Novel protocol significantly improving spermidine content in extracts from wheat germ: Aqueous conversion of polyamines into neutral-base form, solubility in ethanol and acid precipitation as phosphate salts

• Novel 5-step method to obtain wheat germ extract with high spermidine concentration • Simple protocol that exploits the pH-dependent solubility variations of spermidine in ethanol • The 7.6 - 9.6 % spermidine/extract far exceeds previous extraction methods • The efficacy of the protocol was validated in a scaled-up commercial extraction • A comparison to known extraction methods is argumented There is an increased demand for natural spermidine (SPD) supplements, necessitating improved extraction protocols to overcome current limitations in polyamine (PA) extraction protocols from plant-based sources. The present work describes the development and underlying rationale for a five-phase laboratory-scale extraction protocol that exploits the chemical properties of SPD using a novel approach that differs from all previous water-acid methods. Phase 1: Wheat germ (WG) milling, phase 2: alkaline reaction of PAs with Ca(OH) 2 , phase 3: solubilization of PAs in ethanol, phase 4: ethanol-insoluble bio-compound separation by filtration, and phase 5: precipitation of PA salts using H 3 PO 4 . This protocol takes advantage of the high ethanol solubility of SPD in base form, compared to the ethanol-insoluble protonated form (SPDH+). The precipitation step permits the separation and purification of SPD from other components extracted in the natural matrix. In so doing, it was possible to attain a high SPD content extract. The average SPD content of the extract, performed on 10 separate WG 100 g batches, was 7.6 ± 1.9%, significantly exceeding previous methods. The industrial scalability of the protocol using 50 kg WG was implemented with minimal adjustments, yielding 9.6 % SPD/extract. The proposed workable extraction, with no requirement for complex or specialized equipment, has potential towards developing SPD supplements.