Antibiofilm activity of LAE (ethyl lauroyl arginate) against food-borne fungi and its application in polystyrene surface coating.

Several filamentous fungi species as Fusarium oxysporum or Cladosporium sp. can form biofilms by themselves or by participating in polymicrobial biofilms with bacteria. However, despite the high impact of biofilm on the food industry and the high efforts done to control biofilm produced by bacteria in the food area, there has been little study of strategies to control fungal biofilm in this area. In this study, the antibiofilm activity of the safe antimicrobial compound ethyl lauroyl arginate (LAE) was investigated against food spoilage fungi (Cladosporium cladosporioides, Aspergillus ochraceus, Penicillium italicum, Botrytis cynerea and Fusarium oxyspoum). Finally, the efficacy of a varnish-based coating incorporating LAE and coated onto polystyrene microtiter plates has been evaluated as a strategy to reduce fungal biofilm formation. The results of the 2,3-bis-(2-metoxi-4-nitro-5-sulfofenil)-2H-tetrazoilo-5-carboxanilida (XTT) assay, which measure the biofilm metabolic activity of moulds, demonstrated that LAE reduced significantly the formation of fungal biofilm at concentrations from 6 to 25 mg/L. This reduction was confirmed by the micrographs obtained by scanning electronic microscopy (SEM). In addition, LAE also showed antifungal activity against established biofilms. Particularly, it reduced their metabolic activity and viability at concentrations from 6 to 25 mg/L according to results obtained in the XTT assay and observations made by confocal laser scanning microscopy (CLSM). Finally, active coating incorporating from 2% of LAE proved to reduce significantly the biofilm formation in C. cladosporioides, B. cynerea and F. oxyspoum according to the results obtained in the XTT assay. However, the released studies indicated that the retention of LAE in the coating should be improved to prolong their activity.

Ion mobility quadrupole time-of-flight mass spectrometry for the identification of non-intentionally added substances in UV varnishes applied on food contact materials. A safety by design study.

Varnishes are normally applied on printed food packaging to protect it from smearing and scratching. Moreover, they may be applied on the food contact surface in order to improve resistance towards moisture and fat. Some of the compounds that make up the varnish formulation could migrate to the food. In this work, the ion mobility quadrupole time-of-flight mass spectrometry has been used to obtain drift time-aligned mass spectra in which accurate the mass of precursor ions and their fragments are used to identify both intentionally and non-intentionally added substances (NIAS). The compound 2-propenoic acid,1,1'-[2-[[3-[2,2-bis[[(1-oxo-2-propen-1-yl)oxy]methyl]butoxy]-1-oxopropoxy]methyl]-2-ethyl-1,3-propanediyl] ester was identified as a NIAS formed from the varnish monomer 2-propenoic acid, 1,1'-[2-ethyl-2-[[(1-oxo-2-propen-1-yl)oxy]methyl]-1,3-propanediyl] ester. The compound 5, 11-diethyl-7-oxo-4,6,10,12-tetraoxopentadecane-3,13-diyl diacrylate is a NIAS derived from the varnish monomer 2-propenoic acid, 1,1'-[oxybis(methyl-2,1-ethanediyl)] ester, and was found to migrate into the food simulant tested at a level of 0.03 mg kg-1. Finally, the NIAS, 2-{2-[2-(acryloyloxy)-1-methylethoxy]-1-methylethoxy}-1-methylethyl acrylate, an impurity of a photoinitiator used for UV curing of the varnish, was identified, and its migration of 0.14 mg kg-1 exceeded the threshold established as safe for human consumption.

Determination of volatile compounds and their sensory impact in a biopolymer based on polylactic acid (PLA) and polyester.

Polylactic acid (PLA) is one of the most commonly used biopolymers for manufacturing food packaging; its control is very important to ensure consumers' health. In this work, a blend of PLA and polyester was studied and its volatile composition in the polymer and in the migration to food simulants was determined by gas chromatography-mass spectrometry (GC-MS) and atmospheric pressure gas chromatography-quadrupole-time of flight mass spectrometry (APGC-QTOF). The results showed that both techniques provided complementary information, to give complete information on the biopolymer's composition. Some compounds such as lactide or cyclopentanone were detected only by GC-MS while others, such as the cyclic dimer [AA-BD]2 (AA:adipic acid, BD:butanediol), were detected only by APGC-MS. In migration, lactide, AA-BD and [AA-BD]2 were identified in ethanol 95%. A GC-olfactometry study was also carried out. Some compounds showed sensory impact on the polymer odor but not in migration.

Fabric phase sorptive extraction as a reliable tool for rapid screening and detection of freshness markers in oranges.

