Pulsed electric field was employed as a pre-treatment to enhance the recovery yield of bioactive flavonoids and polyphenols from fresh red clover plants (Trifolium pratense L.), a legume crop rich in health-promoting isoflavones. Plants were pre-treated with electrical field strength (1, 1.7 and 2 k Volts per centimetre) with corresponding total specific energy inputs (15, 23, 30 kJ/kg) using the DIL high voltage pulsed 10 kw modular treatment system. Pre-treated samples were extracted using optimised extraction conditions (80 % ethanol, at 40 °C for 45 min). The extracts were analysed for total phenolic content, total flavonoid content, antioxidant activities, followed by ultra-high performance liquid chromatography tandem mass spectrometry analysis. The treatment 1 kV/cm and 15 kJ/kg enhanced total phenolic content to 15.27 ± 0.59 mg of gallic acid equivalents per gram of dry extract (mg GAE/g DE), an 15.68 % increase compared to the control (13.05 ± 4.08 mg GAE/g DE, p = 0.093), while also maintaining total flavonoid content (9.94 ± 3.37 mg quercetin equivalents/g DE, p = 0.085). Ultra performance liquid chromatography mass spectrometry/mass spectrometry analysis identified and quantified thirty-eight polyphenols, with the characteristic red clover flavonoids, notably biochanin A, daidzein, formononetin and cyanidin as dominant compounds in the treated extracts. Collectively, pre-treatment may enhance the efficiency of recovering antioxidant compounds from crops, like red clover. The application of low-energy PEF treatments (15 kJ/kg) successfully and significantly enhanced the extraction of phenolic compounds in fresh red clover, demonstrating its potential as a successful processing technology for recovering valuable bioactive compounds from underutilised agricultural crops.

Grass

Total phenolic content

Isoflavones

Non-thermal processing

UPLC-MS/MS

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