Comparison of LC-ESI, DART, and ASAP for the analysis of oligomers migration from biopolymer food packaging materials in food (simulants).

Biopolymers based on polylactic acid (PLA) and starch have numerous advantages, such as coming from renewable sources or being compostable, though they can have deficiencies in mechanical properties, and for this reason, polyester resins are occasionally added to them in order to improve their properties. In this work, migration from a PLA sample and from another starch-based biopolymer to three different food simulants was studied. Attention was focused on the determination of oligomers. The analysis was first performed by ultraperformance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS), which allowed the identification of the oligomers present in migration. Then, the samples were analyzed by two ambient desorption/ionization techniques directly coupled to mass spectrometry (ADI), direct analysis in real-time coupled to standardized voltage and pressure (DART-MS) and atmospheric pressure solids analysis probe (ASAP-MS). These methodologies were able to detect simultaneously the main oligomers migrants and their adducts in a very rapid and effective way. Nineteen different polyester oligomers, fourteen linear and five cyclic, composed of different combinations of adipic acid [AA], propylene glycol [PG], dipropylene glycol [DPG], 2,2-dibutyl-1,3-propanediol [DBPG], or isobutanol [i-BuOH] were detected in migration samples from PLA. In migration samples from starch-based biopolymer, fourteen oligomers from poly(butylene adipate co-terephthalate) polyester (PBAT) were identified, twelve cyclic and two linear. The results from ADI techniques showed that they are a very promising alternative tool to assess the safety and legal compliance of food packaging materials.

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.

Ambient mass spectrometry as a tool for a rapid and simultaneous determination of migrants coming from a bamboo-based biopolymer packaging.

New bamboo-based biopolymers are used as food packaging materials, but it must be evaluated to ensure consumers safety. In this study, migration from a commercial bamboo-based biopolymer to ethanol 10% (v/v), acetic acid 3% (w/v) and ethanol 95% (v/v) was studied. The migrants were determined from three different perspectives. Volatile and semi-volatile compounds were analyzed by gas chromatography-mass spectrometry (GC-MS). Twenty-five compounds were detected. In addition, a number of phytosterols were detected in ethanol 95%. Non-volatile compounds were identified and quantified by ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-Q/ToF). Twelve non-volatile compounds were detected in migration solutions, mainly melamine and its derivatives, coming from polymer resins present in the biopolymer. Melamine migration was higher than 50 mg/Kg in the third sequential migration test. Finally, the migration samples were analyzed by DART-SVP (direct analysis in real time coupled to standardized voltage and pressure). This methodology was able to detect simultaneously the main volatile and non-volatile migrants and their adducts in a very rapid and effective way and is shown as a promising tool to test the safety and legal compliance of food packaging materials.

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 adhesive acrylates in recycled polyethylene terephthalate by fabric phase sorptive extraction coupled to ultra performance liquid chromatography - mass spectrometry.

This article presents fabric phase sorptive extraction (FPSE) as a simple and effective pre-concentration method for the enrichment of acrylate compounds in different food simulants and subsequent analysis of the extracts by ultra-high-performance liquid chromatography with mass spectrometric detection (UPLC-MS). Acrylate compounds come from acrylic adhesives used commonly for sticking the paper labels on polyethylene terephthalate (PET) bottles and therefore, they may exist in recycled polyethylene terephthalate (rPET). Four acrylates were studied: ethylene glycol dimethacrylate (EGDM), pentaerythritol triacrylate (PETA), triethylene glycol diacrylate (TEGDA) and trimethylolpropane triacrylate (TMPTA). Five different types of FPSE media coated with different sol-gel sorbents were studied and finally sol-gel polyethylene glycol- polypropylene glycol-polyethylene glycol triblock copolymer (PEG-PPG-PEG) coated FPSE media was chosen for its satisfactory results. The optimal conditions affecting the extraction efficiency of compounds were determined in three different food simulants. Statistical evaluation of this method reveals good linearity and precision. Under the optimized conditions, the method provided limits of detection of the compounds in the range of (0.1-1.9 ng g-1, 0.1-1.2 ng g-1, 0.2-2.3 ng g-1) in EtOH 10%, HAc 3% and EtOH 20% and the enrichment factor values (EFs) after applying N2 were in the range of 11.1-25.0, 13.8-26.3, 8.3-21.9, in simulants A, B and C respectively. The optimized method was applied successfully to analyze thirteen types of recycled PET samples. Acrylates were found in some of the samples at ng g-1 levels.

