Fruit ripening

We have tested several type of fruit to verify that our engineered bacteria are able to produce ethylene (cell transformed with BBa_K1065001) and methyl salicylate (cell transformed with BBa_K1065102 and BBa_K1065106) for enhancing or blocking the ripening of fruit, respectively.

The flasks containing the induced culture were kept at 37 degree in agitation and connected to a sealed jar (or a desiccator) where the fruit was exposed to either ethylene or methyl salicylate (MeSA). Negative controls were either a fruit kept in airtight jar or a fruit placed in a jar connected to a flask with non-induced cells.

Within the same experiment we used fruit assumed to be at the same ripening stage (i.e.: the same bunch of bananas).

The results obtained varied according to the fruit tested. We tested: bananas, cherry plums, cherries and heirloom tomatoes, plums, blackberries, kiwifruit, pears and others. Results were evaluated qualitatively by observing skin color changes and pulp texture or firmness.

In some cases we were able to detect different levels of fragrance, however this results were too subjective and we discarded them.

We were also able to perform a iodine coloration test on starch-containing fruit.

We also performed quantitative evaluations of ethylene effects by fruit compression strength test.

In summary: cell transformed with BBa_K1065001 (our ethylene producing device) successfully ripen all the fruit tested when compared to the negative control, except for a few cases (Test with Date Tomatoes) where no significant effects were observed. We believe that in the experiment did not work because the temperature in the laboratory (in those days) was over 30 degrees, causing a decreased activity of lycopene synthesis enzymes. [reference]

Starch accumulates in apples during growing season and is hydrolyzed to sugar in the last stages of maturation and development (Krotkov and Helson 1946). Hydrolysis occurs first in the core area and progresses outwards (Phillips and Poapst 1952). Starch in cut sections of the fruit reacts with a solution of iodine-potassium iodide to produce a blue-black color. The intensity and pattern of the color developed is an indication of the stage of maturation.

A compression test determines behaviour of materials under crushing loads. The specimen is compressed and deformation at various loads is recorded. Compressive stress and strain are calculated and plotted as a stress-strain diagram, which is used to determine: elastic limit, proportional limit, yield point, yield strength and - for some materials - compressive strength. In our case, we were able to perform a compression strength test with kiwifruit and bananas.

Figure 1: Compression test on kiwifruit. After exposure to ethylene produced by BBa_K651001 for XX days the fruit were cut into small cubes of the same size. For each fruit we tested 5 samples. Negative controls were treated and analysed with the same method. Kiwifruit exposed to ethylene (in green) were significantly more ripen than the two negative controls used (no cells in blue, and non induced cells transofrmed with Bba_K1065001 in red), as demonstrated by the lower strength needed to compress the fruit.

Figure 2: Average of ultimate strength for kiwifruit treated with ethylene and negative controls. Kiwifruit exposed to ethylene produced by BBa_K1065001 and negative controls were left for xx days in airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimension from the same fruit. For a comparison we also estimated the strength based the best 3 measurements obtained. A significant difference between the treated and untreated kiwis is evident, indicating that our ethylene producing device (BBa_K1065001) successfully ripen the kiwis exposed to ethylene after xx days. b.

Figure 3: Bananas compression test. After exposure to ethylene produced by BBa_K651001 for XX days the bananas were cut into small cubes of the same size. For each banana we tested 5 samples. Negative controls were treated and analysed with the same method. Banana exposed to ethylene (in green) were slightly more soft than the two negative controls used (no cells in blue, and non induced cells transofrmed with Bba_K1065001 in red).

Figure 4: Average of ultimate strength for bananas treated with ethylene and negative controls. Bananas exposed to ethylene produced by BBa_K1065001 and negative controls were left for xx days in airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimensions from the same fruit. For a comparison we also estimated the strength based the best 3 measurements obtained. Treated bananas are xx fold less firm than untreated bananas.

Color area L*a*b* (CIELAB) The most used color area is the CIELAB area that uses L* (lightness), a* (redness) and b* (yellowness) parameters. This particular color area is the most complete color space and it describes all the colors visible to the human eye and was created to serve as a device-indipendent model to be used as a reference. [16] The L* parameter indicate a range between yields black (0) and diffuse white (100), the a* parameter indicate a range between red (+a*) and green (-a*, complemental color of red), the b* parameter indicate a range between yellow (+b*) and blue (-b*, a complemental color of yellow).
We used this test to quantify the difference between the color of ripe and unripe fruit's pulp.

Figure 5: L* represent the luminosity of the pulp's kiwifruit. We can show that the EFE sample is more dark than the control samples.

Figure 6: B* represent the yellow intensity, A* represent the red intensity. This experiment allow to quantify the normal different color between ripened and unripened kiwifruit. That confirm that the our construct that produce ethylene work in order to ripened more quick a fruit.

Cells transformed with BBa_K1065102 and BBa_K1065106 (our methyl salicylate producing devices) did not show any difference when compared to the negative control. We were not able to determine if MeSA actually inhibited fruit ripening because the experiments were not long enough to allow ripening of the control. We demonstrated by GCMS-FID that YY ml of bacteria culture (induced at O.D.600 equal to 0.6) transformed with our device produce successfully MeSA at a concentration of xx ppm in the culture medium. However, the final concentration of the inhibitor in the gas phase in the jar could be lower than the needed concentration. We are now in the process of better estimating these values.

Exposure to pure MeSA instead caused an excessive ripening when added to the culture medium while it had a toxic effect when the fruit was drenched with it. MeSA was reported to slow down ripening at high concentration (XX) and to accelerate the process at lower concentration (YY). [ref] We think that under the used conditions its concentration was above the threshold required to inhibit ripening.