Team:Paris Bettencourt/Notebook/Anthocyanin


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:

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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:

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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

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

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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.

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Week 11th - 17th July

We extracted anthocyanin from grape skin. We started by peeling grapes and kept the obtained skins to separate it from the fruit, rich in carbohydrate.

For 1g of grape skins, we add 2.5mL of a solution of MeOH with 1% of HCl. We let the maceration overnight at the fridge. The original solution (transparent) became red and the grape skins were decolorized. In a second time, we evaporated the methanol into a bescher at 37°C, 150 rpm overnight. The dry extract was resuspended into 100 mL of water.
During this week we tried first without acide but we rapidly changed because acid form enhance the solubility of anthocyanin.

Week 18th -24th July

Anthocyanins are chemical compounds who change color with pH. We use this property to confirm that our extract contain anthocyanin by observing color swap at several different pH. The anthocyane appear red at pH 1, transparent at pH 4.5, Blue at pH 7 and Yellow at pH 9. We put 100µL of the obtained solution into 900µL of chloridric acid 37% (pH 1, red), Buffer solution (pH 4.5, transparent), water pH(7, violet) Buffer solution pH 12 (Blue) and NaOH 12M (pH 14, Yellow). We confirmed the presence of anthocyanin in our extract.

Week 25th -31th July

We tried to purify our anthocyanin with a liquid-liquid phase extraction. We started by adding toluene to our extract, we observed a biphase solution with the toluen down and the ethanol with anthocyanin. we separated the toluene phase with the anthocyanin. Then we added NaOH until we observed a color-switch from the red to the blue. After that we added again toluen and we observed the blue color into the upper phase. We kept this phase and switch again into acid with water and finally kept this phase.

Week 01st -05th August

We tried again to produce more anthocyanin with the method developed before but we observed that the toluen phase was everytime upper the aqueous phase. We don't understand why and decided to start to work with only extract non purified from protein and carbohydrate. Ethanol extraction, evaporation à 37°C extrait sec non purifié.

Week 08th -12th August

We used the anthocyanin quantification method developed by Lee et Al. in 2005. We quantified the anthocyanin extracted by measuring with the spectrophotometer the absorbance at 510nm and 700nm of a sample at pH 1 and pH 4.5.

Week 15 -19th August

Carbohydrate quantification with Fehling solution We prepared 200µL of solution A, 200µL of solution B and 200µL of our carbohydrated solution into 4.5 pH buffer. Then we measure the decrease of the blue color, consumed by the Fehling reaction. We made an absorbance measuring range of carbohydrate with glucose at 650nm and observed a linearity between 1g/L to 5g/L. We used this curve to quantify the sugar into our samples. We developed an assay to measure the degradation of anthocyanin by making a media M9 + extract for bacteria growth. At the day of measure, we take 600µL of to media and centrifuge 1min at 6000rpm. We take 3x150µL into TECAN 96 wells plates with 3x50µL of HCl 37%. Then we follow during a period the relative decrease of the signal. We started a screening of soil sample from several places in the world. We put 0,2g of soil into 10 mL of media and measure with the TECAN the absorbance to determine if there is into some samples bacteria able to degrade anthocyanin.

Week 23 -26th August

We analysed after 6 days of culture the quantity of anthocyanin for each conditions. We observed a contamination into our blank by bacteria and Funghi, this contamination showed a diminution of the quantity of anthocyanin in time. We still observed interesting data from different samples, E.Coli seems to show a non degradation ability of anthocyanin, which are expected. Sample with Lyon soil seems able to degrade anthocyanin when the same autoclaved soil from Lyon looked to be unable to degrade anthocyanin which is an argument for an organic degradation of anthocyanin. Bacteria 2, SS1.1 seems to be able to degrade anthocyanin. We decided to do again this experiment with a filtration step of anthocyanin to avoid a contamination of our media.

Week 29th August - 2nd September

Manip fail.

Week 5th -9th September

Do again Success Degradation with time

Week 12th -16th September

Rien

Week 19th -23th September

pH 7 media 10 bacteria Candidates Success 3 bacterias

Week 26th -30th September

Identification of the 3 bacterias


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