Team:UST Beijing/Demonstrate
Mixed Fermentation
Because the solid-fermentation is simple operation and low cost, We use this measure to deal with notoginseng. Through the experiment, we know that the rhizopus can effectively break down the cell wall of notoginseng, and release a large amount of reducing sugar. This sugar will become carbon source for E.coli to grow up, and provide enzyme to decompose the saponin of notoginseng.
In this system,it contains notoginseng, rhizopus and synthetic E.coli.The notoginseng is culture medium, the rhizopus can decomposed the cell wall and provide carbon source for E.coli. The E.coli product β-glucosidase to depart the sugar from saponin.
Grain medium
At the beginning of the experiment, we hope to find a new solid medium to ferment the E.coli, and then the use of E. coli hydrolysis the saponin in notoginseng.
Considering the economic factors and practicality, we paid attention to the grain medium to test whether it can provide full of nutrition to E.coli growth and reproduction or not , such as corn meal, millet, rice and so on.
Red fluorescent protein, which is also called RFP, is widely used as indicator or reporter gene in iGEM and other researches because it is easy for observation and measurement. Therefore, we used the BL21(DE3) with RFP to test the medium. The result was exciting that the E.coli can grow on different grain mediums.
But there were still some questions in this experiment. The first and the most fatal shortcoming is that the growth of E. coli required a few weeks. Obviously, it is not practical for us if it’s needed to wait a few weeks for each experiment. Because of the solid medium, it was difficult to be shaken by shaker. Therefore the E.coli can't obtain sufficient oxygen to reproduce rapidly. The second question that the long growth period can rise the risk of contamination by other bacteria also causes a headache.
Notoginseng medium
At this moment, however, we tried a new medium--notoginseng medium. We put the E.coli into paste of notoginseng directly. Fortunately, we had succeeded. That meant we don't need the grain medium to culture the E.coli.
Referring to relevant literature, we knew that the cell wall of notoginsing root is composed of Polysaccharides, such as galactose, glucose, arabinose and so on. After learning of these, we thought if we can hydrolysis its cell wall to release carbohydrates, the paste of notoginseng will become a natural medium and the carbohydrates will be carbon source.
Though, this idea may make the grain medium worthless. That means our two month work was in vain. It is very disappointing, but exciting.
Mixed fermentation
We only turned our attention to focus on how to hydrolysis the cell wall of notoginseng. We contacted the reality that the rhizopus and yeast were widely used in home fermentation. The principle of these process is similar with our experiment aim for cellulose hydrolyzed. So we brought edible rhizopus and yeast.
The experimental procedure is very simple. At first, we would boil the notoginseng powder with some water about 20~30min. The aim of this step was breaking some of glucosidic bond in carbohydrates and sterilization. During the boiling, we would prepare a glass jar disinfected by 70% ethanol. Then we dropped the paste of notoginseng into the glass jar waiting for cooling down. When the fermentation system cooled to room temperature, we sprinkled the rhizopus, yeast or both of them.
About a week later, the paste was beginning liquefaction, and a fragrance of wine was produced.
In two months’ time, we carried out four groups of fermentation with different bacteria. At the same time, we used the DNS to test the reducing sugar concentration.
DNS would react with reducing sugar resulting a red product after 2min at 100℃. The product can be measured at 540nm wavelength by spectrophotometer. Through drawing the standard curve by different gradient of glucose solution, we could know the concentration of reducing sugar in the glass jar. Finally, the concentration would be achieved 10g/L after two month fermentation.
At this time, we would boiling the paste again to kill the rhizopus, yeast and other bacteria if it existed. After cooling down, we poured the E.coli with double-plasmid and E.coli with RFP into two groups of paste to go on fermentation.
Three weeks later, we used the Microporous resin to adsorb the saponin in paste of notoginseng overnight. The second day, we eluted resin by water and ethanol . When the ethanol dried down, we tested how many glycosyls of saponins had been hydrolyzed.
Future plans
Because solid fermantation is easy to operate and cheap, it's available to families. As for its good performance of health caring, notoginseng will became a good health product if families can decompose its cell wall by solid fermentation to make it easier for people to absorb saponin. And more, notoginseng tastes bitter, but it will smell like wine and sugar and taste great after solid fermentation. Also, rhizopus can be bought in supermaket, and it's non-toxic to eat, so this method is possible to practice.
