The motivation lies partly in regulation. In 2011, the European Food Safety Authority concluded that there is a cause-and-effect relationship between consuming olive oil phenolics and protecting low-density lipoprotein particles, the cholesterol carriers in blood, from oxidative damage. European regulation permits health claims for olive oils rich in these compounds, but only if the oil contains at least 5 mg of hydroxytyrosol and its derivatives, including oleuropein complexes and tyrosol, per 20 g of oil, equivalent to 250 mg/kg. Because phenolic concentrations vary considerably between oil batches and often fall below that threshold, producers have an incentive to enrich their oils, and olive leaves, an abundant waste stream, are an obvious candidate source.

The scale of that waste stream is substantial. Pruning generates roughly 25 kg of twigs and leaves per olive tree each year, and oil extraction separates a further quantity of leaves amounting to about 10% of the fruit weight. Olive leaves contain higher oleuropein levels than any other part of the tree, with reported concentrations of 60 to 90 mg/g in dried leaves. The research team, working with leaves collected from trees in Tauranga and Hamilton, steeped whole leaves in boiling water for 30 minutes to produce an olive leaf aqueous extract, deliberately using intact leaves to avoid triggering the leaves’ own enzymes, which would otherwise hydrolyse oleuropein prematurely.

Drying conditions proved to be a powerful lever over the extract’s composition. When leaves were dried gently at 24 degrees Celsius for 14 days, the extract contained 23% more oleuropein than extract from fresh leaves, along with dramatically more hydroxytyrosol, a 134% increase, and more tyrosol and elenolic acid. The likely explanation is that slow drying bursts cell walls and gives endogenous beta-glucosidase time to convert some oleuropein into aglycone and then into smaller phenolics. When a second batch of leaves was dried rapidly at 70 degrees Celsius for just 3 days, oleuropein and ligstroside concentrations were higher than in the room-temperature-dried leaves, up 40% and 94% respectively, because heat denatures the endogenous enzymes before they can degrade the parent glycosides. Across both batches, oleuropein in the extract followed the order fresh, then room-temperature dried, then 70-degree dried.

Stability testing showed that storage temperature matters just as much. Extracts held at minus 20 degrees Celsius kept all their compounds stable for the full 189-day trial. At room temperature, oleuropein starting at 343 mg/kg fell by 26% over 189 days, while at 40 degrees it dropped 29% in just 50 days and 84% by the end, apparently through chemical oxidation, bond cleavage and side reactions such as ring closure and polymerisation rather than enzymatic action, since the boiling step had inactivated the leaf enzymes. The hydrophobic oleuropein aglycone, detected at low levels in the aqueous extract, behaved differently: at 40 degrees it initially rose 86% by day 79, presumably from thermal degradation of oleuropein, before declining as it broke down into oleacein and elenolic acid. The practical message is clear: freeze the extract to preserve oleuropein for controlled conversion.

The centrepiece of the study was the use of almond products as a food-compatible source of beta-glucosidase, the enzyme that cleaves the sugar from oleuropein. Purified almond beta-glucosidase is expensive, and purification buffers such as ammonium sulfate would make the product unsuitable for human consumption, so the team tested ordinary sweet almond flour and freshly milled almond meal instead, an approach the authors report has not previously been described. Adding 100 mg of almond flour to 1 mL of extract containing 3668 mg/kg oleuropein achieved a greater than 99% reduction in oleuropein within 12 hours at 40 degrees, with ligstroside falling below the detection limit. Freshly prepared almond meal outperformed the oldest commercial almond flour by 4.5-fold over 30 minutes, likely because the blanching step used to make flour, above 100 degrees for 2 to 5 minutes, partially denatures the enzyme.

Temperature optimisation revealed a delicate trade-off. Using filtered almond milk as the enzyme source, hydrolysis ran faster at higher temperatures: after 120 minutes, oleuropein had fallen by 3175 mg/kg at 60 degrees versus 1499 mg/kg at 30 degrees. But the aglycone product is fragile. Its concentration peaked at about 10 minutes and then declined, with less accumulating at higher temperatures, 613 mg/kg at 40 degrees versus 357 mg/kg at 60 degrees at the 10-minute mark. In stability experiments in a 50:50 extract-methanol mixture, the aglycone’s half-life collapsed from 256 minutes at 30 degrees to 125 minutes at 40 degrees and just 16 minutes at 60 degrees, following first-order kinetics. The authors attribute the loss partly to oxidation of the reactive ortho-diphenol group to an ortho-quinone. For contrast, the aglycone is far more stable in an oil matrix, with a reported half-life of 405 days at 25 degrees, underscoring the need to transfer it quickly into oil.

That transfer, however, is where the process stalled. In preliminary enrichment experiments combining extract, almond flour and oil at 40 degrees, oleuropein in the extract was 94% hydrolysed, yet only 0.3 mg/kg of oleuropein aglycone appeared in the oil phase. Raising the starting oleuropein concentration, switching to almond milk to remove lipid-coated particles, and shortening incubation to 10 minutes lifted the oil concentration only to 6.5 mg/kg, far below the 1786 mg/kg that olive oils can naturally contain, showing the system was nowhere near saturation. Vigorous homogenisation to create stable emulsions did not help, and neither did testing two extra virgin olive oils with different total phenolic contents, 23 and 118 mg/kg, to see whether phenolics acting as surfactants could lower interfacial tension.

The most telling clue came from filtration. If the aglycone were merely adsorbed onto solid particles, filtering them out should change the measured concentration, but samples filtered at 0.45 and 0.2 micrometres showed no significant difference from unfiltered samples, around 454 to 474 mg/kg. Since micelles typically measure 10 to 100 nanometres and would pass through such filters, the result supports the hypothesis that almond milk components stabilise the lipophilic aglycone in micelle-like structures within the aqueous phase, effectively trapping it and blocking mass transfer into the oil. Salting-out with sodium chloride and sugaring-out with glucose, applied at water activities expected to preserve enzyme function, also failed to improve transfer.

The study’s conclusions are candid about both achievement and limitation. Almond-derived beta-glucosidase reliably and completely hydrolyses oleuropein from a waste-derived aqueous extract, and drying temperature and duration can be tuned to steer the extract’s compound profile. But under every condition tested, the movement of oleuropein aglycone into the oil phase remained minimal, with micelle formation, degradation and binding to solids implicated as barriers. The authors note that enrichment itself is not prohibited under the International Olive Council definition of virgin olive oil, though oil with added exogenous phenolics could not be marketed as virgin olive oil and would need a different product designation. Turning olive leaf waste into functional, secoiridoid-enriched oil therefore remains a promising but unfinished project, hinging on new strategies to overcome the aqueous-oil interface.

Subject of Research: Enriching olive oil with oleuropein aglycone from olive leaf extract using almond-derived beta-glucosidase

Article Title: Using Olive Leaves to Enrich Olive Oil With Oleuropein Aglycone

Article References: te Bogt, J., Speed, D., Schlothauer, R. C., Fahey, J. W., & Grainger, M. N. C. (2026). Using Olive Leaves to Enrich Olive Oil With Oleuropein Aglycone. Food Science & Nutrition, 14(10), Article e72421. https://doi.org/10.1002/fsn3.72421

Image Credits: AI Generated

DOI: 10.1002/fsn3.72421

Keywords: olive oil, oleuropein, oleuropein aglycone, olive leaves, beta-glucosidase, almond flour, secoiridoids, phenolic compounds, hydroxytyrosol, food chemistry, micelles, mass transfer