A simple, fast and sensitive analyte extraction method based on fabric phase sorptive extraction (FPSE) followed by gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) analysis was developed for the analysis of 12 volatile compounds that represent most of the principal chemical families possessing different polarities and volatilities. Five FPSE media coated with different sol-gel sorbent chemistries having different polarities and selectivities were studied: long chain poly(dimethylsiloxane) (PDMS), short chain poly(tetrahydrofuran) (PTHF), Carbowax 20M (CW20M), short chain poly(dimethyl siloxane) (SC PDMS) and polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer (PEG-PPG-PEG). CW20M coated FPSE media was found to be the most efficient extraction media for the analytes of interest in the intended study. The developed methodology was applied to the analysis of orange juice obtained from fresh oranges and oranges after storing at 5°C for two months in order to identify the best chemical markers, both volatiles and non-volatiles, attributed to the freshness of orange. For this purpose, aliquots of the same juice extracts were analysed by GC-MS as well as by UPLC-QTOF-MS. Monoterpenes and terpenoids, such as terpinene, citronellal or estragole were among the volatile compounds that endured the biggest decrease after the extended storage period. Three non-volatile compounds including one amide (subaphyllin) and two flavanoids (tangeretin and nobiletin) also showed a clear decrease in signal intensity (>70%) after orange stored for two months.

Antioxidant effect of an innovative active plastic film containing olive leaves extract on fresh pork meat and its evaluation by Raman spectroscopy.

An antioxidant food packaging material was developed and applied to fresh minced pork meat. The material consists of a multilayer polyethylene film in which 4 different concentrations (2%, 5%, 10%, and 15%) of olive leaves (OL) extract were immobilized in an adhesive formula used to build the multilayer. The antioxidants were not in direct contact with the meat. The packaged meat was kept at 4°C during 16days and finally analyzed by two methods: Raman spectroscopy and thiobarbituric acid reactive substances (TBARS). Raman demonstrated a higher sensitivity for antioxidant evaluation than TBARS. Color of fresh meat packaged with the active film was also measured to evaluate the shelf life of packaged meat. The results showed that active film containing natural antioxidants efficiently enhanced the stability of fresh meat against oxidation processes, thus being a promising way to extend the shelf life of fresh minced meat for about two days.

Fabric phase sorptive extraction: An innovative sample preparation approach applied to the analysis of specific migration from food packaging.

Additives added to food packaging materials can migrate to food in contact with them during storage and shelf life. A novel simple, fast and sensitive analyte extraction method based on fabric phase sorptive extraction (FPSE), followed by analysis using ultra-high performance liquid chromatography and mass spectrometry detection (UPLC-MS) was applied to the analysis of 18 common non-volatile plastic additives. Three FPSE media coated with different sol-gel sorbents characterized with different polarities including sol-gel poly(dimethylsiloxane), sol-gel poly(ethylene glycol) and sol-gel poly(tetrahydrofuran) were studied. All three FPSE media showed very satisfactory results. In general, compounds with low logP values seemed to have higher enrichment factors (EFs), especially with poly(tetrahydrofuran) and poly(ethylene glycol) media. For compounds with high logP values, the use of sol-gel poly(dimethylsiloxane) improved the enrichment capacity. Sample preparation time was optimized at 20 min for sample extraction and 10 min for solvent desorption. Acetonitrile was selected as desorption solvent since recoveries were over 70% for 13 out of 18 selected compounds in all FPSE media. The best extraction recovery values were obtained when compounds were dissolved in aqueous acetic acid solution (3%), where 17 out of 18 compounds showed improvement in their signal intensity after FPSE extraction and 10 obtained enrichment factors above 3 for all the tested FPSE media. When FPSE extracts were concentrated under nitrogen, 11 out of 18 compounds reached EFs values above 100.

Phenolic content and antioxidant activity of olive by-products and antioxidant film containing olive leaf extract.

The antioxidant activity of olive leaf (OL) and cake (OC) extracts with different solvents was evaluated. 70% of aqueous ethanol extract of OL was chosen as the most antioxidant extract based on antiradical activity (DPPH) (95.4±0.3%) and oxygen radical absorbance capacity (ORAC) (0.82±0.07g equivalent Trolox per g of solution) assays. This OL extract was incorporated in two multilayer materials consisting of (i) polyethylene/polyethylene (PE/PE) film and (ii) polyethylene/paper (PE/P). These multilayers were exposed to a gas stream enriched in free radicals to evaluate the scavenging capacity of both materials. PE/PE film exhibited the highest scavenging activity of free radicals (78.8%). Migration of the phenolic compounds from olive by-products into two simulants was performed and demonstrated a non-migrating behavior. The limits of detection and quantification for oleuropein were 0.5µgkg(-1) and 1.7µgkg(-1) and for Luteolin-7-O-glucoside 1.3µgkg(-1) and 4.3µg kg(-1) respectively.

