Raman Imaging Spectroscopy as a Tool To Investigate the Cell

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Raman imaging spectroscopy as a tool to investigate the cell damage on Aspergillus ochraceus caused by an antimicrobial packaging containing benzyl isothiocyanate Isabel Clemente, Margarita Aznar, and Cristina Nerin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00116 • Publication Date (Web): 31 Mar 2016 Downloaded from http://pubs.acs.org on March 31, 2016

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Analytical Chemistry

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Raman imaging spectroscopy as a tool to investigate the cell damage on Aspergillus ochraceus caused by an antimicrobial packaging containing Benzyl isothiocyanate

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Isabel Clemente1, Margarita Aznar1, and Cristina Nerín1*

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1) Departamento de Química Analítica, Instituto de Investigación en Ingeniería de Aragón (I3A), Grupo GUIA, Universidad de Zaragoza, Zaragoza, Spain.

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*Corresponding author:

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Tel: +34976761873

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E-mail address: [email protected]

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Abstract

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Raman imaging spectroscopy is a non-destructive 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 mould producer of ochratoxin A (OTA), in order to investigate the cell damage caused on it by the action of the antimicrobial benzyl isothiocyanate. The study was performed in both direct contact and vapour phase, in order to check the use of BITC as active agent in food packaging material.

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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 mould 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 vapour phase.

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Keywords

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Raman imaging spectroscopy, active packaging, Benzyl isothiocyanate, cell damage mould, mechanism of action, vapour phase, principal component analysis, antimicrobial, chemometrics.

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1. Introduction

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Aspergillus ochraceus is a foodborne mould which is able to produce mycotoxins as secondary metabolites, in this case ochratoxin A (OTA), which poses a serious health risk and has a great diversity of toxic effects, acting as a neurotoxic, immunosuppressive, genotoxic, carcinogenic and teratogenic agent 1. Mycotoxins 2,3 are commonly present in food , especially in cereals and cereal products, whose OTA content represents almost the 70% of the overall OTA consumed in the diet 4. Coffee, beer, wine, meat products, vegetables and spices are also affected but its contribution to the OTA consumption is much lower5,6. The levels of OTA in food are closely related with the production and conservation conditions. Agricultural practices and environmental conditions (temperature and humidity) during storage and transport directly affect OTA content in foodstuffs. Furthermore, it has been shown that a high water activity facilitates the production of OTA in foodstuffs 7.

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Giving its toxicity, the OTA content in humans food (1.2-14 ng/kg b.w. per day) and animal feed is regulated by EFSA (European Food Safety Authority) 8,9 that allows very low OTA concentration levels in. For this reason, there is the need to find new ways to control, inhibit and/or eliminate those moulds responsible of its formation from foods and feeds. Recently, it has been shown that the use of an active packaging based on the incorporation of essential oils caused an inhibition or decrease in the production of mycotoxins, both in direct contact as well as in vapour phase10. Some of the essential oils more commonly used as active agents in active packaging are cinnamon, carvacrol, tymol or rosmarinus11-13. In this work, benzyl isothiocyanate (BITC), a major antibacterial component present in Salvadora persica extracts from Brassicaceae family, is going to be studied as active agent. This compound, has a rapid effect against microorganisms such as Salmonella enterica, Pseudomonas aeruginosa, Streptococcus pyogenes or Staphylococcus aureus14. Furthermore, BITC, used by Kassie et al. as natural chemopreventive agent, demonstrated to have genotoxic effects on Escherichia coli and human-derived HepG2 cells, where the induction of DNA damage by BITC was observed15. BITC as well as compounds from the isothiocyanate family (ITCs) have been positively evaluated by European Food Safety Authority (EFSA) with maximum allowed levels of daily intake, 96 µg/person/day in the case of BITC 16.

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Several techniques can be used to study the cell damage caused by antimicrobial agents and most of them are based on proteomics. However, the great advance in spectroscopic techniques has recently shown that these techniques are a powerful non-destructive tool for this task. Among them, Raman micro-spectroscopy has recently attracted much attention as a powerful, rapid, non-destructive vibrational spectroscopic and high specific technique for the acquisition of information on the molecular composition of a sample17. In biological samples, it does not require any special dyes or specific excitation wavelengths, since it determines the compound chemical bonds and allows the association between the obtained bands and specific molecules such as proteins, carbohydrates, nucleic acids and other biomolecules and their functional groups18. Living cells were successfully studied in some cases without additional requirements19.


