Migration of oligomers from a food contact biopolymer based on polylactic acid (PLA) and polyester.

Polylactic acid (PLA) is a biopolymer commonly used in food packaging due to its good characteristics, similar to PET. To evaluate the safety of this material, the analysis of the non-intentionally added substances (NIAS) is required. Oligomers are NIAS and their behavior needs a deep study, especially if they migrate to the food. In this work, the analysis of the polymer and the migration to food simulants was carried out. A total dissolution/precipitation procedure was applied to PLA pellets and films, using dichloromethane and ethanol as solvent and antisolvent system respectively. The migration tests were carried out in three liquid simulants to mimic any kind of food. Since oligomers are not present in the positive list of the Directive 10/2011/EC, their concentration must be below the 0.01 mg/kg of food. UPLC-QTOF-MS, with and without ion mobility (IM), was used for the analysis. Thirty-nine different PLA oligomers made of repeated monomer units of [LA] (C3H4O2) and with different structures were identified. They corresponded to cyclic oligomers with [LA]n structure and two groups of linear oligomers, one with an hydroxyl group, OH-[LA]n-H, and the other one with an ethoxy group, CH3-CH2-O-[LA]n-H. Cyclic oligomers only appeared in the material and were not present in migration solutions. Linear oligomers HO-[LA]n-H were already present in the pellets/film and they migrated in a higher extension to aqueous food simulants (EtOH 10% and AcH 3%). However, linear oligomers CH3-CH2-O-[LA]n-H were not present initially in the pellets/film, but were detected in migration to simulants with ethanol content, EtOH 95% and EtOH 10%. Furthermore, 5 cyclic polyester oligomers were identified in migration. Ethanol 95% and ethanol 10% migration solutions were also analyzed by scanning electron microscopy (SEM), and the presence of microstructures that could be attributed to the oligomers migration was found. They could be seen as microplastics.

Determination of oligomers in virgin and recycled polyethylene terephthalate (PET) samples by UPLC-MS-QTOF.

An oligomer is a molecule that consists of a few monomer units. It can be formed during polymer manufacturing and also due to polymer degradation processes or even during use conditions. Since oligomers are not included in chemical databases, their identification is a complex process. In this work, the oligomers present in 20 different PET pellet samples have been determined. Two different sample treatment procedures, solvent extraction and total dissolution, were applied in order to select the most efficient one. The analyses were carried out by UPLC-MS-QTOF. The use of high resolution mass spectrometry allowed the structural elucidation of these compounds and their correct identification. The main oligomers identified were cyclic as well as lineal from the first, second, and third series. All of them were composed of terephthalic acid (TPA), diethylene glycol (DEG), and ethylene glycol (EG). Quantitative values were very different in both procedures. In total dissolution of PET samples, the concentration of oligomers was always, at least, 10 times higher than in solvent extraction; some of the compounds were only detected when total dissolution was used. Results showed that the oligomers with the highest concentration values were dimers and trimers, cyclic, as well as lineal, from the first and second series. The oligomer with the maximum concentration value was TPA2-EG-DEG that was found in all the samples in a concentration range from 2493 to 19,290 ng/g PET. No differences between virgin and recycled PET were found. Migration experiments were performed in two PET bottles, and results showed the transference of most of these oligomers to a fat food simulant (ethanol 95%). Graphical abstract Graphical abstract of the two procedures developd and optimized for identifying oligomers in PET pellets and in migration form PET bottles.

Fabric phase sorptive extraction for specific migration analysis of oligomers from biopolymers.