Multiple headspace-solid phase microextraction for the determination of migrants coming from a self-stick label in fresh sausage.

Most fresh sausages are sold with a self-stick adhesive label stuck directly on it. Because of that, the substances in the adhesive could migrate into the fresh sausage. In this work, the multiple headspace-solid-phase microextraction technique has been optimized to quantify the migrants found in the fresh sausage. All the compounds could be analyzed by this technique since its concentration decay exponentially with the number of extractions with good correlation coefficients (0.8258-0.9987). Then, migration assays were carried out and an evaluation of the potential risk for the human health was undertaken with the conclusion that the migration of the compounds from the label does not endanger human health. The results were compared those obtained in migration to casing filled with isooctane used as fat food simulant by Canellas et al. (2014). The values obtained for isooctane (10-600 ng/g) were much higher than the migration values found in the meat stuffed in casing expressed as ng/g of fat content (ranged from 0.02 to 3.3 ng/g of fat content). This finding shows that in some scenarios, it is difficult to simulate the intended contact of materials used in food packaging with simulants.

Risk assessment derived from migrants identified in several adhesives commonly used in food contact materials.

Adhesives are used to manufacture multilayer materials, where their components pass through the layers and migrate to the food. Nine different adhesives (acrylic, vinyl and hotmelt) and their migration in 21 laminates for future use as market samples have been evaluated and risk assessment has been carried out. A total of 75 volatiles and non volatile compounds were identified by gas chromatography-mass spectrometry and ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Most of the compounds migrated below their specific migration limit (SML), lowest observed adverse effect level (LOAEL), no observed adverse effect level (NOAEL) and values recommended by Cramer. Six compounds classified as high toxicity class III according to Cramer classification, migrated over their SML and exposure values recommended by Cramer, when they were applied in the full area of the packaging. Nevertheless, these adhesives fulfill the threshold in the real application as they are applied in a small area of the packaging.

Performance of an active paper based on cinnamon essential oil in mushrooms quality.

The antioxidant capacity of two active papers (based on solid and emulsion paraffin) with cinnamon essential oil was studied. Mushroom samples were introduced in macroperforated PET trays covered with the active papers, and weight loss and browning monitored for 9 days. The antioxidant capacity of the different papers was evaluated based on scavenging 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and tyrosinase inhibition kinetics, and the release of aromatic volatile oils was determined by HSPME-GC-MS. Differences in performance were observed: the active papers were more efficient at avoiding weight loss and mushroom browning when compared to the non-active paraffin-based papers. The efficiency increased when the bottom and walls of the trays were covered rather than the bottom alone. Better results were observed when cinnamon was incorporated as emulsion paraffin instead of a solid.

Compounds from multilayer plastic bags cause reproductive failures in artificial insemination.

High levels of reproductive failure were detected in some Spanish sow farms in the Spring of 2010. Regular returns to estrus and variable reductions in litter size were observed. The problem started suddenly and did not appear to be related to the quality of the ejaculates, disease, alterations of body condition or any other apparent reasons. Subsequent studies determined that the problem was the origin of the plastic bags used for semen storage. Chemical analysis of the suspicious bags identified unexpected compounds such as BADGE, a cyclic lactone and an unknown phthalate that leached into the semen at concentrations of 0.2 to 2.5 mg/L. Spermatozoa preserved in these bags passed all of the routine quality control tests, and no differences were observed between storage in the control and suspicious bags (p > 0.05). In vitro fecundation tests and endocrine profiler panel analysis (EPP) did not show any alterations, whereas the in vivo tests confirmed the described failure. This is the first described relationship between reproductive failure and toxic compounds released from plastic bags.

The challenge of identifying non-intentionally added substances from food packaging materials: a review.

Packaged food can contain non-intentionally added substances (NIAS) as a result of reaction and degradation processes or the presence of impurities in the raw materials used for the packaging production. This manuscript reviews the evidence of NIAS and their possible origin. One of the most challenging and difficult tasks when a sample of packaging materials arrives at the laboratory is knowing the procedure to apply for identifying the unknown compounds. This work proposes an analytical procedure for sample treatment, applicable to polymers as well as to migration samples, and for NIAS identification. The identification protocol comprises the determination of both volatile and non-volatile compounds. A review is presented of the most novel analytical techniques used for identification purposes, particularly high resolution mass spectrometry (HRMS).

