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The secret of mature fruits

By studying the metabolism of fruit after collection, a team has unmasked the biochemical processes that make them sweet and soft. The research opens up the doorway for changes that will slow down ripening

Could it be that still life paintings actually reflect stillness in life?  The negative response inevitably comes up as we enter into the laboratory of Professor Franco Lajolo, Head of the Department of Food and Experimental Nutrition of the Pharmaceutical Science School of the University of São Paulo (USP): here, bananas and papaya breath normally in containers with tubes, from which they absorb oxygen and breath out carbon dioxide gas.

This is the main setting of the thematic project Post Harvest Biochemical Transformations and the Quality of Food and Raw Materials. Kicked off two years ago with financial support from FAPESP, it covers two themes: the sweetening and the texture of vegetables. The results could lead to the production of sweeter (or less sweet) bananas. In the case of texture, the study of the cellular walls could help to prevent problems with the reverse processes: the softening of the papaya fruit and the hardening of the bean.

Fruit are kept under controlled conditions of humidity and temperature, while samples are periodically taken to study their metabolism during ripening. For their preservation, the samples remain frozen, surrounded by a container with liquid nitrogen at – 80o C (Negative 80 degrees Celsius).

Better quality
Fruit are “alive”, both when whole and cut up, because their physiological processes go on after harvesting, making them change color, smell and softness. Coordinated by Lajolo, a pharmaceutical biochemist, and a doctor in Food Science, the project aims to uncover the metabolic processes that occur during this phase – or that is to say, to discover everything that happens to fruit after its harvesting.

The fruit are of known origin and the researchers follow its development right from flowering. “We have control of the life of the fruit, so that we can carry out all the comparisons necessary”, says the chemist Dr. Beatriz Rosana Cordenunsi. Controls of humidity, temperature and breathing are carried out, as well as studies of the chemical compounds and the levels of carbohydrates, sugars, starch, sucrose and fructose, among other substances.

Lajolo is optimistic about the results already obtained. He explained that the fruit becomes sweet because its carbohydrates change and soften because of alterations in the cellular walls of the pulp. The biochemical transformations being studied are diverse. Highly coordinated, these modifications are related to the action of enzymes and have the participation of hormones.

“The idea of the project”, he explains “is to find out what controls these transformations, what is the mechanism and the molecular bases involved in this process. For this reason we’re studying more deeply the mechanisms associated with the carbohydrates (for sweetening) and the cellular walls (for texture), which are the important quality parameters.” When we learn all about this process, it will be possible to develop technologies that increase the quality and the durability of fruit.

Enzymes sweeten banana
For the sweetening process, the research concentrated on bananas (Musa paradisiaca), considered to be a good example of the metabolism changes of the carbohydrates: it is easy to follow its ripening, for example, through the successive changes in color. While it is maturing, its breathing accelerates. Furthermore, its oxygen intake increases as the changes increase. By measuring its respiration, the researchers can capture the signs of the internal work of maturing.

The team has already made important discoveries. The banana is composed of around 20% starch, which during ripening transforms itself into sugars: this occurs because various enzymes act upon the starch grains and break them down, while other enzymes transform them, synthesizing sucrose (one of the natural forms of sugar). “One of these enzymes, the SPS (sucrose – phosphate synthase), particularly interests us. What we see – and what we don’t understand – is that, during the process of ripening, this enzyme has its quantity increased though the activation of its respective gene: thus it is the gene that can control the synthesis of sucrose”, Lajolo tells.

He reveals that another enzyme, named sucrose synthase (SS), can also act in the formation of sucrose, but they don’t know how it interferes in the process. Now it is known that in the banana the SS doesn’t take part in the sucrose synthesis: while the gene of the SPS enzyme is active, that of the SS is switched off.

Sequenced genes
There was another conquest that led to these conclusions. “One of the unprecedented results of the work was the cloning and the sequencing of genes of the SPS enzyme of the banana, as well as that of the SS”, highlights João Roberto Oliveira do Nascimento, also a pharmaceutical biochemist with his doctorate in Food Science and who worked in the area of molecular biology during the project. He isolated and cloned a part of the DNA (deoxyribonucleic acid) that contains a code for these enzymes. The fragments of DNA were sequenced and in this manner they could determine the formula of these genes.

