Ball milling of avocado seed residues: Defining an operational space for preserving bioactive molecules and antioxidant functionality

Avocado seeds are phenolic-rich by-products, but dry comminution may alter powder structure and extractable phenolics. This study evaluated avocado seed flour after 120 min of ball milling at 30–90 rpm and ball-to-powder ratios (BPR) of 1.0–2.0, with milling time and grinding-media mass fixed. Particle size and morphology were assessed by laser diffraction and scanning electron microscopy; total phenolics, ortho-diphenols, flavonoids, ABTS, DPPH, FRAP, and targeted high-performance liquid chromatography with diode-array detection (HPLC-DAD) markers were measured. An operational low-degree-proanthocyanidin index (ΣPAC) was calculated. Process-level replication was available only at the center point (60 rpm, BPR 1.5; three independent runs). Samples 1–4 were single milling runs, so their statistical lettering reflects extraction and analytical variability and treatment comparisons are exploratory. Milling reduced D50 from 17.10 μm to 9.80–12.15 μm. Among the tested single-run factorial batches, Sample 1 (30 rpm, BPR 2.0) had the highest ΣPAC retention (0.975); the other tested batches showed retention of 0.323–0.532 and lower antioxidant responses. The algebraic retention analysis indicated that assay decline was not described by ΣPAC retention alone and may also reflect extractability, matrix interactions, assay responsiveness, and unmeasured transformations. The results identify Sample 1 as a candidate for confirmatory testing, not an optimized operating window, and support direct measurement of internal temperature, oxygen exposure, enzyme activity, and media wear in future replicated studies.