**Background:** Olive pomace (OP) is a by-product of olive oil production, particularly from two-phase extraction systems, which is rich in bioactive compounds such as hydroxytyrosol, α-tocopherol, oleic acid, and dietary fiber. However, its use as a fresh food ingredient requires removal of stone fragments to obtain olive pomace paste (OPP) and application of heat treatment to ensure microbial safety. This study aimed to characterize OPP and evaluate the impact of four different time/temperature binomials on its nutritional quality, phytochemical content, antioxidant activity, and natural microbial load, to identify the optimal treatment for producing a safe functional ingredient.
**Methods:** OP (5.3 kg) from a two-phase system in Trás-os-Montes, Portugal (mixture of Cobrançosa, Cordovil, Madural, and Verdeal Transmontana varieties) was manually sieved to remove stones, yielding 2.3 kg of OPP (43% yield). The OPP was divided into 250 g portions and subjected to four heat treatments: OPPA (65°C/30 min), OPPB (77°C/1 min), OPPC (88°C/15 s) in a Thermomix, and OPPD (120°C/20 min) in an autoclave. Untreated OP and OPP served as controls. All samples were lyophilized and analyzed for proximate composition (moisture, ash, fat, protein, fiber, carbohydrates), vitamin E profile (HPLC-DAD-FLD), fatty acid profile (GC-FID), total phenolics (Folin-Ciocalteu), total flavonoids (colorimetric), hydroxytyrosol content (HPLC-DAD-FLD), antioxidant activity (FRAP and DPPH•-SA), and total microbial count (pour-plate method at 22°C and 37°C). Statistical analysis used one-way ANOVA with Tukey's HSD (p < 0.05).
**Key Results:** Fresh OPP had higher moisture (73.1 g/100 g fw), ash (1.3 g/100 g fw), and total fat (2.4 g/100 g fw) compared to OP, but lower carbohydrates and fiber. All heat treatments significantly reduced protein content, with OPPD showing the greatest loss (27% dw). Ash content increased significantly only in OPPD (1.2-fold dw). Fat content decreased significantly in OPPC (25% dw) and OPPD (27% dw). Total fiber remained high (43.5–44.6 g/100 g dw) but decreased slightly, with OPPD showing a 9% loss. Total vitamin E ranged from 4.4 to 6.1 mg/100 g dw; all treatments caused significant losses (6–13% dw), with OPPD showing the smallest reduction (6%). α-Tocopherol was the major vitamer. Fatty acid profile was dominated by oleic acid (73–75%), palmitic (11%), and linoleic (9–10%) acids, and was not significantly affected by any treatment. Total phenolics (TPC) in OP was 3.08 g GAE/100 g dw; OPP production increased TPC by 1.3-fold. All treatments reduced TPC (7–15% dw), with OPPA and OPPB showing the highest losses (15% and 14%, respectively) and OPPC and OPPD the lowest (7% each). Total flavonoids (TFC) in OP was 2.69 g CE/100 g dw; OPP increased TFC by 1.3-fold. Treatments reduced TFC by 3–19% dw, with OPPD showing the smallest loss (3%) and OPPA the largest (19%). Hydroxytyrosol content (HTC) in OP was 0.35 g/100 g dw; OPP increased HTC 1.8-fold. Treatments reduced HTC by 17–46% dw, with OPPD showing the smallest loss (17%) and OPPA the largest (46%). FRAP and DPPH•-SA values were highest in OPP and OPPD (around 6 g FSE/100 g dw and 2 g TE/100 g dw, respectively). All treatments reduced antioxidant activity, with OPPA and OPPB showing the greatest reductions. Microbiological analysis showed that all treatments effectively reduced microbial load; OPPA, OPPC, and OPPD completely eliminated microorganisms at both 22°C and 37°C, while OPPB reduced but did not eliminate them at 37°C.
**Clinical Implications:** Although this is a food science study, the findings have indirect clinical relevance. The high content of dietary fiber (43.5–44.6 g/100 g dw) in heat-treated OPP could support glycemic control, cholesterol reduction, and colon health. The presence of oleic acid (73–75%) and linoleic acid (9–10%) may contribute to cardiovascular benefits, including inhibition of cholesterol synthesis and blood pressure regulation. The significant levels of hydroxytyrosol (0.35–0.65 g/100 g dw) and α-tocopherol (major vitamer) provide antioxidant, anti-inflammatory, and neuroprotective potential. The 88°C/15 s treatment (OPPC) was identified as optimal because it preserved most bioactive compounds, maintained high fiber and protein content, and completely eliminated microbial load, while being fast and industrially scalable. Incorporating heat-treated OPP into food products could offer a sustainable, functional ingredient that meets consumer demand for health-promoting foods and supports circular economy goals in the olive oil industry.