Interlaboratory Study to Evaluate a Testing Protocol for the Safety of Food Packaging Coatings.

According to European regulations, migration from food packaging must be safe. However, currently, there is no consensus on how to evaluate its safety, especially for non-intentionally added substances (NIAS). The intensive and laborious approach, involving identification and then quantification of all migrating substances followed by a toxicological evaluation, is not practical or feasible. In alignment with the International Life Sciences Institute (ILSI) and the European Union (EU) guidelines on packaging materials, efforts are focused on combining data from analytics, bioassays and in silico toxicology approaches for the risk assessment of packaging materials. Advancement of non-targeted screening approaches using both analytical methods and in vitro bioassays is key. A protocol was developed for the chemical and biological screening of migrants from coated metal packaging materials. This protocol includes guidance on sample preparation, migrant simulation, chemical analysis using liquid chromatography (LC-MS) and validated bioassays covering endocrine activity, genotoxicity and metabolism-related targets. An inter-laboratory study was set-up to evaluate the consistency in biological activity and analytical results generated between three independent laboratories applying the developed protocol and guidance. Coated packaging metal panels were used in this case study. In general, the inter-laboratory chemical analysis and bioassay results displayed acceptable consistency between laboratories, but technical differences led to different data interpretations (e.g., cytotoxicity, cell passages, chemical analysis). The study observations with the greatest impact on the quality of the data and ultimately resulting in discrepancies in the results are given and suggestions for improvement of the protocol are made (e.g., sample preparation, chemical analysis approaches). Finally, there was agreement on the need for an aligned protocol to be utilized by qualified laboratories for chemical and biological analyses, following best practices and guidance for packaging safety assessment of intentionally added substances (IAS) and NIAS to avoid inconsistency in data and the final interpretation.

Early Life to Adult Brain Lipidome Dynamic: A Temporospatial Study Investigating Dietary Polar Lipid Supplementation Efficacy.

The lipid composition of the brain is well regulated during development, and the specific temporospatial distribution of various lipid species is essential for the development of optimal neural functions. Dietary lipids are the main source of brain lipids and thus contribute to the brain lipidome. Human milk is the only source of a dietary lipids for exclusively breastfed infant. Notably, it contains milk fat globule membrane (MFGM) enriched in polar lipids (PL). While early life is a key for early brain development, the interplay between dietary intake of polar lipids and spatial dynamics of lipid distribution during brain development is poorly understood. Here, we carried out an exploratory study to assess the early postnatal temporal profiling of brain lipidome between postnatal day (PND) 7 and PND 50 using matrix-assisted laser desorption ionization as a mass spectrometry imaging (MALDI-MSI) in an in vivo preclinical model. We also assessed the effect of chronic supplementation with PL extracted from alpha-lactalbumin-enriched whey protein concentrate (WPC) containing 10% lipids, including major lipid classes found in the brain (37% phospholipids and 15% sphingomyelin). MALDI-MSI of the spatial and temporal accretion of lipid species during brain development showed that the brain lipidome is changing heterogeneously along time during brain development. In addition, increases in 400+ PL supplement-dependent lipids were observed. PL supplementation had significant spatial and temporal effect on specific fatty esters, glycerophosphocholines, glycerophosphoethanolamines, and phosphosphingolipids. Interestingly, the average levels of these lipids per brain area tended to be constant in various brain structures across the age groups, paralleling the general brain growth. In contrast, other lipids, such as cytidine diphosphate diacylglycerol, diacylglycerophosphates, phosphocholines, specific ether-phosphoethanolamines, phosphosphingolipids, glycerophosphoinositols, and glycerophosphoserines showed clear age-dependent changes uncoupled from the general brain growth. These results suggest that the dietary PL supplementation may preferentially provide the building blocks for the general brain growth during development. Our findings add to the understanding of brain-nutrient relations, their temporospatial dynamics, and potential impact on neurodevelopment.

Effects of interesterified lipid design on the short/medium chain fatty acid hydrolysis rate and extent (in vitro).