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 volatile non intentionally added substances coming from a starch-based biopolymer intended for food contact by different gas chromatography-mass spectrometry approaches.

The rapid growth of polymer technology in the field of food contact materials (FCMs) needs to be supported by continuous improvement in material testing, in order to ensure the safety of foodstuff. In this work, a range of different starch-based biopolymer samples, in the shape of pellets and retail samples (cups and dishes) were studied. The optimized extraction process was performed on three different pellet shapes: pellets with no modification (spherical), pellets shattered under high pressure (lentils), and pellets cryogenically ground (powder). The analysis of unknown volatile and semi-volatile compounds was carried out by gas chromatography-mass spectrometry, using both electron ionization with a single quadrupole mass analyzer (GC-EI-MS), and atmospheric pressure gas chromatography with a quadrupole/time of flight mass analyzer (APGC-Q/ToF). The identification process was implemented using the latest advances in the understanding of APGC ionization pathways. Chemical migration was also assessed on prototype samples using the food simulants: ethanol 10% v/v, acetic acid 3% w/V, ethanol 95% v/v, isooctane, and vegetable oil. Each migration test was performed three consecutive times, as recommended for materials intended for repeated use.

Synergistic properties of mustard and cinnamon essential oils for the inactivation of foodborne moulds in vitro and on Spanish bread.

This work was performed to evaluate the antifungal effect of cinnamon and mustard essential oil (EO) alone and in combination against a range of mould strains. A wide range of resistance levels was observed among different mould species, being R. stolonifer the most resistant one. Mustard EO showed the biggest antifungal effect, and for this reason it was selected to study its effect in vapour phase on bread. The shelf life of bread inoculated with R. stolonifer and non- inoculated increased by 3-4 days at 25 °C, while at 4 °C the shelf-life increased beyond 50 days. However, the results from the acceptability test were negative. Based on this, the antifungal effect of cinnamon/mustard EO in combination was evaluated, with results mostly additive and synergistic. The ratio of the combination was defined taking into account the most resistant strain (100:8, cinnamon and mustard respectively) and their application was performed in vapour phase. This combination maintained the antifungal activity presented by mustard EO by itself and masked the mustard flavour, providing positive results in the acceptability tests. It was therefore suggested to be used at industrial scale.

Identification of key odorant compounds in starch-based polymers intended for food contact materials.

Biopolymers used for food contact materials must be evaluated in order to ensure food safety and quality. In this work, the aroma profile of starch-based packaging materials, presented as pellets and films has been characterized. Headspace solid phase microextraction (HS-SPME) technique and gas chromatography coupled to both mass spectrometry and a sniffing port (GC-MS-O) were used for identification. In total, 35 odorant compounds were detected. The results showed that aldehydes were the odorants with the highest aromatic impact in starch-based films. Eight odorant compounds, such as trimethylamine, 1-octen-3-one, sotolon, (Z) and (E)-2-nonenal, p-vinylguaiacol, eugenol and 1-undecanol, defined as aroma-impact compounds, obtained modified frequency values (MF%) above 60% in at least 3 out of 4 films. A sensory panel evaluated the toasted, sweet/fruity, green, flower, distasteful, fat and spices notes in the films and it was observed that the quality of all samples decreased when the toasted and spices notes increased.

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.

Development and validation of a LC-MS/MS method for the analysis of bisphenol a in polyethylene terephthalate.

Bisphenol A (BPA) is widely recognized being an endocrine disrupter and it is employed in many food packaging applications. Although it is not intended to take part in the manufacture of polyethylene terephthalate (PET) food grade, the presence of BPA in recycled PET should not be neglected. To satisfy the increasing need to ensure "BPA-free" articles, a liquid chromatography-tandem mass spectrometry method was developed. The crucial step in the sample preparation was the total dissolution/reprecipitation of the polymer. The repeatability of the method (RSD%, n = 6) was lower than 7.6%, while HorRat values ranged between 0.3 and 0.5. Limits of detection and quantitation were 1.0 and 3.3 ng g-1, respectively. Recovery ranged from 89 to 107%. The method was applied to 23 samples of virgin and recycled pellets, preforms and bottles. Migration tests were also carried out. Results shown significantly higher levels of BPA in recycled PET.