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Raman imaging has long been used to demonstrate the chemical nature of a sample, providing information on molecular orientation, symmetry and structure with submicron spatial resolution.

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The study of cell damage is essential to understand how BITC will presumably inhibit OTA production. Since Raman microscopy is a non-destructive technique, it can be explored for this task. Thus, the effects produced by active compounds on foodborne mould will be evaluated in a molecular level with the use of new spectroscopic techniques20.

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The aim of the present work was to explore Raman Imaging Spectroscopy as cell damage measuring tool for the study of the effects caused by benzyl isothiocyanate on A. ochraceus cells. A principal component analysis was performed with the aim of identifying patterns on the date set and establishing the main differences between the Raman spectra of control and samples.

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2. Materials and Methods

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2.1 Microbial culture

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Aspergillus ochraceus CECT 2093 was the mould selected; it was supplied by the Spanish Type Culture Collection (CECT). Potato dextrose agar (PDA) as solid media and yeast extract broth (YEB) as liquid media were employed, both supplied by Scharlab (Barcelona, Spain).

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2.2 Antimicrobial agents

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As active agent, benzyl isothiocyanate (BITC) 98% of quality (CAS 622-78-6) was provided by Sigma-Aldrich Química (Madrid, Spain).

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2.3 Antifungal activity in liquid medium

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To study the antifungal properties in direct contact, a broth macrodilution method was used. A mould inoculum of 106 CFU/mL was prepared in NaCl 0.9% and confirmed by plate counting21. Samples were prepared as follows: first, serial dilutions of essentials oil in ethanol were prepared in the range of 160000-10µg/mL. Tubes containing 1780 µL of YEB were inoculated with 200 µL of fungal suspension and 20 µL of essential oil dilution, so that the final essential oil concentration in sample tubes was 100-fold diluted. Solvent controls with 20 µL of ethanol were also included in the assay. Samples were incubated for 48 h at 25 °C with continuous shaking. After incubation, the MIC (minimal inhibitory concentration) was determined as the lowest EO concentration that yielded no visible growth to the naked eye22,23. For the Raman experiments, samples corresponding to 1xMIC and 1/2xMIC (sub-inhibitory) values were used.

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2.4 Antifungal activity in vapour phase



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To study the antifungal activity in vapour phase, an active package approach was developed. Active stickers of 1x1.5 cm2 were made using a water-based adhesive adhered to a filter paper; quantity of BITC incorporated in active stickers was of 2.16 g/m2. More details about the packaging system and the formula cannot be disclosed because of confidential reasons. Plates with PDA placed at the top were inoculated with 100 µL of a physiological saline solution containing 106 (CFU)/mL of the microorganism under study11. The active stickers (with BITC) were placed at the bottom of the plate. Blank stickers were prepared following the same procedure described above without BITC addition.

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2.5 Sample preparation for Raman analysis

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The concentration of essential oil selected for the preparation of Raman samples in the case of liquid media (macro dilution method), was the last concentration of antimicrobial that enabled the growth of mould (sub-MIC). In the case of vapour phase experiments, the concentration of essential oil selected was the the sub-MIC concentration corresponding to the delay area of the plate, The collection of the samples was performed using a sterilised swab (figure S-1).

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Samples and controls were transferred to sterile 2 mL Eppendorf and centrifuged at 11000 rpm for 10 minutes. Afterwards, the pellet was washed with 2 mL of physiological saline solution and this procedure was repeated three times. After the final washing step, the pellet was resuspended in sterile water to a final volume of 1 mL. Finally, 20 µL of each sample were placed in a BaF2 cell and dried in an oven at 40 °C.

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2.6 Raman Analysis

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Raman spectroscopic analysis was performed using a Thermo Scientific (Madison, WI, USA) DXRTMxi Raman imaging microscope equipped with a 532 nm laser. Laser power was set at 10 mW (measured at the sample) on the moulds through a 100x or 50x objective, depending on the sample analysed. Raman spectra of each sample were collected over a simultaneous wavenumber shift range from 3400 to 50 cm-1 in extended mode. 5 Hertzios Aperture was set to 25 µm pinhole, Fluorescence correction and cosmic ray rejection were applied. Data analysis and spectral interpretation were made with Thermo Scientific™ OMNIC™xi Software.