Short/medium chain fatty acids have well known health effects such as gut immune regulation and ketogenesis. The ability to realise these health effects is potentially limited by their rapid gastro-intestinal lipolysis. It was proposed that synthesising novel interesterified lipids via an interesterification reaction to generate a combination of short/medium and long chain fatty acids would modulate their gastrointestinal digestion. Using in vitro gastric and gastro-intestinal digestion models, the effect of the fatty acid chain length and interesterification on the rate and extent of lipolysis was analysed. Overall, "pure" (consisting of a single fatty acid) lipids of ≤C8 underwent rapid lipolysis releasing three fatty acids after intestinal hydrolysis while lipids of ≥C10 released two fatty acids after intestinal hydrolysis. The most interesting observation is that the extent of gastric lipolysis of C4 fatty acids was much lower when they were interesterified with longer chain fatty acids compared to that with the pure C4 triglyceride. Tributyrin underwent ∼60% lipolysis by gastric lipase as indicated by a decrease in total fatty acid release during SIF lipolysis after pre-exposure to rabbit gastric lipase (RGL) in SGF. In comparison, the C4-C8 interesterified lipid exhibited only a 18.1% decrease, and the C4-C18:1 interesterified lipid a 6.1% decrease in total fatty acid release in SGF-SIF. These results suggest that interesterification modulates the digestion of butyric acid from within the stomach to later in the intestine. This study reveals that the design of interesterified lipids alters the timing, but not the extent of short chain fatty acid delivery in the gastrointestinal tract. Such understanding has likely benefits for designing novel interesterified lipids which may have unique applications in various dietary and therapeutic modalities.

Synthesis, identification and quantification of oligomers from polyester coatings for metal packaging.

Polyester can coatings protect both food and packaging from mutual contamination. Even though, can coatings may release Non-Intentionally Added Substances (NIAS) in addition to Intentionally Added Substances (IAS). As NIAS are mainly constituted by cyclic or linear side products that are formed during the polymerization process, we focused our attention on these oligomeric species of molecular weight <1000 Da. These oligomers were obtained from two different polyester resins, each synthesized from four monomers (two phthalic acids and two diols), and from the corresponding final enamel can coatings using ethanol at 95% and 50% at 60 °C for 4 h and 10 days, respectively, as food simulants. HPLC-ESI-MS analysis on the extracts allowed identifying various cyclic and linear oligomers. For the conclusive identification of the different oligomers and their isomeric structures, ad hoc standards were synthesized by acylation reaction between alkyl diols and phthaloyl chlorides. By comparison of 1H NMR spectra, linear and cyclic oligomers were characterized by finding the major presence of 2 + 2 cyclic compounds. The 16 synthesized standards, 4 linear and 12 cyclic compounds were used to establish a method for quantification of linear and cyclic oligomers in enamel migration samples by micro HPLC-high-resolution MS (HRMS). The results showed no significant differences between the amounts of cyclic oligomers extracted with both ethanol concentrations (50 and 95%) and time contact. The extracts showed only a small amount of linear compounds and a prevalence of 2 + 2 cyclic oligomers. The work shows the great importance of the synthesis of specific standards to allow exact quantification in food contact material migrates.

Integrating bioassays and analytical chemistry as an improved approach to support safety assessment of food contact materials.

Food contact materials (FCM) contain chemicals which can migrate into food and result in human exposure. Although it is mandatory to ensure that migration does not endanger human health, there is still no consensus on how to pragmatically assess the safety of FCM since traditional approaches would require extensive toxicological and analytical testing which are expensive and time consuming. Recently, the combination of bioassays, analytical chemistry and risk assessment has been promoted as a new paradigm to identify toxicologically relevant molecules and address safety issues. However, there has been debate on the actual value of bioassays in that framework. In the present work, a FCM anticipated to release the endocrine active chemical 4-nonyphenol (4NP) was used as a model. In a migration study, the leaching of 4NP was confirmed by LC-MS/MS and GC-MS. This was correlated with an increase in both estrogenic and anti-androgenic activities as measured with bioassays. A standard risk assessment indicated that according to the food intake scenario applied, the level of 4NP measured was lower, close or slightly above the acceptable daily intake. Altogether these results show that bioassays could reveal the presence of an endocrine active chemical in a real-case FCM migration study. The levels reported were relevant for safety assessment. In addition, this work also highlighted that bioactivity measured in migrate does not necessarily represent a safety issue. In conclusion, together with analytics, bioassays contribute to identify toxicologically relevant molecules leaching from FCM and enable improved safety assessment.