Oligomers are potential migrants from polymers or biopolymers intended to food packaging and they have to be under control. In order to comply with European regulation 10/2011, their concentration in migration must be below 0.01 µg g-1. In this work, fabric phase sorptive extraction (FPSE) was explored as an effective method for extraction and pre-concentration of oligomers migrated from a blend PLA-polyester material. Both food simulant B (3% acetic acid) and juice, as real food, were used for migration experiments. The parameters of FPSE were optimized and the analysis was done by UHPLC-QTOF and UHPLC-QqQ. A total of 21 oligomers were identified, 9 of them coming from PLA and 12 oligomers from the polyester part. These oligomers were formed by adipic acid (AA), phthalic acid (PA) and/or butanediol (BD), ten were cyclic and 11 were linear molecules. Using the optimized FPSE procedure in 3% acetic acid as food simulant, it was possible to identify 3 new compounds that were not detected by direct injection of the simulant into UHPLC-QTOF. In addition, 2 extra compounds, cyclic PA-BD4-AA3 and cyclic PA2-BD3-AA, were only identified in juice samples after FPSE extraction. Besides, in order to quantify the compounds identified, an isolation procedure for PLA oligomers was carried out. Two oligomers were isolated: cyclic (LA)6 and linear HO-(LA)4-H, both with a purity higher than 90% (LA: lactic acid). The highest concentration value was found for the cyclic oligomer [AA-BD]2, that showed 22.63 µg g-1 in 3% acetic acid and 19.64 µg g-1 in juice. The concentration of the total amount of remaining oligomers was below 7.56 µg g-1 in 3% acetic acid as well as in juice.

Migration studies and toxicity evaluation of cyclic polyesters oligomers from food packaging adhesives.

Multilayer materials used in food packaging are commonly manufactured with a polyurethane adhesive layer in its structure that may contain cyclic esters oligomers as potential migrants. However, little is known about their toxicity. In this work, two cyclic esters of polyurethane are evaluated in migration from 20 multilayer packaging samples. They were composed by adipic acid (AA), diethylene glycol (DEG) and isophthalic acid (IPA) and their structure was AA-DEG and AA-DEG-IPA-DEG. The concentration of these compounds in migration exceeded the maximum level established by Regulation EU/10/2011 (10 ng g-1). Bioaccessibility of both compounds was evaluated by studying gastric and intestinal digestion. The studies showed that the concentration of the compounds decreased during digestion and that their hydrolysed molecules increased. Furthermore, endocrine activity in vitro assays were performed. A weak androgen receptor antagonism was identified, whereas no arylhydrocarbon receptor activity or binding to the thyroid hormone transport protein was found.

Determination of non-volatile components of a biodegradable food packaging material based on polyester and polylactic acid (PLA) and its migration to food simulants.

Bioplastic materials are increasingly used due to its benefits for the environment preservation. Among them, food packaging materials based on polylactic acid (PLA) are among the most employed. In this work, a sample treatment methodology based on dissolution/precipitation has been optimized, selecting finally dichloromethane/ethanol as solvent/antisolvent system. The extracts obtained were analysed by UPLC-MS(QTOF), that allowed the identification of the main PLA non-volatile components. The recovery results were between 100.9 to 114.0%. The methodology was applied to the analysis of pellets and films of a PLA-polyester blend sample. A total of 37 different compounds were detected, where the four compounds with the highest intensity in pellet samples were cyclic oligomers coming from the polyester part of the blend and composed by adipic acid (AA), phthalic acid (PA) and butanediol (BD). Migration experiments to 3 food simulants were also performed: ethanol 95% (v/v), ethanol 10% (v/v) and acetic acid 3% (w/v). The results showed that in addition to those compounds previously detected in the film, new compounds coming from the reaction of PLA components with food simulants were present in migration solutions.

Identification of non-volatile migrants from baby bottles by UPLC-Q-TOF-MS.

Baby bottles made of polypropylene, Tritan® and silicone were evaluated regarding the migration of non-volatile compounds using UPLC-QTOF-MS. Twenty-seven compounds were identified. In all polypropylene samples the migration of 2.2'-(tridecylimino)bis-ethanol and derivatives thereof were detected in concentrations below the specific migration limit (1.2 mg.kg-1). Furthermore, clarifying agents and glycerol derivatives were detected. Tritan baby bottle showed the migration of one slip additive. On the other hand, twenty compounds were detected in silicone baby bottles. Most of them were unknown compounds derived from acrylates. Once the migrants were identified, the risk assessment was carried out using the Threshold of Toxicological Concern (TTC) approach. The risk assessment of migrants coming from silicone samples showed levels above the threshold recommended as safe for babies.

