**Background:** Cholesterol oxidation products (COPs), particularly non-enzymatic oxysterols such as 7β-hydroxycholesterol (7βOHC), 7-ketocholesterol (7KC), and various epoxides, form during food production and storage. These compounds are biologically active, with demonstrated pro-inflammatory, cytotoxic, and pro-apoptotic effects in human tissues, including the intestinal epithelium. They have been proposed as biomarkers of food freshness and quality. This study investigated how packaging type affects COP accumulation in milk chocolate over 12 months of simulated market storage.
**Methods:** Three prototype milk chocolates were produced using whole milk powders (WMPs) with increasing shelf-lives: 20 days (Chocolate A), 120 days (Chocolate B), and 180 days (Chocolate C). Each chocolate was packaged in two ways: standard packaging (STD)—unsealed laminated paper and aluminum foil providing light barrier only—and PLUS packaging—hermetically sealed biaxially-oriented polypropylene (BOPP) and metallized polypropylene providing complete oxygen and moisture barrier. Chocolates were stored in dark conditions at 18°C and analyzed at 0, 3, 6, 9, and 12 months. Oxysterols were quantified by gas chromatography-isotope dilution mass spectrometry (GC-MS). Seven non-enzymatic COPs (7αOHC, 7βOHC, 7KC, α-epoxy, β-epoxy, triol, 25OHC) and one exclusively enzymatic oxysterol (27OHC) were measured. Statistical significance was assessed using ANOVA with repeated measures and Student's t-test (significance set at p < 0.05).
**Key Results:** At time 0, total non-enzymatic COPs increased with WMP age: Chocolate A = 368.84 ± 10.24 ng/g, Chocolate B = 456.31 ± 17.41 ng/g (1.2-fold increase), Chocolate C = 651.3 ± 20.98 ng/g (1.8-fold increase) (p < 0.001). The non-enzymatic COPs/cholesterol ratio similarly rose from 1.63 ± 0.04 ng/μg (A) to 2.05 ± 0.07 ng/μg (B) and 3.26 ± 0.1 ng/μg (C) (p < 0.001). The dominant COPs at baseline were 7αOHC, 7βOHC, and 7KC, constituting 86–87% of total non-enzymatic COPs.
Over 12 months in STD packaging, total non-enzymatic COPs in Chocolate A increased 4.5-fold (to 1673.33 ± 39.28 ng/g), in Chocolate B 4.2-fold (to 1908.63 ± 54.79 ng/g), and in Chocolate C 4.3-fold (to 2792.36 ± 55.56 ng/g) (all p < 0.001). The PLUS packaging significantly reduced COP accumulation at every time point (p < 0.001). The quenching effect (percent reduction versus STD) ranged from 16–21% for Chocolate A, 15–18% for Chocolate B, and 23–34% for Chocolate C. Notably, chocolates in PLUS packaging at 12 months had COP levels comparable to those in STD packaging at just 6 months.
A qualitative shift in COP profile occurred after 6 months: at 3 months, 7-series oxysterols (7αOHC, 7βOHC, 7KC) comprised 84–87% of total non-enzymatic COPs while epoxides (α-epoxy, β-epoxy) were only 8–11%. From 6 months onward, epoxides rose sharply to 37–45% of total, at the expense of 7-series oxysterols (49–57%). The enzymatic oxysterol 27OHC decreased progressively during storage in all conditions, with PLUS packaging slightly but consistently quenching this loss.
**Clinical Implications:** Non-enzymatic COPs, particularly 7βOHC and 7KC, have been shown to exert pro-oxidant and cytotoxic effects on intestinal epithelial cells, upregulate pro-inflammatory cytokines, and disrupt tight junction proteins (ZO-1, occludin, JAM-A), potentially increasing gut permeability to microbiota and pathogens. Cholesterol epoxides, which markedly increased after 6 months, have been linked to NADPH oxidase-1 activation and apoptotic cell death in Caco-2 cells, and have been implicated in carcinogenesis. These findings suggest that limiting COP accumulation through fresh ingredient selection and oxygen-barrier packaging is important for food safety. The study demonstrates that non-enzymatic COPs serve as reliable biomarkers for both food freshness and the effectiveness of anti-oxidation strategies in manufacturing and packaging. The authors recommend more stringent shelf-life limitations for milk chocolate unless effective oxygen-barrier protection is provided.