Team:UNITN-Trento/Project/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_K1065106) for enhancing or slowing down 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 a 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, plums, cherries and heirloom tomatoes, blackberries, kiwifruit 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 and by a fruit color measurement test.

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Ripened plums become red

Type of fruit Plums Duration of experiment 4 days Exposition to Ethylene 4 days Container Fruit in 1 L jar

Cavendish bananas are successfully ripened

Type of fruit Cavendish banana Duration of experiment 6 days Exposition to Ethylene 6 days Container Essicator (10.3L)

Heirloom tomatoes ready to be eaten

Type of fruit Heirloom tomatoes Duration of experiment 9 days Exposition to Ethylene 9 days Container Fruit in 1 L jar

Cherry tomatoes are successfully ripened

Type of fruit Cherry tomatoes Duration of experiment 8 days Exposition to Ethylene 1 days Container Fruit in 0.5 L jar

Cherry tomatoes are successfully ripened

Type of fruit Cherry tomatoes Duration of experiment 7 days Exposition to Ethylene 1 day Container Fruit in 0.5 L jar

"Fried Green Tomatoes"

Type of fruit Date tomatoes Duration of experiment 7 days Exposition to Ethylene 2 day w/, 3 days w/o, 2 days w/ Container Fruit in 0.5 L jar Note: Lycopene synthesis probably inhibited by the high temperature (>30 degrees) in the lab as suggested in the literature

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 was used to determine the maturation stage of kiwifruit and bananas. All these measurments were done in the laboratory of dr. Alessandro Pegoretti in the engineering department. In this test the fruit is subjected to a crushing load to measure the resistence of a sample to compression strength. The test allows to measure the strength necessary to break the material analyzed, as an indication of maturation stage.

Figure 4: In the left panel bananas compression test. After exposure to ethylene produced by BBa_K651001 for 5 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). In the right panel Bruno show with curiosity the functioning of the machine.

Figure 5: In the left panel, a banana's sample between the plate of machinery. In the right panel 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 5 days in a 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 1.5 fold less firm than untreated bananas.

The test helped us to quantify the stage of maturation of kiwifruit and bananas, as another confirmation that our ethylene producing device works as predicted. However the test worked better on kiwis rather than bananas probably because the bananas used started already from a more advanced maturation stage.

Fruit color measurement test

We used this test to quantify the difference between the color of ripe and unripe fruit's pulp. All measurments were done in the laboratory of dr. Sara Spilimbergo in the engineering department.

We estimated the color of kiwifruit using a high resolution miniature spectrometer, connected to a optic fiber.

The instrument measures three parameters L* (lightness), a* (redness) and b* (yellowness). The L* parameter indicate a range between black (0) and 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). A shift in the color toward a darker green and a decrease in lightness are an indication of kiwi ripening.

Figure 6: In the left panel lightness measurament of kiwifruit treated for 8 days with ethylene (BBa_1065001 induced, green) and negative controls (No cells, blue and BBa_1065001 not induced, red). Treated kiwifuit have a lower L* value as a proof of advanced stage of ripening. In the right panel the instrument used to acquired the data. We note the high resolution miniature spectrometer which a fiber optic reflection probe connected.

Figure 7: In the left panel a detail of the fiber optic reflection probe and a slice-sample of kiwifruit. In the right panel the color measurement of kiwifruit. Average B* (yellow intensity) and average A* (red intensity) were calculated on 5 slices of the same kiwifruit. Kiwi treated with ethylene (BBa_1065001 induced, blue) show a consistent shift toward a darker green shade respect to controls (no cells, green and BBa_1065001 not induced, red). Methyl salicylate

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Date tomatoes stay green

Type of fruit Date tomatoes Duration of experiment 7 days Exposition to MeSA (BBa_K1065106) 2 day w/, 3 days w/o, 2 days w/ Container Fruit in 0.5 L jar

Cherry tomatoes unripen

Type of fruit Cherry tomatoes Duration of experiment 7 days Exposition to MeSA (BBa_K1065106) 7 days Container Fruit in 0.5 L jar Note: One of the control was probably at a different maturation state ad day1.

Pure MeSA has a toxic effect on cerry tomatoes

Type of fruit Cherry tomatoes Duration of experiment 8 days Immersion in MeSA pure 8 days Container Fruit in 0.5 L jar Note: The immersion in MeSA pure killed the fruit.

Cells transformed with 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 1 ml (analized GCMS-FID) of bacteria culture (induced at O.D.600 equal to 0.6) transformed with our device produce successfully MeSA at a concentration of 0.4mM 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 coused a toxic effect on cherry tomatoes. We also tested the effect of pure MeSA dissolved in LB but we obtain inconsistent results. For example in a few cases we observed an unexpected change color in the skin of plums and banana. MeSA was reported infact to slow down ripening at high concentration (0.5mM) and to accelerate the process at lower concentration (0.01mM). [The dual effects of methyl salicylate on ripening and expression of ethylene biosynthetic genes in tomato fruit. Ding, C. and Wang, Y. 164, 2003, Plant Science, pp. 589-596.] We think that under the used conditions its concentration was above the threshold required to inhibit ripening.

We conclude that our device (BBa_1065001) worked as expected, and successfully ripened fruit...and of course we TASTED IT!!!