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For each control sample (spores/vapour, spores/liquid, hyphae/vapour and hyphae/liquid) between 7 and 9 maps were collected, and for each BITC treated sample (spores/vapour, spores/liquid, hyphae/vapour and hyphae/liquid) between 11 and 13 maps were collected. Each map corresponds to one spore or one hyphae and in each map a Raman spectra was taken every 0.5 µm; an average of 80 measures were performed in each map point.

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Acquisition time for each spectrum was 0.20 seconds. Final acquisition time will depend on the map size since spectra were taken every 0.5 µm. For an standard map,


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110 collect points (10 x 11), final time would be 110 point x 80 exposures x 0.2 seconds each exposure = 1760 s = 29.3 minutes

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2.7 Data analysis

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With the aim of seeking differences in the Raman spectra between samples and controls, the average spectrum of each collected map was performed, assuming the heterogeneity of each map. Final data for the average spectrum of each map was obtained by calculating the average of all the sample map points (excluding background points). Once each mapping-related spectra was obtained, the values were normalized, in this way, in the average spectrum of all the maps for the same sample, all spectra had the same weight on it. This data normalization was made using the OriginPro8 SR0® program (OriginLab corporation, Northampton USA). The representativeness of each spectrum peak type was set with the OMNIC 9® program (Thermo Fisher Scientific Inc, Spain). For PCA, the average spectrum of each map was used. For Raman imaging spectra (figure 1) the average of all the maps corresponding to each kind of sample was calculated. Regarding to sample deviations, RSD values among the same type of samples were in most cases below 10%.

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2.8 Statistics

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Principal components analysis (PCA) was carried out with the software Unscrambler X 10.3® (Camo Software AS).

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3. Results and discussion

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The mode of action behind ITCs antimicrobial activity is not yet fully understood, but since it might penetrate membranes and no single site of action has been described, it is generally regarded as a non-specific inhibitor of periplasmic or intracellular targets. It is not yet clear if ITC rapidly crosses membranes and enters the cytoplasm of prokaryotic and eukaryotic cells, or if it has an effect on cell membranes 24. Delaquis and Luciano proposed that the antimicrobial mode of action of ITC is related to its general inhibition of enzymes and alteration of proteins by oxidative cleavage of dissulfide bonds. The reaction of the ITC with proteins is probably occurring between the R (primary amine) of lysine, sulfhydryl group of cysteine and the amino group of the isothiocyanate 25,26.

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3.1 Antifungal activity of BITC in liquid medium (direct contact)

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The last concentration of antimicrobial agent that enabled the growth of mould (subMIC) was selected. The aim of this experiment was to evaluate the biological targets of BITC action on fungal cell by assessing the differences between Raman spectra collected for treated and control samples (table 1) of spores and hyphae.

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Only one type of spectral profile was found for BITC-treated spores from the broth macrodilution experiment (BITC spore). These treated spores presented a higher number of differences respect to control spores, visually and by spectroscopy. In the same way, for treated hyphae only one spectral profile was found.



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3.1.1. Effects of BITC on spores

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Microscope images showed that there were morphological visible differences between treated and non-treated spores. BITC-treated spores had a regular morphology but a larger size (figure S-2c) than control spores (figure S-2a), mean size for non-treated spores was 2.67µm ± 0.05 and for treated spores 5.18 µm ± 0.66. These differences were later on explained with the results of Raman spectra.

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The biggest difference between non-treated and treated spores was the appearance of a high intensity peak at 987cm-1(figure 1a) when spores were in contact with BITC, which could be due to phosphate (Pi). In the spectra of treated spores, other bands also appeared at 451 cm-1, 617 cm-1, 1577 cm-1, 779 cm-1 and 929 cm-1 (table 1). On the contrary, the band generally assigned to amino acids at 1002 cm-1 and to DNA at 670 cm-1 disappeared and the C-H stretching band at 2932 cm-1 corresponding to CH2 and CH3 groups present in proteins, lipids, nucleic acids, and carbohydrates was reduced drastically.

