“Both protein and phenolic compounds can be extracted from okara. As a food engineer, I’m always interested in ways of using such residues. In Brazil, I know of no more promising source than soybeans to obtain the extract on an industrial scale, although in principle equol can be obtained from all isoflavone-rich plants,” she said.
The study was supported by FAPESP via a postdoctoral scholarship awarded to Cintia Rabelo e Paiva Caria, second author of the article.
Methods
The researchers produced a soy milk extract with the right initial concentration of isoflavones for their experiments. “The industrial process used to obtain the extract for soy-based drinks sold in supermarkets doesn’t have sufficient phenolic content because they’re designed to serve as a source of non-animal protein,” Macedo explained.
The group used different processes to obtain equol by mimicking metabolization of the isoflavones in the extract. “In the first, we deployed enzymes [a mixture of molecules, including beta-glucosidase] to remove glucose from glycosylated isoflavones. In this case, we performed measurements to find out whether a metabolite of interest could be obtained using enzymes and to quantify the glycosylated and aglycone isoflavones at the end of the process,” Macedo said.
The second strategy entailed fermenting the extract with a mixture of lactobacilli. The researchers inoculated the extract with lactic bacteria in the absence of oxygen (anaerobiosis) and analyzed the isoflavones before and after fermentation.
In the third process, they combined enzymes with probiotics. “After the enzymatic action, I inoculated the mix of probiotics. The aim was to facilitate the work of the lactobacilli by meeting them halfway with the enzymes in an attempt to speed up the process and obtain more metabolites at the end. We found that this combination did indeed work better. It enhanced anti-oxidant capacity, production of metabolites, and conversion of glycosylated into aglycone isoflavones. Both treatments combined had synergistic effects on soy-based products,” Macedo said.
The group made a point of testing processes that can be replicated on an industrial scale, working with commercial enzymes and probiotics. “We have to develop solutions that make sense from a technological standpoint,” she said.
Effects and contraindications
Because equol is very similar to estrogen, receptors of this hormone in the ovaries, womb and breasts do not detect any difference, and in menopausal women the organism does not react to lack of estrogen, which appears to be present, minimizing the symptoms. “This is the point of obtaining compounds that mimic estrogen. They’re also found in blackberry leaves, yams and other plants. I don’t know if they’re more bioavailable or also require some kind of transformation to be absorbable by the organism,” Macedo said.
According to existing knowledge, she added, phytoestrogen acts similarly to estrogen taken for hormone replacement therapy, albeit in far smaller doses. “There are already products on the market based on soy extract or soy milk, and some are indicated for menopause symptoms, but they aren’t effective for everyone. Every gut microbiota is different. We aim to develop a product to treat menopause symptoms that’s based on phytoestrogen and sufficiently bioavailable to be digested easily,” Macedo explained.
Some cancer patients cannot take the hormone, however: breast, ovarian and endometrial (uterine) cancer rely on estrogen to develop and grow. “Some types of prostate cancer also respond to estrogen. People with these ailments won’t be able to use the products we’re developing,” she noted.
Next steps
According to Macedo, equol can exist in two forms, R-equol and S-equol. “Only the latter is highly absorbed, but in the process used to obtain the metabolite we couldn’t find a way to separate them, so we opted for two routes. One was identification by chromatography, which distinguishes between equol and other metabolites of interest. However, the concentration is too low to enable us to detect the two forms of the molecule separately. The other was in vitro studies involving human cancer cells to test the estrogenic effects of the extracts obtained,” she said.
The cells were treated with soy extract processed using enzymes and fermentation. If they multiplied, the extract was deemed to have estrogenic effects. “The findings from the assays involving cancer cells aren’t included in the latest article in Foods, which wasn’t our first on this topic, but we plan to publish them soon,” she said.
Nevertheless, the results of in vitro simulation of digestion confirmed that the anti-oxidant effects and other benefits of isoflavones persisted. “We wanted to find out which process was most efficient in terms of isoflavone biotransformation, and whether it was good enough to assure bioavailability and absorption by the organism.
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