Cholesterol (μg/g) and COPs¹ (ng/g) in prototype milk chocolate bars made with WMP of 120 days of shelf-life (Milk Chocolate B), packed with laminated paper and aluminum, unsealed (STD) or with two laminated layers of biaxially-oriented polypropylene (BOPP) and metallized polypropylene (PP), sealed (PLUS), then kept on virtual market for 0, 3, 6, 9, 12 months.

Values are expressed as means ± SD (n = 3). At any time point, the levels of non-enzymatic COPs in the Milk Chocolate B resulted to be significantly higher than those measured in the Milk Chocolate A (p < 0.001). On the contrary, at any time point, the levels of non-enzymatic COPs in the Milk Chocolate B in packaging PLUS resulted to be significantly lower than those estimated in the Milk Chocolate B in STD packaging (§ p < 0.001).</p

Fig 2 -

Distribution of non-enzymatic cholesterol oxidation products (COPs) (ng/g) in prototype milk chocolate bars made with whole milk powder (WMP) of 20, 120 and 180 days of shelf-life (Milk Chocolate A, B and C, respectively), packed with laminated paper and aluminum, unsealed (STD) or with two laminated layers of BOPP and metallized polypropylene (PP), sealed (PLUS), kept on virtual market for 0, 3, 6, 9, 12 months. Values are expressed as means ± SD (n = 3).</p

Ingredients, fat composition as indicated by the manufacturer, cholesterol (μg/g), and COPs (ng/g) content of the commercial samples of milk chocolate included in the study.

Ingredients, fat composition as indicated by the manufacturer, cholesterol (μg/g), and COPs (ng/g) content of the commercial samples of milk chocolate included in the study.</p

List of sterols and oxysterols quantified by isotope dilution mass spectrometry.

List of sterols and oxysterols quantified by isotope dilution mass spectrometry.</p

Cholesterol (μg/g) and COPs¹ (ng/g) in prototype milk chocolate bars made with WMP of 20 days of shelf-life (Milk Chocolate A), packed with laminated paper and aluminum, unsealed (STD) or with two laminated layers of biaxially-oriented polypropylene (BOPP) and metallized polypropylene (PP), sealed (PLUS), then kept on virtual market for 0, 3, 6, 9, 12 months.

Values are expressed as means ± SD (n = 3). At any time point, the levels of non-enzymatic COPs in the Milk Chocolate A in packaging PLUS resulted to be significantly lower than those estimated in the Milk Chocolate A in STD packaging (§ p < 0.001).</p

Experimental research design.

Non-enzymatic cholesterol oxidation products (COPs) are nowadays receiving increasing attention in food technology for their potential use as biomarkers of freshness and safety in raw materials and complex food matrices, as well as markers of cholesterol oxidation during the production and shelf-life of end products. Here reported is the investigation of how long three prototype milk chocolates containing whole milk powders (WMPs) of increasing shelf-lives (i.e. 20, 120, and 180 days), could be safely stored in the market by adopting the non-enzymatic COPs as a quality markers. In addition, the protective effect of two different primary packaging, sealed and unsealed ones, in mitigating the generation of non-enzymatic COPs in three prototype milk chocolates after 3, 6, 9, 12 months of shelf-life was assessed to simulate two real storage conditions. Quantifying oxysterols’ levels by mass spectrometry, the oxygen impermeable packaging (PLUS) resulted to significantly quench the non-enzymatic COPs production up to 34% as to that found in the same product but with unsealed standard packaging (STD). This study represents one practical application of non-enzymatic COPs as a reliable tool for corrective strategies to prevent food oxidation.</div

Distribution of non-enzymatic cholesterol oxidation products (COPs) in commercial and prototype milk chocolate tablets made with whole milk powder (WMP) of 20, 120, and 180 days of shelf-life, respectively).

Commercial milk chocolate tablets: samples 1–9. Prototype milk chocolate tablets: sample A (20 days of shelf-life), sample B (120 days of shelf-life), sample C (180 days of shelf-life). Values are expressed as means ± SD (n = 3).</p

Cholesterol (μg/g) and COPs¹ (ng/g) in prototype milk chocolate bars made with WMP of 180 days of shelf-life (Milk Chocolate C), packed with laminated paper and aluminum, unsealed (STD) or with two laminated layers of biaxially-oriented polypropylene (BOPP) and metallized polypropylene (PP), sealed (PLUS), then kept on virtual market for 0, 3, 6, 9, 12 months.

Values are expressed as means ± SD (n = 3). At any time point, the levels of non-enzymatic COPs in the Milk Chocolate C resulted to be significantly higher than those measured in the Milk Chocolate B (p < 0.001). On the contrary, at any time point, the levels of non-enzymatic COPs in the Milk Chocolate C in packaging PLUS resulted to be significantly lower than those estimated in the Milk Chocolate C in STD packaging (§ p < 0.001).</p

Cholesterol (μg/g) and COPs (ng/g) in the three prototype milk chocolate bars and the ingredients used for their production.

Cholesterol (μg/g) and COPs (ng/g) in the three prototype milk chocolate bars and the ingredients used for their production.</p

Analysis of the two main TGFβ1 receptors in tissue samples of adenomas and adenocarcinomas.

<p>TGFβ1RI: TGFβ1 receptor I; TGFβ1RII: TGFβ1 receptor II.</p><p>TA: tubular adenoma; TVA: tubulovillous adenoma; I-IV AC: malignant stages of adenocarcinoma.</p><p>In brackets percentages of patients with alteration of the TGFβ1 receptor system. Minimal or low grade immunostaining was taken as an index of receptors’alteration.</p