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The appearing of the phosphatase (Pi) band might be related with the inhibition of ATPase due to the effect of BITC. Inhibition of ATPase would produce an accumulation of phosphate (Pi) and consequently the interruption of the transport chain of electrons, thereby an accumulation of NADH (1577 cm-1) would be generated. Furthermore, this phosphate band might be related with the release of phosphate due to the degradation of DNA and/or RNA backbones and the disappearance of the band of 670 cm-1 indicates also this effect 15. A number of isothiocyanates, including BITC, has been found to induce cell cycle arrest in cultured cells 27. BITC may be affecting the metabolism of lipids, proteins, carbohydrates, DNA and RNA on A. ochraceus, which would explain the decrease or disappearance of the bands corresponding to these biomolecules in the treated samples such as 670 cm-1, 1002 cm-1 and 2932 cm-1.

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The presence of oxoiron species such as Fe(IV)=O along with NADH band might be related with the Cytochrome P450 enzymes superfamily 28. As Goosen and Mills showed, BITC is an efficient inhibitor of P45029. The inactivation of this enzyme inhibits the defence mechanism of BITC degradation, and as consequence, BITC can reacts with their different targets, as it was previously shown. Furthermore, the presence of these bands could also be explained by the degradation of cytochrome a3, a part of the cytochrome c which is involved in the oxidative phosphorylation and electron transport chain30. The interruption of this process would involve the accumulation of NADH, as a consequence of the cellular respiration inhibition, which also further justifies the appearance of the Pi band mentioned before.

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As a result of all these cellular modifications, some alterations in the exchange of Na+Ca2+ and in the intracellular homeostasis could take place, which could be associated with the larger size observed for treated spores. All these changes could indicate that, on treated samples, an apoptosis was the final consequence of the exposure to BITC . Xiao et al observed that, in human breast cancer cells exposed to BITC, a rapid disruption of the mitochondrial membrane potential and a subsequent apoptosis took place 31. Also,


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the accumulation of intracellular calcium can trigger apoptosis through the release of cytochrome c from the mitochondria.

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The reaction of ITCs with proteins and enzymes leads to the loss of its tertiary structure and to a partial or total loss of its function. All the proteins mentioned before are enzymes of crucial importance in the cell and have lysines in their active sites. This fact is in accordance with what it was argued by Tiznado et al, who observed enzymes with lysine in their active sites such as pyrophosphatases, H+ pumps enzymes, cytochrome P450 or ATPase 32.

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The values obtained for BITC-treated spores in direct contact in liquid medium are indicative that these treated spores were no longer functional. BITC was accumulated within the mould cells and attacked the active site of enzymes by bindings to thiol or amine groups 24, thus impacted enzymatic activities such as respiration, metabolism and gene transcription.

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3.1.2. Effect on BITC on hyphae

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A hypha consists of one or more cells surrounded by a tubular cell wall. In most mould, hyphae are divided into cells by internal cross-walls called septa. Microscope images showed that non-treated hyphae presented a normal morphology with a long branching filamentous structure of a mould, in this case without a visible septum, but with defined cellular wall and a homogenous colour along it; being an indicator of a normal cellular activity (figure S-2b), while treated hyphae presented a very different morphology (figure S-2d) with the formation of clumps inside the hypha, disruptions in the cell wall and no homogenous colour along it.

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As it was observed for treated spores, in treated hyphae only one spectral profile pattern was observed. When comparing non-treated and treated hyphae, the spectral profiles obtained of both kinds of samples was similar but the intensity of the bands was reduced drastically in treated samples (figure 1b).

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It was seen a drastic reduction of the bands at 747 cm-1, 1002 cm-1 , 1127 cm-1 , 1171 cm-1 , 1227 cm-1 ,1309 cm-1, 1337 cm-1, 1448 cm-1, 1583 cm-1 and 1657 cm-1(table 1). The formation of clumps inside the hyphae might be explained by protein aggregation and accumulation of these aggregates inside the cell, as indicated by the reduction of the bands shown before 747 cm-1, 1002 cm-1, 1171 cm-1, 1227 cm-1, 1448 cm-1 which are related to the protein (table 1) . This clump formation can be seen as a result of conjugation of BITC with the thiol groups of proteins that promotes the aggregation of misfolded proteins. These results are in agreement with those reported by Dufour et al 24 .

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The reduction of 1227 cm-1, 1337 cm-1 and 1583 cm-1 bands were related to DNA and RNA indicated modifications on gene transcription, which could also be related to the loss of cellular viability produced by the conjugation with amino acids. This may



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promote a stringent response by the depletion of free amino acids 24 and by the reaction with the DNA/RNA backbone 15.