Analysis of isophthalaldehyde in migration samples from polyethylene terephthalate packaging.

In the present work, different pre-concentration strategies were evaluated for the analysis of isophthalaldehyde in migration samples from food packaging materials. This compound is a potential migrant in several copolymers used for food packaging, and since it is considered a non-intentionally added substance, its concentration in migration samples must be determined. Derivatisation was the first sample treatment evaluated. o-(2,3,4,5,6-Pentafluorobenzyl)hydroxylamine was tested as derivatisation agent, but no satisfactory results were obtained. Then, hollow-fibre liquid-phase microextraction (HF-LPME) and solid-phase microextraction were optimised. The HF-LPME method showed the highest sensitivity, achieving an enrichment factor of 60-fold. The limit of detection of the method was 10 ng g-1, the limit of quantification was 30 ng g-1 and the relative standard deviation was 6.1%. Finally, the method was applied to migration studies to evaluate the safety in use of a poly(ethylene terephthalate) packaging material. The content of isophthalaldehyde was determined in two aqueous food simulants: 10% ethanol (v/v) and 3% acetic acid (w/v). Different migration conditions were tested. The results obtained showed a considerable rise in the concentration of isophthalaldehyde when increasing the time and temperature of the migration experiment.

Overall and specific migration from multilayer high barrier food contact materials - kinetic study of cyclic polyester oligomers migration.

Most multilayer high barrier materials used in food packaging have a polyurethane adhesive layer in their structures. In order to assess the safety of these materials, it is important to determine the compounds intentionally added to the adhesives (IAS) as well as those non-intentionally added substances (NIAS). During the manufacture of polyurethane adhesives, some by-products can be formed, such as cyclic polyester oligomers coming from the reaction between dicarboxylic acids and glycols. Since these compounds are not listed in the Regulation 10/2011/EU, they should not be found in migration above 0.01 mg/kg of simulant. In this study two flexible multilayer packaging materials were used and migration was evaluated in simulant A (ethanol 10% v/v), simulant B (acetic acid 3% w/v) and simulant ethanol 95% v/v during 10 days at 60ºC. Identification and quantification of non-volatile compounds was carried out by UPLC-MS-QTOF. Most of migrants were oligomers such as cyclic polyesters and caprolactam oligomers. Overall migration and specific migration of adipic acid-diethylene glycol and phthalic acid-diethylene glycol were monitored over time and analysed by UPLC-MS-TQ. In most cases, ethanol 95% v/v was the simulant with the highest concentration values. Overall migration kinetics followed a similar pattern than specific migration kinetics.

Engineering new mycobacterial vaccine design for HIV-TB pediatric vaccine vectored by lysine auxotroph of BCG.

In this study, we have engineered a new mycobacterial vaccine design by using an antibiotic-free plasmid selection system. We assembled a novel Escherichia coli (E. coli)-mycobacterial shuttle plasmid p2auxo.HIVA, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism for plasmid selection and maintenance based on glycine complementation in E. coli and lysine complementation in mycobacteria. This plasmid was first transformed into glycine auxotroph of E. coli strain and subsequently transformed into lysine auxotroph of Mycobacterium bovis BCG strain to generate vaccine BCG.HIVA(2auxo). We demonstrated that the episomal plasmid p2auxo.HIVA was stable in vivo over a 7-week period and genetically and phenotypically characterized the BCG.HIVA(2auxo) vaccine strain. The BCG.HIVA(2auxo) vaccine in combination with modified vaccinia virus Ankara (MVA). HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. Polyfunctional HIV-1-specific CD8+ T cells, which produce interferon-γ and tumor necrosis factor-α and express the degranulation marker CD107a, were induced. Thus, we engineered a novel, safer, good laboratory practice-compatible BCG-vectored vaccine using prototype immunogen HIVA. This antibiotic-free plasmid selection system based on "double" auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth.