Experimental Study On The Liquid Fermente Degradation Of Tea Saponin By Mixed Bacterias

Tea saponin is extracted from Camellia Linn plant roots, stems, leaves, seeds and flowers in a class oleanane type pentacyclic triterpenoid saponins mixture. In our study, tea saponin as the sole carbon source, with tea saponin degradation rate as the evaluation index, the use of microbial fermentation liquid shaker to study it.Seven kinds of bacterias including Penicillium citrinum, Aspergillus niger, Candida tropicalis,Bacillus licheniformis, Pseudomonas aeruginosa,Bacillus subtilis and Pseudomonas stutzeri were dominant bacteria screened on the liquid fermente degradation of Tea Saponin, by fermentation time, fermentation temperature, rotation speed and inoculum size single factor screening experiments,etc. Quantitative determination of their degradation rate by high performance liquid chromatography. The best actual degradation of the mix species were choosed to continue the next liquid bacteria fermentation experiments.The dominant bacteria screened by mixed bacteria combinations, by mixing ratio of inoculations, fermentation time, fermentation temperature, rotation speed and inoculum mixed with bacterias and the other single factors were considered to evaluate the effection of degradation. Through Plackett-Burman experiments and Box-Behnken experimental single factor for mixed fermentation, conditions were optimized to get the best fermentation conditions.The solid phase extraction small column on the fermentation products of extracellular and intracellular were gradient eluted by n-hexane, toluene, dichloromethane, n-butanol, acetone and ethyl acetate,. Finally the collected eluent would be detected product compositions by gas chromatography- mass spectrometry analysis. In this study, the conclusions were as follows:(1) Through dominant strains of screening results, three groups of mixed bacterias such as Penicillium citrinum mixed with Aspergillus niger, Bacillus licheniformis mixed with Pseudomonas aeruginosa, Bacillus subtilis mixed with Pseudomonas stutzeri were choosed to liquid fermentation experiments.(2) By single factor experiment and response surface experiments, the best fermentation conditions of Penicillium citrinum mixed with Aspergillus niger as: mixed inoculation ratio of 2: 1, fermentation time 7.8d, fermentation temperature 36.7 ?, rotation speed 150 r / min, 1.20% inoculated quantity. Under optimum conditions of the mixed liquid bacterias fermentation experiments, get mixed tea saponin degradation rate(78.53 ± 0.23)%. The best fermentation conditions of Bacillus licheniformis mixed with Pseudomonas aeruginosa as: mixed inoculation ratio of 1: 2, fermentation time 12.8d, fermentation temperature 30.7 ?, rotation speed 150 r / min, 8.81% inoculated quantity. Under optimum conditions the mixed liquid bacteria fermentation experiments, get mixed tea saponin degradation rate(73.76 ± 0.63)%. The best fermentation conditions of Bacillus subtilis mixed with Pseudomonas stutzeri as: mixed inoculation ratio of 2: 3, fermentation time 11.8d, fermentation temperature 30.8 ?, rotation speed 150 r / min, 8.69% inoculated quantity. Under optimum conditions the mixed liquid bacteria fermentation experiments, get mixed tea saponin degradation rate(74.08 ± 0.51)%. Penicillium citrinum mixed with Aspergillus niger in combination with other groups, the required fermentation time was shorter, and the degradation rates of tea saponin was higher.(3) Each combination of mixed bacteria fermentation under the optimum fermentation conditions extracellular, intracellular product was analyzed by gas chromatography- mass spectrometry for analysis, the results were showed: Penicillium citrinum and Aspergillus niger mixed liquid fermentation had received 24 kinds of extracellular fermentation products and 19 kind of intracellular fermentation products. Bacillus licheniformis and Pseudomonas aeruginosa mixed liquid fermentationg had received 17 kinds of extracellular fermentation products and 23 kind of intracellular fermentation products. Bacillus subtilis and Pseudomonas stutzeri mixed liquid fermentationg had received 21 kind of extracellular fermentation products and 18 kind of intracellular fermentation products. Mostly extracellular fermentation products including acids, esters, alcohols and hydrocarbons. Intracellular fermentation products mainly contained acids, esters, alcohols, methadones, phenols and ethers.