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Furthermore, it can also be observed an intensity reduction in the bands of, 1309 cm-1, 1448 cm-1 and 1657 cm-1, that are related with lipids, phospholipids and triglycerides. This might indicate the reduction of the cell fluidity as a result of the BITC action on cell wall components. This fact, in addition to the homeostatic pressure, can result in the visible disruptions of the cell wall. Dufour et al. also noted that conjugations of ITCs may affect cell redox homeostasis 24.

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Besides, a disappearance of the bands at 1360 cm-1 and 1393 cm-1 was also observed. These disappearances are related with the cellular effect explained before.

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All these results obtained in treated-hyphae indicated that the cellular functionality had been reduced because there were large reductions in the bands of peptides, lipids, DNA and/or RNA, saccharides as a consequence of the BITC action on the hyphae.

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The biggest difference with the profile of the treated spores is the absence of the phosphate bands, meaning that the effect of the BITC was less aggressive than in hyphae.

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3.2 Antifungal activity of BITC in vapour phase

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In the vapour phase experiment, performed with stickers containing 8% BITC, three different areas were found in the plate: an inhibition area (1), a delay area (2) and a normal growth area (3) (figure S-1c-d). The blanks correspond to figures S-1a-b. It is important to point out that the samples collected from the delay area revealed a great decrease in sporulation, as it has been observed by optical microscopy.

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The sample was collected from the delay halo, where visible growth was observed but no sporulation was visible (figure S-1c-d). As in the previous experiment, non-treated and treated spores and hyphae, were studied

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The results obtained using the developed sticker, were compared with the ones obtained previously for the direct contact in liquid medium experiment. Two different profiles of treated spores and hyphae were found with vapour phase treatment. One of the profiles was similar to that obtained for the direct contact in liquid medium experiment.

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3.2.1. Effect of BITC on spores

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In contrast to what it was seen for treated spores in liquid medium experiment (directcontact), in the case of vapour phase, two different spectral profiles patterns were detected treated spores: one of them presented a similar profile to that obtained in the broth macrodilution method (BITC1-treated) while the other one, had a significantly distinct profile (BITC2-treated) (figure 1c).

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Morphologically differences between non-treated and treated spores were observed in the microscope images. BITC1-treated spores had a much more irregular, deformed


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morphology and a more irregular distribution of cellular content (figure S-2g), while BITC2-treated spores had an irregular morphology and a larger size (figure S-2i) than non-treated spores (figure S-2e). Furthermore, there were larger differences when comparing the spectral profiles of non-treated and treated spores, which could also explain (figure 1c) the different morphologies, obtained in both cases.

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The profile obtained for BITC1-treated spores (figure 1c) was the same as the one found for treated spores in the liquid medium experiment (figure 1a), which indicates that the mode of action of BITC was similar in both liquid and vapour phase, and therefore, the molecular alterations caused in both assays were similar.

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In contrast to what it was seen for BITC1-treated spores (figure 1c), the most visible differences when comparing the spectra of treated and non-treated samples, was the presence of the bands at 1061 cm-1, 1294 cm-1 [methylene twisting] 33 and 1437 cm-1. There was a random conformation of C-C and deformation of acyl chain, and as a consequence, a loss of the structures which were directly related to a loss of functionality.

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It was observed a drastic reduction of the bands of the 1500-1700 cm-1 region [protein (amide I), lipids, fatty acids, phospholipids and amino acids (phenylalanine and tyrosine)] including the disappearance of many of them. Reduction of the signal of all this cellular components resulted in a loss of the cell structure that might be related with the fluidity of the cellular wall. This could explain the irregular morphology observed. In addition, the disappearance of bands related to amino acids indicated the loss of protein functionality.

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It was also observed the disappearance of bands at 1392 cm-1, 1002 cm-1 proteins, 782 cm-1 [DNA-RNA] and finally the modification of the profile between 300 and 600 cm–1, corresponding to saccharides region. The loss of the band corresponding to DNA/RNA indicated the loss of genetic functionality.

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The biggest difference between the profiles obtained for BITC2-treated spores and the profiles for BITC1-treated spores was the appearance of the phosphate band and a drastic reduction of the band corresponding to CH2 and CH3 groups, commonly present in proteins, lipids, nucleic acids, and carbohydrates. This meant that BITC effect on A. ochraceus spores was not homogeneous and that a stronger or weaker action can be observed depending on the spore analysed.

