Pyrolysis-gas chromatography-isotope ratio mass spectrometry for monitoring natural additives in polylactic acid active food packages.

Compound-specific isotope analysis (CSIA) usually requires preparative steps (pretreatments, extraction, derivatization) to get amenable chromatographic analytes from bulk geological, biological or synthetic materials. Analytical pyrolysis (Py-GC/MS) can help to overcome such sample manipulation. This communication describe the results obtained by hyphenating analytical pyrolysis (Py-GC) with carbon isotope-ratio mass spectrometry (IRMS) for the analysis of a polylactic acid (PLA) a based bio-plastic extruded with variable quantities of a natural plant extract or oregano essential oil. The chemical structural information of pyrolysates was first determined by conventional analytical pyrolysis and the measure of δ13C in specific compounds was done by coupling a pyrolysis unit to a gas chromatograph connected to a continuous flow IRMS unit (Py-GC-C-IRMS). Using this Py-CSIA device it was possible to trace natural additives with depleted δ13C values produced by C3 photosystem vegetation (cymene: -26.7‰±2.52; terpinene: -27.1‰±0.13 and carvacrol: -27.5‰±1.80 from oregano and two unknown structures: -23.3‰±3.32 and -24.4‰±1.70 and butyl valerate: -24.1‰±3.55 from Allium spp.), within the naturally isotopically enriched bio-plastic backbone derived from corn (C4 vegetation) starch (cyclopentanones: -14.2‰±2.11; lactide enantiomers: -9.2‰±1.56 and larger polymeric units: -17.2‰±1.71). This is the first application of Py-CSIA to characterize a bio-plastic and is shown as a promising tool to study such materials, providing not only a fingerprinting, but also valuable information about the origin of the materials, allowing the traceability of additives and minimizing sample preparation.

A subchronic 90-day oral toxicity study of Origanum vulgare essential oil in rats.

Oregano essential oil (Origanum vulgare L. virens) (OEO) is being used in the food industry due to its useful properties to develop new active packaging systems. In this concern, the safety assessment of this natural extract is of great interest before being commercialized. The European Food Safety Authority requests different in vivo assays to ensure the safety of food contact materials. One of these studies is a 90 days repeated-dose oral assay in rodents. In the present work, 40 male and 40 female Wistar rats were orally exposed to 50, 100 and 200 mg/kg body weight (b.w.) OEO during 90 days following the OECD guideline 408. Data revealed no mortality and no treatment-related adverse effects of the OEO in food/water consumption, body weight, haematology, biochemistry, necropsy, organ weight and histopathology. These findings suggest that the oral no-observed-adverse-effect level (NOAEL) of this OEO is 200 mg/kg b.w. in Wistar rats, the highest dose tested. In conclusion, the use of this OEO in food packaging appears to be safe based on the lack of toxicity during the subchronic study at doses 330-fold higher than those expected to be in contact consumers in the worst scenario of exposure.

Development of PLA films containing oregano essential oil (Origanum vulgare L. virens) intended for use in food packaging.

Consumers' concerns about the environment and health have led to the development of new food packaging materials avoiding petroleum-based matrices and synthetic additives. The present study has developed polylactic acid (PLA) films containing different concentrations of essential oil from Origanum vulgare L. virens (OEO). The effectiveness of this new active packaging was checked for use in ready-to-eat salads. A plasticising effect was observed when OEO was incorporated in PLA films. The rest of the mechanical and physical properties of developed films did not show much change when OEO was included in the film. An antioxidant effect was recorded only for films containing the highest percentages of the active agent (5% and 10%). In addition, films exhibited in vitro antibacterial activity against Staphylococcus aureus, Yersinia enterocolitica, Listeria monocytogenes, Enterococcus faecalis and Staphylococcus carnosus. Moreover, in ready-to-eat salads, antimicrobial activity was only observed against yeast and moulds, where 5% and 10% of OEO was the most effective.

Toxicological evaluation of an Allium-based commercial product in a 90-day feeding study in Sprague-Dawley rats.

Proallium AP(®) is a commercial Allium extract intended to be used in active food packaging as the antibacterial and antioxidant effects of some organosulfur compounds are well known. However, there is little information on its toxicity and the Scientific Committee on Food (UE) requires the safety assessment of substances used in food contact materials. Thus, the aim of this study was to conduct for the first time a subchronic oral toxicity study of Proallium AP(®) with groups of 10 males and 10 females Sprague-Dawley rats fed a diet containing 0, 25, 100, 400 mg/kg/d for 90 days. No treatment-related clinical signs or mortality were noted. Besides, no treatment-related effects with regard to any of the toxicological biomarkers considered were observed, including biochemical, haematological and histopathology parameters. In conclusion, the non-observed-adverse-effect-level (NOAEL) for Proallium AP(®) in rats was determined to be a dietary dose of 400 mg/kg/d under the present experimental conditions, a value 500-fold higher than the exposure derived from its potential use in active packaging.

Genotoxicity assessment of propyl thiosulfinate oxide, an organosulfur compound from Allium extract, intended to food active packaging.

Essential oils from onion (Allium cepa L.), garlic (Allium sativum L.), and their main components, such as propyl thiosulfinate oxide (PTSO) are being intended for active packaging with the purpose of maintaining and extending food product quality and shelf life. The present work aims to assess for the first time the potential mutagenicity/genotoxicity of PTSO (0-50 µM) using the following battery of genotoxicity tests: (1) the bacterial reverse-mutation assay in Salmonella typhimurium (Ames test, OECD 471); (2) the micronucleus test (OECD 487) (MN) and (3) the mouse lymphoma thymidine-kinase assay (OECD 476) (MLA) on L5178YTk(+/-), cells; and (4) the comet assay (with and without Endo III and FPG enzymes) on Caco-2 cells. The results revealed that PTSO was not mutagenic in the Ames test, however it was mutagenic in the MLA assay after 24 h of treatment (2.5-20 µM). The parent compound did not induce MN on mammalian cells; however, its metabolites (in the presence S9) produced positive results (from 15 µM). Data from the comet assay indicated that PTSO did not induce DNA breaks or oxidative DNA damage. Further in vivo genotoxicity tests are needed to confirm its safety before it is used as active additive in food packaging.