Migrants determination and bioaccessibility study of ethyl lauroyl arginate (LAE) from a LAE based antimicrobial food packaging material.

Ethyl lauroyl arginate (LAE, ethyl-N-dodecanoyl-L-arginate hydrochloride) is a strong antimicrobial agent that was included as an active compound in an antimicrobial food packaging material. The potential existence of non-intentionally added substances (NIASs) such as impurities must therefore be checked before launching any food contact material onto the market. For this reason, an untargeted analysis of the migration was performed in both food simulants and fresh chicken breast fillets wrapped with the active material. The analysis was performed by liquid chromatography coupled to mass spectrometry detection with a quadrupole-time-of-flight analyzer, LC-MS(QTOF), for the identification of nonvolatile substances. The migration values found for LAE were 0.94±0.14 and 1.62±0.70 µg/g in ethanol 10% v/v (simulant A) and in ethanol 95% v/v (simulant D), respectively, and 0.93±0.17 µg/g in chicken. Other migrants such as dipropylene glycol methyl ether or tributyl-o-acetylcitrate, both coming from the coating were also found, but none of them have potential adverse effects. Bioaccessibility studies showed that after a simulated gastrointestinal digestion, LAE was not available anymore for subsequent intestinal absorption and new toxic compounds were not formed.

Role of catechins in the antioxidant capacity of an active film containing green tea, green coffee, and grapefruit extracts.

The oxygen radical absorbance capacity (ORAC) method was used to characterize the antioxidant capacity of natural extracts of green tea, green coffee, and grapefruit. These natural extracts were incorporated into a plastic film layer, which was subsequently subjected to a free radical gas stream in order to determine the antioxidant capacity directly in the active film. The green tea extract (GTE) afforded the strongest antioxidant activity. To identify the active compounds in the extract, concentration of the diverse catechins in samples were determined by HPLC-UV analysis. The results showed that the content of catechins in the GTE is around 77% (w/w), the major components being (-)-epigallocatechin gallate, (-)-epicatechin gallate, and (-)-epicatechin. A variation in the concentration profile of catechins was detected during the oxidation process. The chromatographic study demonstrated that (-)-gallocatechin, (-)- epigallocatechin, (+)-catechin, and (-)-catechin gallate exhibited the most radical scavenging.

UPLC-Q-TOF-MS analysis of non-volatile migrants from new active packaging materials.

Ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry (MS) is a useful tool in the analysis of non-volatile compounds, and the use of a quadrupole-time-of-flight (Q-TOF) mass analyzer allows a high sensitivity and accuracy when acquiring full fragment mode, providing a high assurance of correct identification of unknown compounds. In this work, UPLC-Q-TOF-MS technology has been applied to the analysis of non-volatile migrants from new active packaging materials. The materials tested were based on polypropylene (PP), ethylene-vinyl alcohol copolymer (EVOH), and poly(ethylene terephthalate) (PET). The active packaging materials studied were one PP film containing a natural antioxidant, and two PP/EVOH films, two PET/EVOH films and one coextruded PP/EVOH/PP film containing natural antimicrobials. The chemical structure of several compounds was unequivocally identified. The analysis revealed the migration of some of the active substances used in the manufacture of active packaging, such as caffeine (0.07 ± 0.01 µg/g), carvacrol (0.31 ± 0.03 µg/g) and citral (0.20 ± 0.01 µg/g). Unintentionally added substances were also found, such as citral reaction compounds, or citral impurities present in the raw materials.

Atmospheric pressure gas chromatography coupled to quadrupole-time of flight mass spectrometry as a powerful tool for identification of non intentionally added substances in acrylic adhesives used in food packaging materials.

Acrylic adhesives are used to manufacture multilayer laminates that are used in food packaging to form the geometric shape of the package as well as to stick labels on the packages. Once applied on the packaging adhesives can supply potential migrants that could endanger the packaged food. Adhesives are complex matrices where intentionally and non intentionally added substances are present, but the identification of the migrants is required by law. In this study atmospheric pressure gas chromatography coupled to a quadrupole hyphenated to a time of flight mass spectrometer (APGC-MS/Q-TOF) has been explored for identification of unknowns coming from three different acrylic adhesives. The results are compared to those obtained by conventional GC-MS-Q (quadrupole). Sixteen compounds were identified by GC-MS/Q and five of them were confirmed by APGC-MS/Q-TOF as their molecular ions were found. Moreover, additional three new compounds were identified and their structure was elucidated working with the spectra obtained by APGC-MS/Q-TOF. This finding was very relevant as these compounds were biocides suspected to be allergenic and cytotoxic in humans. Migration studies were carried out using Tenax as solid food simulant and the results showed that the three acrylic adhesives tested in this work were safe for being used in food packaging materials since the migration of compounds previously identified was below the limit established in the current legislation.