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.

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.

Effect of an active label based on benzyl isothiocyanate on the morphology and ochratoxins production of Aspergillus ochraceus.

The aim of this work was the study of the main effects of benzyl isothiocyanate (BITC) on A. ochraceus morphology and on its production metabolism of ochratoxins. This compound was evaluated as active agent of an antimicrobial label in food packaging. Microbiological studies showed a slowdown in mould growth when the active material was applied to A. ochraceus and the presence of three different areas of growth. Scanning electron microscopy was successfully used to demonstrate the mode of action of BITC on this strain. A. ochraceus exhibited modifications in morphology compared to the control samples such as the disappearance of sclerotia or cleistothecia An extraction protocol and an analytical method by UPLC-MS/MS to determine ochratoxins (OTs) was developed. The results showed that all these morphological changes were related to a decrease on OTs production, both ochratoxin A (OTA) and ochratoxin B (OTB). The presence of BITC caused a great decrease on OTA that modified the OTA/OTB ratio, increasing the OTB proportion. Furthermore, the active packaging also modified the production of other secondary metabolites. The morphological and metabolic effects observed, as well as the relationship between them, are of great interest since they have not been reported before for A. ochraceus.

Toxic compounds from tobacco in placenta samples analyzed by UPLC-QTOF-MS.

Polycyclic aromatic hydrocarbons (PAHs), tobacco-specific nitrosamines (TSNAs) and aromatic amines are carcinogens present in cigarette smoke. These compounds are distributed in the human body and they could be transferred to the foetus during the pregnancy. Placenta is the main barrier to these toxic compounds and its study is the objective of this work. A method based on solid-phase extraction (SPE) with ultra-performance liquid chromatography-tandem quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MS) has been examined and optimized for the analysis of 9 target analytes (4 tobacco-specific nitrosamines and some of their metabolites, 3 aromatic amines, nicotine and cotinine) in 26 placenta samples from smoking and non-smoking women. Limits of detection (LODs) were in the range of 3-27ng/g of placenta. Nicotine, cotinine, N-nitrosoanatabine (NAT) and 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK) metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) were detected in the placenta samples of smoking woman. Nicotine was detected in 3 out of 8 placentas from smoking women, always below the limit of quantification (88ng/g). This could be expected, as the half-life of nicotine in the body is limited to about 0.5-3h. Cotinine, the main metabolite from nicotine, was detected in all placentas from smoking women at concentrations between 17.2 and 61.8ng/g, reaching the highest values for those women that smoked the highest number of cigarettes. NAT and NNAL were detected in all placentas from smoking women, always below the limit of quantification (40ng/g and 33ng/g respectively).

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.

Safety by design of printed multilayer materials intended for food packaging.

Printing inks are commonly used in multilayer plastics materials used for food packaging, and compounds present in inks can migrate to the food either by diffusion through the multilayers or because of set-off phenomena. To avoid this problem, the right design of the packaging is crucial. This paper studies the safety by design of multilayer materials. First, the migration from four different multilayers manufactured using polyethylene terephthalate (PET), aluminium (Al) and polyethylene (PE) was determined. The structural differences among materials such as the presence of inks or lacquer coatings as well as the differences in layers position allowed the study of a safety-by-design approach. Sixty-nine different compounds were detected and identified; 49 of them were not included in the positive list of Regulation EU/10/2011 or in Swiss legislation and 15 belong to Cramer class III, which means that they have a theoretical high toxicity. Some of the compounds related to ink composition were pyrene, a compound commercially used to make dyes and dye precursors and the antioxidant Irganox 1300. The application of external lacquers decreased the concentration of some migrants but also brought the potential for new migrants coming from its composition. A final risk assessment of the material allowed evaluating food safety for different food simulants and confirm it.

Raman spectroscopy, electronic microscopy and SPME-GC-MS to elucidate the mode of action of a new antimicrobial food packaging material.