“What we realized about the SPS is that, when the banana is maturing and producing lots of sugar, during two to three days, we see an increase in the quantity of RNA (ribonucleic acid), a sign that that gene is being activated”, sums up Nascimento. This result was confirmed through the analysis with antibodies produced against the proteins of the fruit previously purified. The group keeps on studying the function of these other genes that break up the molecules of the starch grain, and providing substrates for the synthesis of sugar.

As well, the sequences of the genes of phosphorylase a and b amylase have already been partially obtained. Initial results with probes of DNA and antibodies show that the activity of some enzymes depends on the activation of genes during ripening, while that of others (such as the phosphorylase) does not.

In the cell
The basic methodology is advanced: electronic microscope, DNA probes, antibodies, mass spectrometer. But not everything needs to be so sophisticated, and good examples are the models that the group has developed.

Beatriz Cordenunsi, a doctor in Food Science, and responsible for the biochemical studies during ripening, explains: “We took some bananas, cleaned them well on the outside to make that there was no contamination, and placed them in special containers under controlled temperature. In the container there are series of tubes to let the air on and out. The outgoing air passes through a piece of equipment where the respiration of the fruit is measured.”

Another study model is using slices of banana. The researchers infiltrate into the slices substances that interfere with the metabolism, so later they can study the reactions. From this, another discovery popped up: the infiltration with the vegetal hormones indole acetic acid and gibberellic acid slowed down the ripening of the banana. It was verified that they affected the expressing of the genes linked to the break down of the starch (a and b amylase).

Papaya texture
The studies on the texture are being carried out starting with the papaya (Carica papaya), which softens very quickly, as well as with the bean (Phaseolus vulgaris), which tends to harden after it has been gathered.

The papaya was considered to be a good model because of the evolution of its texture: as in other tropical fruit, it changes rapidly after having been collected and too quick softening can cause large economic loses.

Little is known about the biochemical basis of this softening, but the team decided to concentrate on its cellular wall. “We’re associating the chemical structure of the cellular wall and its chemical organization with the enzymes that carry out all of these transformations. And we’ve already discovered that there is at least one important enzyme synthesized during the process – the beta galactosidase (b-gal)”, Lajolo adds.

The researchers amplified part of the gene of this enzyme, which was cloned and sequenced. It was discovered that during the ripening there is an increase in its activity and that of other enzymes – pectinmethylesterase (PME), polygalacturonase (PG) and cellulase. The conclusion was that ionization radiation (with gamma rays) held back for two days the beginning of ripening and also retarded the increase in the presence of these enzymes (all except cellulase)  – or that is to say, retarded softening.

Immunolocalization studies, carried out in collaboration with the John Innes Center (in the United Kingdom), are being done with specific antibodies and using an electronic microscope. They are showing the locations on the cellular wall and in the lamella medium, where the structural modifications associated with these enzymes occur.

On the other hand, with the bean, frequently stored under the humid and hot conditions that predominate in a large part of the country, a post harvest takes place. Consequently, the reintroduction of water becomes difficult and cooking time increases. “There are sensorial, nutritional and economic loses”, explains Lajolo. In this case, the evolution of the processes in the cellular walls was studied, whose polysaccharides were isolated for the study of its chemical composition. There were no decisive conclusions, but the research pointed to ferulic acid and extensin as possibly involved in the process of hardening.

“Through these studies we’re preparing a basis so that new processes of conservation will able to be developed and to enhance fruit quality, and even develop new varieties through genetic engineering,” admits Lajolo. He said that he personally has nothing against transgenic products, as he considers it important to make use of the advances available in order to create new varieties, to increase their useful life and to improve the quality of foods. “From the safety point of view, there is no scientific evidence of any health risk.”

The work could also have an influence on the sector of semi-processed foods – the case of already peeled, cut and packed vegetables –, that are much more perishable. Thus, it is important to know how the fruit matures not only in the inside, but also when it is semi-processed, in order to increase its useful life and its quality.

Dr. Franco Maria Lajolo, 59 years of age, graduated in Pharmacy and Biochemistry from USP, did his post doctorate in the Biochemistry of Foods at Massachusetts Institute of Technology (MIT) in the United States, and is a professor of Food Science and Experimental Nutrition, and is responsible for various discipline courses at graduate and post-graduate levels in USP.
Project: Post-Harvest Biochemical Transformations and Quality of Food and Raw Materials
Investment: R$ 180,245.97 and US$ 345,645.00