New UPLC coupled to mass spectrometry approaches for screening of non-volatile compounds as potential migrants from adhesives used in food packaging materials.

The objective of this study was to identify the non-volatile compounds as potential migrants from adhesives used in food packaging. A number of the current acrylic adhesive formulations were extracted and prepared for analysis. The extracts were screened using ultra-performance liquid chromatography coupled to a time-of-flight mass spectrometer detector (UPLC-TOF-MS). This approach allowed the identification of several components by a combination of exact mass and in-source collision induced dissociation (CID). Due to the lack of freely available information on adhesive formulations further analyses were undertaken using ultra-performance liquid chromatography coupled to high definition mass spectrometry (UPLC-HDMS). Using the Mass Fragment tool to interrogate fragmentation data, a wide series of compounds were identified, demonstrating the usefulness and importance of these tools for difficult problems. Moreover, using several packaging materials containing adhesives, qualitative migration tests were performed with Tenax as a food simulant. Several non-volatile compounds were identified as well in the Tenax which emphasizes the importance of this work and demonstrates that even the non-volatile compounds have the potential to migrate into food which is in contact with packaging materials. The main characteristics of the screening study and the results obtained are shown and discussed.

Analytical methods for the screening of potential volatile migrants from acrylic-base adhesives used in food-contact materials.

Two different analytical techniques were studied for screening the volatile compounds present in pure adhesives and those from the adhesives in different laminates. Three different adhesive formulations were used for the study, all acrylic-based and supplied by different producers. Laminates with polypropylene and paper, polypropylene and polyethylene, and aluminium and polyethylene as substrates were prepared and studied. Adhesives themselves were acetonitrile extracted and volatiles identified by time-of-flight mass spectrometry based on accurate mass measurement of molecular and main fragments. The volatiles in the films themselves were determined by a headspace solid-phase microextraction analysis followed by gas chromatography-mass spectrometry. Significant differences were found within the adhesive formulations. Compounds detected in the screening were assessed in terms of migration through the laminate polypropylene and paper into polyethylene used as a matrix-simulating food. The concentration of the compounds in the polyethylene ranged from 0.04 to 1.6 microg dm(-2) in the polypropylene side, and from 0.27 to 28 microg dm(-2) in the paper side. The most toxic compound detected in the screening, 2,4,7,9-tetramethyl-5-decyne-4, was not found in any of the sides. Analytical features were also calculated to provide the conditions for quantitative purposes. Sensitivity was at low ng dm(-2) of polyethylene and the relative standard deviation was below 10%.

Adaptation of the ORAC assay to the common laboratory equipment and subsequent application to antioxidant plastic films.

The oxygen radical absorbance capacity (ORAC) method has been adapted to the instrumental laboratory and optimized for the determination of the antioxidant capacity of a novel active packaging. As the ORAC assay requires the monitorization of a reaction at controlled temperature by means of the fluorescence signal decrease over time, specific instrumental is usually necessary. In this work, a common liquid chromatographic device has been adapted to perform the ORAC assay, leaving it accessible to any laboratory. Using this adaptation, five different essential oils have been determined resulting in the following antioxidant order: clove (2.66 g Trolox per gram of essential oil), oregano (2.25), cinnamon (1.93), rosemary (1.66), and ginger (1.47). After incorporating the essential oils to the film, its antioxidant capacity has also been checked and related to the concentration of essential oil as well as the thickness of the active film. The results point out that for the same amount of essential oil incorporated measured as grams per square meter, thicker films have more antioxidant capacity than the thinner and more concentrated ones. Furthermore, the antioxidant capacity found in the films was always higher than expected taking into account the amount of essential oil incorporated. Some likely explanations have been proposed, leading to the improvement of the antioxidant film under development.

Critical review on recent developments in solventless techniques for extraction of analytes.

The most recent contributions on solventless extraction techniques have been reviewed. This paper deals with those techniques that use solid phases, such as solid-phase microextraction, liquid phases, such as single-drop microextraction and hollow-fibre liquid-phase microextraction, and subcritical fluids, such as subcritical water extraction. In all cases, the most recent publications have been critically studied. Direct extraction and derivatization processes to facilitate the extraction of analytes in different areas have been included. Hyphenated approaches, if available, are also included in this review. Comparison of techniques organized by analytes and matrices also enhances this critical overview of solventless techniques.