Modulation of (-)-epicatechin metabolism by coadministration with other polyphenols in Caco-2 cell model.

Widely consumed beverages such as red wine, tea, and cocoa-derived products are a great source of flavanols. Epidemiologic and interventional studies suggest that cocoa flavanols such as (-)-epicatechin may reduce the risk of cardiovascular diseases. The interaction of (-)-epicatechin with food components including other polyphenols could modify its absorption, metabolism, and finally its bioactivity. In the present study we investigate (-)-epicatechin absorption and metabolism when coexposed with other polyphenols in the intestinal absorptive Caco-2 cell model. Depending on the type of polyphenols coadministered, the total amount of 3'-O-methyl-epicatechin and 3'-O-sulfate-epicatechin conjugates found both in apical and basal compartments ranged from 19 to 801 nM and from 6 to 432 nM, respectively. The coincubation of (-)-epicatechin with flavanols, chlorogenic acid, and umbelliferone resulted in similar amounts of 3'-O-methyl-epicatechin effluxed into the apical compartment relative to control. Coincubation with isorhamnetin, kaempferol, diosmetin, nevadensin, chrysin, equol, genistein, and hesperitin promoted the transport of 3'-O-methyl-epicatechin toward the basolateral side and decreased the apical efflux. Quercetin and luteolin considerably inhibited the appearance of this (-)-epicatechin conjugate both in the apical and basolateral compartments. In conclusion, we could demonstrate that the efflux of (-)-epicatechin conjugates to the apical or basal compartments of Caco-2 cells is modulated by certain classes of polyphenols and their amount. Ingesting (-)-epicatechin with specific polyphenols could be a strategy to increase the bioavailability of (-)-epicatechin and to modulate its metabolic profile.

In vitro digestion of citric acid esters of mono- and diglycerides (CITREM) and CITREM-containing infant formula/emulsions.

CITREM is an emulsifier used in the food industry and contains citric acid esters of mono- and diglycerides (GCFE). It is generally recognized as safe but no publication on its digestibility under gastrointestinal conditions and impact on fat digestion was available. It was shown here that fatty acids are released from CITREM by gastric lipase, pancreatic lipase, pancreatic-lipase-related protein 2 and carboxyl ester hydrolase. A two-step in vitro digestion model mimicking lipolysis in the stomach and upper small intestine of term and preterm infants was then used to evaluate the digestibility of CITREM alone, CITREM-containing infant formula and fat emulsions, and isolated GCFE fractions. Overall, it was shown that fat digestion is not significantly changed by the presence of CITREM, and fatty acids contained in CITREM compounds are released to a large extent by lipases. Nevertheless, undigestible water-soluble compounds containing glycerol and citric acid units were identified, indicating that the ester bond between citric acid and glycerol is not fully hydrolyzed throughout the proposed digestion.

Highly diastereoselective Diels-Alder reactions of Baylis-Hillman adducts.

[reaction: see text] Baylis-Hillman adducts were found to be excellent dienophiles in Diels-Alder reactions, providing essentially complete diastereocontrol (although mixtures of endo/exo isomers) with all dienes.

Highly stereocontrolled synthesis of natural barbacenic acid, novel bisnorditerpene from Barbacenia flava.

Barbacenic acid, a bisnorditerpene with five contiguous asymmetric centers (four fully substituted), has been prepared for the first time through a highly stereocontrolled route in 5.2% overall yield from a known octalone. The synthesis serves to define the absolute configuration of the natural product.