Week 27th June - 3rd July

Week 4th - 10th July

We implement the protocol based on Zhang Hua et al. 2013, this protocol fits in our expectations of not to use acids and dangerous solvents. Here a aqueous two phases systems is used to extract the anthocyanins. We chose the ratios of different solvents and sample based in the figure 2 and 3 of the paper, where the partition coefficient is maximum at 28% of ethanol and 20% of amonium sulfate.
We decide to follow the protocol called C in the paper since this wasthe one with the highest partition coefficient. (Figure 4)
The protocol can be found at the protocol section, and it is called Aqueous Two Phase Extraction.
The separation of the anthocyanins is done by its solubility in ethanol. The amonium sulfate disolved in the water stabilize the interaction between water molecules and it reduces its solutibilty in ethanol, therefore the anthocyanins which where in water at the begining will be finally disolved into the ethanol, then by recovering the upper face, composed of ethanol and some water, we can harvest the anthocyanins which can be concentrated by evaporation of the ethanol by shaking an overnight in the incubator a 37C, since anthocyanins become unstable and are degraded up to 45C.
We also implemented a method to measure the quantity of anthocyanin after the extraction where the anthocyanins where concentrated. In principle the method is useful to quantify in an aproximative way, or at least relative way, since the extinction coefficient of the complex mix that we obtain is not the proper one because we dont really know the composition of the mix regarding the quantity of anthocyanins.
Such protocol is based on the paper written by Ronald E. Wrolstad in 1976. The principle is to measure the difference of absorbance between the extracted mix a pH 4.5 and pH 1. It is because at pH 4.5 the anthocyanins are transparent, whereas a pH 1 they are red and therefore they have a pick of absorbance at 520nm. By computing the difference of absorbance at this wavelength is possible to quantify the relative quantity of anthocyanins respect to other samples. In the future, if we can make experiments with known concentrations of anthocyanins we could know the extinction coefficient and quantify in an absolute way the quantity of anthocyanins.
The protocol can be found at the protocol section, and it is called Anthocyanin quantification.
We start by carrying out the protocol Aqueous Two Phase Extraction in order to extract the anthocyanin content of blakberries. The origin of the sample comes from a commertial stabliment (Picard), and the fruits are frozen at -20C.
Instead of following the part of the protocol in which the sample is prepared, we used all the fruit to extract the anthocyanidins.
Using 5 grams of grinded blackberries as sample.

Extraction seems to be succesful:

Here is possible to see the two phases, the upper one, kind of red, containing the anthocyanins and the lower one with water. In principle both phases contains impurities, but it won't be a problem for some of the assays, although it will be for the problems of microorganism isolations.
In order to ascertain if we have anthocyanins in the upper phase we decide to change the pH and see if the color of the sample changes according with the expected change in the anthocyanins absorbance:

In this figure we can see the pH values below the tubes. The color correspond with the expected values, from red at low pHs to yellow when the pH increases.
Once we realized that the procol worked we decided to work directly with blak grapes, in order to do that we also follow the protocol Aqueous Two Phase Extraction.
We used 200g of black grapes from Italy, in this case we follow all the protocol from the begining, including the preparation of the sample where the skin of the fruit is harvested.
Finally we extract 45g of skin sollution to do the extraction.
Therefore the weight of the solvents is 900g:
- 180g of Ammonium sulfate + 468g H2O
- 252g of pure ETOH but we use 96% ETOH so we need to weight 262g

Once we recover the upper phase (the red one in the figure above), we adjust the pH at different values:

In this case the values of the colors were different as for the blackberries, since the yellow color is reached before and after pH 8 the color goes to green.
We quantified following the protocol Anthocyanin quantification, in the TECAN INFINITE M200 PRO reader, 200ul of sample and we read the spectra of each pH value from 350nm to 700nm.
We have a nice peack at 530nm and with the expected pH, pH 1.
By this method we can quantify the antocyanin in a relative way. Therefore we can quantify the anthocyanin before and after and assay, and it is for instance useful to mesure the consumption of it.

Week 11th - 17th July

We started a new method of anthocyanin extraction from grape skin directly, which is the most concentrated tissue in anthocyanin. those grape skin macerated into 10 mL of a methanol solution with 1% of Chloridric Acid.
We observed an intense red coloration of the solution and in the same time a décoloration of grape skin. We confirmed the presence of anthocyanin by observing color switch at different pH, characteristic of anthocyanin. We then started to think about a purification process.

Week 18th -24th July

Anthocyanins are chemical compounds who react with pH. At pH 1, the anthocyanes appear red and blabla pH variation et LogP ; confirmation avec photos.

Week 25th -31th July

Methanol + Toluene fail phase rouge en bas, pas d'explication

Week 01st -05th August

Ethanol extraction, evaporation à 37°C extrait sec non purifié.

Week 08th -12th August

Methode de quantification des anthocyanes Screening 1

Week 15 -19th July

Methode de quantification des sucres, fehling reaction avec titrage indirect. Screening 2

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France

+33 1 44 41 25 22/25

igem2016parisbettencourt@gmail.com
2016.igem.org

Team:Paris Bettencourt/Notebook/Anthocyanin