One critical challenge when developing a new antimicrobial packaging material is to demonstrate the mode of action of the antimicrobials incorporated into the packaging. For this task, several analytical techniques as well as microbiology are required. In this work, the antimicrobial properties of benzyl isothiocyanate, allyl isothiocyanate and essential oils of cinnamon and oregano against several moulds and bacteria have been evaluated. Benzyl isothiocyanate showed the highest antimicrobial activity and it was selected for developing the new active packaging material. Scanning electron microscopy and Raman spectroscopy were successfully used to demonstrate the mode of action of benzyl isothiocyanate on Escherichia coli. Bacteria exhibited external modifications such as oval shape and the presence of septum surface, but they did not show any disruption or membrane damage. To provide data on the in vitro action of benzyl isothiocyanate and the presence of inhibition halos, the transfer mechanism to the cells was assessed using solid-phase microextraction-gas chromatography-mass spectrometry. Based on the transfer system, action mechanism and its stronger antimicrobial activity, benzyl isothiocyanate was incorporated to two kinds of antimicrobial labels. The labels were stable and active for 140 days against two mould producers of ochratoxin A; Penicillium verrucosum is more sensitive than Aspergillus ochraceus. Details about the analytical techniques and the results obtained are shown and discussed. Graphical Abstract Antimicrobial evaluation of pure compounds, incorporation in the packaging and study for mode of action on S. coli by Raman, SEM and SPME-GC-MS.

Progress in mass spectrometry for the analysis of set-off phenomena in plastic food packaging materials.

In most cases, food packaging materials contain inks whose components can migrate to food by diffusion through the material as well as by set-off phenomena. In this work, different mass spectrometry approaches had been used in order to identify and confirm the presence of ink components in ethanol (95%) and Tenax(®) as food simulants. Three different sets of materials, manufactured with different printing technologies and with different structures, were analyzed. Sample analysis by ultra performance liquid chromatography mass spectrometry (UPLC-MS), using a quadrupole-time of flight (Q-TOF) as a mass analyser proved to be an excellent tool for identification purposes while ion mobility mass spectrometry (IM-MS) shown to be very useful for the confirmation of the candidates proposed. The results showed the presence of different non-volatile ink components in migration such as colorants (Solvent Red 49), plasticizers (dimethyl sebacate, tributyl o-acetyl citrate) or surfactants (SchercodineM, triethylene glycol caprilate). An oxidation product of an ink additive (triphenyl phosphine oxide) was also detected. In addition, a surface analysis technique, desorption electrospray mass spectrometry (DESI-MS), was used for analyzing the distribution of some ink components (tributyl o-acetyl citrate Schercodine L, phthalates) in the material. The detection of some of these compounds in the back-printed side confirmed the transference of this compound from the non-food to the food contact side. The results also showed that concentration of ink migrants decreased when an aluminum or polypropylene layer covered the ink. When aluminum was used, concentration of most of ink migrants decreased, and for 5 out of the 9 even disappeared.

Raman Imaging Spectroscopy as a Tool To Investigate the Cell Damage on Aspergillus ochraceus Caused by an Antimicrobial Packaging Containing Benzyl Isothiocyanate.

Raman imaging spectroscopy is a nondestructive analytical method that can be a useful tool to obtain detailed information about the molecular composition and morphology of biological samples. Its high spatial resolution was used to collect spectra of Aspergillus ochraceus, a mold producer of ochratoxin A (OTA), in order to investigate the cell damage caused on it by the action of the antimicrobial benzyl isothiocyanate (BITC). The study was performed in both direct contact and vapor phase, in order to check the use of BITC as active agent in food packaging material. The results showed that there were morphologic alteration and a characteristic Raman spectrum on spore and hyphae exposed to BITC. BITC was accumulated in the mold cells where it caused an enormous amount of alterations in cellular components (lipids, proteins, saccharides, amino acids...) and cellular functions (cell cycle, respiration, metabolism, transcription of genes, fluidity of the cellular wall). All these structural, composition, and metabolic changes will affect the production of OTA. Pattern recognition with chemometrics using principal component analysis (PCA) demonstrated an excellent separation between control and BITC treated samples, both in spores and hyphae. PCA results also showed two different affection levels when samples were exposed to BITC in the vapor phase.