Study On The Reduction Of Harmful Components In Smoke Aerosol And The Quality Improvement Through Fermentation By Microorganisms

Tobaccos are consumption goods which can satisfy people's physiological and psychological needs through the intake of smoke from burning. However, the burning of macromolecules in tobaccos, such as proteins and carbohydrates, can produce large quantities of harmful substances, including carcinogenic components like PAHS, TSNA, free radicals in gaseous phase and CO.A large number of research has proved that the harmful components is mainly generated after the burning of some macromolecular compound in tobacco such as protein and carbohydrate under high temperature which can not only produce harmful material but also has great impact on tobacco quality.Based on the chemical composition of tobaccos, this study aimed to screen the microorganisms which could effectively degrade the macromolecular substances in tobaccos, such as cellulose, proteins, pectin and starch. By adding such microorganisms in an optimized ratio, proteins and carbohydrates, which produced harmful substances, were degraded and transformed into micromolecular substances which improved tobaccos' quality. This process not only decreased the harmful components in tobaccos but also improved their interior quality.Totally, twenty-four bacterial strains were isolated from eleven cigarette brands, and were appraised in terms of their individual and colonial morphology and 16S rDNA. The twenty-four strains belonged to six species under the two genera Bacillus and Pseodomonas, respectively. These six species were Bacillus subtilis, Bacillus megaterium, Bacillus mucilaginosus, Bacillus cereus, Bacillus coagllan and Pseudomonas fluorescens. Thirty-four bacterial strains were isolated from aged tobaccos. Then, all these fifty-eight strains underwent the screening of cellulose-, protein-, pectin- and amylase-producing microorganisms, and three strains (QLB6, SHB1 & YTB4), which were characterized by strong reducing ability of nitrates and nitrites, were obtained. The cellulase activity of QLB6 was , the protease activity of SHB1 was , and pectinase activity of YTB4 was. Additionally, other enzyme activities were observed. These three strains were appraised in terms of their individual and colonial morphology and 16S rDNA. Results showed that QLB6 and SHB1 belonged to Bacillus subtilis while YTB4 belonged to Bacillus licheniformis.To enhance the enzyme production of these strains, He-Ni laser treatment was performed on QLB6, SHB1 and YTB4, with the aim to obtain positive mutants with higher enzyme activity and stable inheritability. Through mutagenesis, screening of enzyme activities and nitrate/nitrite reduction test, three strains (QLB6F, SHB1D & YTB4A) were screened out of the thirty positive mutants. Compared with the parental generation, their activities of cellulase, protease and pectinase rose by. Their stability of inheritance was proved by the ten-generation propagation test. Besides, no antagonism was observed among these three strains, and therefore they were suitable for mixed culture fermentation.QLB6F,SHB1D and YTB4A were optimized in terms of mixed fermentation medium and conditions. Orthogonal experiment involving 4 factors at 3 levels was carried out to determine their carbon source, nitrogen source, pH and cultivation time. In addition, remedy experiment was also performed to rectify pH and cultivation time. Finally, the optimal fermentation media and conditions for the mixed fermentation were determined respectively as follows: corn flour 1.5%, soy flour 1.0%, pH 7.0, cultivation time 24h. Based on the culture media and techniques of individual strains, the optimal strain combination determined by complex testing was 2:1:2. Cultivated after 24h, the quantity of the microbes could reach 13.8×10~8CFU/mL, and cellulose activity, protease activity, pectinase activity, amylase activity could reach 1486.71U/mL, 797.31U/mL, 643.25U/mL and 56.55U/mL. Tobacco fermentation experiments were carried out with mixed culture products of high enzyme activity. The results showed that enzyme and live microbes in mixed fermentation culture could effectively decrease the macromolecular compound in such a short time such as cellulose, proteins, pectin and starch. The research into the changes of microbe number can guide the utilization of enzyme and mixed fermentation products. One kilogram tobacco filaments were used in laboratory experiment. The orthogonal experiment determined that the optimal fermentation condition was: inoculation 2.5%, temperature 30℃, cultivation time 24h. After spraying, the cellulose, protein, pectin and starch decreased by 5.6%, 1.2%, 2.3% and 3.1% respectively. In addition, 0.2% Saccharomyces cerevisiae B181 was added together. The appraisement of tobacco proved that the stimulating flavor had been obviously lowered and the aroma improved. Both the quality and quantity of tobacco filaments had been improved greatly with much to spare. The 10kg tobacco test proved that after spraying into tobacco leaf, this mixed fermentation products from three strains could reduce the content of PAHs as Benzo[a]pyrene, TSNA as NNK and free radical in smoke aerosol dramatically overall.