Fungus discovered in Brazil nuts may have bactericidal activity

brazil nuts
Glass plates with colonies of Penicillium excelsum, a new species of fungus discovered in Brazil nuts (photo: Marta Taniwaki)

Brazil nuts are being investigated by several research groups across the country. One of their goals is to understand how to prevent the contamination of Brazil nuts by species of mold that produce toxic substances.

An unexpected by-product of these research projects is the description of a new species of fungus, Penicillium excelsum, one of more than 350 species in the genus Penicillium, from which penicillin, the first antibiotic, was obtained. The description was published in the journal PLoS ONE. Its lead author is biologist Marta Hiromi Taniwaki, a researcher at the Campinas Food Technology Institute (ITAL), part of the São Paulo State Department of Agriculture & Food Supply. The research was supported by FAPESP.

The Brazil nut tree, Bertholletia excelsa, is native to the Amazon rainforest. It is a tall, stately tree that can reach 50 m or more in height. Its fruit is a large capsule resembling a coconut, with a hard woody shell. When the capsule matures, it falls to the forest floor, splits open and releases tens of triangular seeds packed like the segments of an orange. These seeds are Brazil nuts.

The world’s largest producer is Brazil, where Acre is the state with the highest output. It is illegal to fell Brazil nut trees, and B. excelsa is classified as vulnerable by the International Union for Conservation of Nature & Natural Resources (IUCN), which included the tree in its Red List of Threatened Species in 1998.

The nuts themselves are valuable on both the domestic and world export markets. Brazil produced 38,000 metric tons in 2013, according to IBGE, the national statistics bureau. The world’s highest consumers are the United States and China. The European Union banned imports of Brazil nuts in 2000, when aflatoxins were detected in them at higher concentrations than those permitted by EU health regulations.

Aflatoxins are toxic fungal metabolites (mycotoxins) produced by certain molds of the genus Aspergillus that grow on a number of raw food commodities. Research has shown that high levels of aflatoxin can damage the liver, causing necrosis, cirrhosis, edema and cancer.

The formation of toxins by fungi in Brazil nuts is caused by the high humidity in the forest and the relatively long stockpiling period required for the seeds to reach a safe level of moisture content. Brazil nuts that are dried at 60 degrees Celsius and stored under appropriate conditions have less fungal growth and are less likely to contain toxins compared with when such precautions are not taken.

After all this research and effort throughout the production chain, from extraction to processing, the EU ban was lifted in 2011, and European imports were resumed. At the same time, ANVISA, the Brazilian health surveillance agency, also set ceilings for aflatoxins in Brazil nuts to protect consumers.

According to Taniwaki, the chief aim of her research was to verify the occurrence of aflatoxin-producing fungi in Brazil nuts. “We were also interested in investigating the mycobiota,” she said, meaning all the fungi present in Brazil nuts and their ecosystem.

The research found that species from the genera Aspergillus and Penicillium predominated in B. excelsa’s ecosystem. “Several species of Aspergillus were found to produce toxins both in the lab and in the wild,” she added. “Some of the findings have been published, and others are forthcoming.”

The first unexpected dividend from the research came in 2012 with the description of a new fungus, Aspergillus bertholletius. The surprises have continued with the description of P. excelsum. “The discovery of a new species of Penicillium in the Amazon forest was unforeseen,” Taniwaki said. “It’s a small example of the Amazon’s vast biodiversity, whose surface we have barely scratched.”

P. excelsum was isolated in samples collected from forests, farms, markets and nut processors in the Brazilian states of Amazonas and Pará. The new fungus was detected practically everywhere in B. excelsa’s ecosystem. It was found in samples of leaves, bark, seeds, capsules, flowers and soil. It was also found in the bees that pollinate the Brazil nut tree’s flowers and in ants. “The fungus propagates throughout the environment surrounding the tree,” Taniwaki said.

To isolate the new species, more than 200 samples were collected, placed on glass slides in an appropriate culture medium and incubated at 25 degrees Celsius for seven days. “We performed more than 1,000 isolations,” Taniwaki said. “After a week’s incubation, the fungi developed and formed a colony.”

Several processes were deployed to identify the species of mold, but none produced conclusive results. “Colony morphology and microscopy showed it was different from all known species,” she explained. “It appeared to be a new species.” Definitive proof came from molecular studies performed by Maria Helena Fungaro at the State University of Londrina, Paraná, Brazil, and Jens Frisvad at the Technical University of Denmark. The final description of the new species was produced by John Pitt at CSIRO Food & Nutrition, part of Australia’s national science agency.

The new species of Penicillium is not the first to be detected in the Brazil nut tree ecosystem. According to Taniwaki, P. glabrum and P. citrinum had already been isolated in Brazil nut trees. P. citrinum produces the mycotoxin citrinin, which can damage the human kidney. P. glabrum produces citromycetin, which has bactericidal activity. “The difference between antibiotics and mycotoxins is that the former combat microorganisms, whereas the latter are harmful to animals and humans,” she said.

Several species of fungus belonging to the genus Penicillium produce antibiotics capable of combating infections caused by viruses, bacteria and other fungi. The most famous, of course, is penicillin, the first antibiotic.

In 1928, Sir Alexander Fleming, a Scottish biologist, pharmacologist and botanist, was studying the properties of Staphylococcusbacteria in his laboratory at St. Mary’s Hospital Medical School in London. On returning from a month’s vacation in September, Fleming noticed that one of his dishes of Staphylococcus had been contaminated by a fungus while he was away, and the bacterial colonies around the fungus had been killed.

Fleming decided to investigate the mystery. He isolated the substance produced by the Penicillium fungus, found that it was a bactericide, and named it penicillin.

“We don’t know whether this new species produces any metabolic substances of interest to the pharmaceutical industry, but we mean to find out,” Taniwaki said.

The article “Penicillium excelsum sp. nov from the Brazil Nut Tree Ecosystem in the Amazon Basin” by Taniwaki et al., published in PLoS ONE (doi:10.1371/journal.pone.0143189), can be read at http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143189.

The article “Aspergillus bertholletius sp. nov from Brazil Nuts” by Taniwaki et al., also published in PLoS ONE(doi:10.1371/journal.pone.0042480), can be read at http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042480.