Analysis Of Bacterial Sources Reconstituted Tobacco Production Products And The Research On Application Of RIPs In Antibacterial And Anti-cancer

Tobacco,as one of important economic crops,has made many contributions to economic development of China.In order to ensure the safe production of cigarettes and improve the quality of cigarettes,we must strictly control the production process of cigarettes.The dominant strain in the production is Bacillus subtilis,which is beneficial to the improvement of tobacco quality.However,penicillium was introduced into the pre-swath pool slurry,which is unfavorable to tobacco quality.Camphor seeds ribosome inactivating proteins(RIPs)have an inhibitory effect on the growth of the introduced Penicillium,but have no effect on the growth of the isolated and purified Bacillus subtilis from the production of papermaking reconstituted tobacco leaves.In addition,camphor seed ribosome inactivating proteins(RIPs),which can inhibit the growth and reproduction of lung cancer cells H446,have an anti-cancer effect.The research results obtained are as follows:(1)We Collected and isolated the bacteria during Paper-making process reconstituted tobacco.We Counted and determined the total number of bacteria in each production process,separated and purified the obtained strain,and then sent the strain to BGI for 16 S rDNA sequence analysis,so as to analyze the source of bacteria in the production process of Paper-making process reconstituted tobacco.The results show that,nutrients and bacteria in tobacco raw materials enter in the extract and pulp respectively during the process of Paper-making reconstituted tobacco production.And the bacteria is continuously propagating,so the quantity value of total bacteria is increasing in these processes.Otherwise,in the paper-making reconstituted tobacco production raw materials,different production processes and the air they are exposed,the main strains under isolated and identified are basically the same.We found that the dominant strain is Bacillus subtilis,which is benefit to tobacco quality.It is worth noting that in the process of production,although the whole system is in an open air,the bacteria have produced dominant strains through adaptation and competition process,in that it does not affect the quality and safety of reconstituted tobacco products.Through the separation and identification of the main bacteria in the tobacco raw materials and each process reconstituted tobacco production,we showed that the bacteria in the process of paper-making reconstituted tobacco production were mainly derived from tobacco raw materials,and no other species of bacteria were introduced in.Moreover,there is no pathogenic bacteria exist in the production process of reconstituted tobacco,so the production process does not pose a potential safety hazard to the quality of reconstituted tobacco products.(2)After repeated low temperature and high speed centrifugation,we receive the crude protein of camphor tree seeds without impurities,lipids and some pigments.Otherwise we use Sephrose6 B coordination affinity chromatography to separate and purify the RIPs in the seeds,and then dialysis and concentrate the RIPs in cold storage.The molecular weight and concentration effect of RIPs from camphor tree seeds were estimated and observed by SDS-page.Last,we use the RIPs gained on the above and purified Bacillus subtilis and Penicillium in the production process of reconstituted tobacco to the next bacteriostatic experiment,the RIPs to the anti-cancer experiment on Lung cancer cell.The results showed that after optimizing the extraction method of RIPs crude protein from camphor tree seeds,the complex protein precipitation process could be avoided and the extraction time of RIPs target protein could be shortened.The degradation rate of 65 KDa Cinnamomin to 43 KDa Cinphorin protein was reduced,and the content of Cinnamomin in the purified target protein was increased.The results of bacteriostatic and anticancer experiments showed that RIPs extract from camphor tree seed has no obvious inhibitory effect on the growth of Bacillus subtilis.The growth inhibition of paecilomyces sp.by RIPs extract from Cinnamomum camphora seeds was significant.When the concentration of Tris-A protein extraction buffer solution was 5 mg/ml,the growth of lung cancer cells was the least affected;The growth and proliferation of lung cancer cells at the concentration of 10.3 mg/ml camphor seed RIPs was affected that the living cells gradually decreased,and apoptotic cells gradually increased.Camphor seeds RIPs have far-reaching research significance in the fields of reducing harmful bacteria in cigarettes,improving the quality of cigarettes,and improving the health value of cigarettes.In the analysis of bacteria during the production process of reconstituted tobacco leaves from paper-making,Bacillus subtilis is the dominant strain in the main isolates identified.The presence of this strain is beneficial to the improvement of tobacco quality,and the presence of Penicillium is unfavorable to the improvement of tobacco quality.Through the antibacterial and anti-cancer experiment results of camphor seed RIPs,we can see that the RIPs of eucalyptus seeds can inhibit the growth of lung cancer cells.The seed RIPs extract can inhibit the growth of Penicillium,and has no obvious inhibitory effect on the growth of Bacillus subtilis.This study is the first application of camphor seed RIPs in the production of reconstituted tobacco leaves from paper-making.The results of this study provide a good theoretical basis and technical support for the future antibacterial and anti-cancer research of camphor seed RIPs in the process of paper-making reconstituted tobacco leaves.