-O-4, β-5 and 5-5 linkages respectively. The results showed that laccase led to the partial cleavage of the β-O-4 bond of phenolic lignin model compound(I). But for nonphenolic β-O-4 dimer compound(II) and containing β-5, 5-5 bonds phenol tetrameric model compound(IV), laccase could not break their bonds. Therefore, it concluded that the β-O-4 linkages in phenolic lignin was cleaved by laccase, resulting in the partial degradation and removal of lignin.