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3.2.2. Effect of BITC on hyphae

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Microscope results showed that, in terms of morphology, there was a big difference between non-treated and treated hyphae in the vapour diffusion assay. For example, non-treated hyphae presented a normal morphology (Figure S-2f), while treated hyphae presented two distinct morphologies: one of them presented greater staining regions (BITC1-treated) (Figure S-2h) while the other one showed agglutination regions (BITC2- treated) (Figure S-2j).



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Spectra results showed two different profile patterns for treated samples as it was observed in spores. Nevertheless, they were more similar between them than in comparison with the profile of the non-treated hyphae (figure 1d). The main difference observed between the two profile patterns of treated samples was the band 987 cm-1, corresponding to phosphate.

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The signals obtained in the BITC1-treated hyphae and BITC2-treated hyphae (figure 1d) were compared. Clear differences were observed between them due to the emerging bands at 987 cm-1 [PO4-3], 617 cm-1 [protein], 647 cm-1 [tyrosine (skeletal) (C-C twist Tyr)] and to the disappearance of the band corresponding to amino acids (1002 cm-1). The formation of agglutination zones in this case could be also explained by an increase of aggregation due to a more pronounced effect of BITC in the BITC1-treated hyphae samples, as it can be observed by the appearance of the phosphate band. The biggest difference of this profile compared to the rest of the profiles that presented these bands related to phosphates was the maintenance in the intensity of the 2932 cm-1 band instead of the reduction observed in the other cases. In this case, no disruptions in the cell wall were observed, although the colour along the hypha was heterogeneous, in the same way as in the other experiments.

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The profile of BITC1-treated hyphae in comparison with non-treated hyphae was showed several differences.

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Drastic reduction of bands at 747 cm -1, 780 cm-1, 1127 cm-1, 1171 cm-1, 1245 cm -1, 1309 cm -1, 1337 cm-1, 1360 cm-1, 1392 cm-1, 1454 cm-1 , 1583 cm-1 , besides a decrease of the signal corresponding to the region of the saccharides (300- 600 cm–1). These results indicated large modifications in the bands corresponding to protein, lipids, saccharides, DNA and/or RNA signal as a consequence of the BITC action, and therefore a drastically decrease on cellular functionality. The reduction of the 1127 cm-1 , 1245 cm -1, 1360 cm-1, 1392 cm-1 and 1583 cm-1 bands indicated the accumulation of protein aggregated and cellular material, what in general can be seen by the formation of staining aggregates inside the hyphae (figure S-2h). The modified bands related to lipids, phospholipids and saccharides (1127 cm-1, 1309 cm -1, 1454 cm-1 and 300-600 cm–1 bands) might be indicative of a reduction in cell fluidity, as it was shown before for the treated hyphae in the direct contact experiment. This effect added to homeostatic pressure changes can result in the visible disruptions of the cell wall (figure S-2j).

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All controls, both hyphae and spores, in the two experiments presented virtually the same profile. The small differences that were observed between the different spores or hyphae in the non-treated samples could be due to the fact that the growing conditions for broth dilution and vapour activity were different. All samples treated with BITC in both experiments showed a reduction or disappearance of the bands corresponding to proteins, lipids and nucleic acids, which are in agreement with the results previously described by Kassie and Poll-Zobel et al., who demonstrated these BITC effects in Escherichia coli and human HepG2 cells 15. The variations of bands indicated the loss or changes in cellular components. The similarities on the obtained profiles for broth


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macrodilution and vapour phase activity indicate that the mode of action of BITC on A. ochraceus mould cells was similar both in solution and in vapour.

387 388 389 390 391 392 393 394

Overall, it can be concluded that the mode of action of BITC was due to its isothiocyanate group. Previous works made by Zhang and Verma showed that the central carbon atom of isothiocyanate (R−N=C=S) is highly electrophile and reacts readily with oxygen-, sulfur-, or nitrogen-centered nucleophiles 34,35. Thus, within the cell, ITC reacts with several biomolecules, as it was explained in the last sections, saccharides, amino acids, proteins and lipids. These results are in agreement with those obtained by Kawasaki and Cejpek 36-38. This behaviour was observed in all the samples treated with BITC in this work using both conditions, liquid and vapour contact.

395

3.3 PCA analysis

396 397 398 399 400

A principal component analysis was carried out in order to check if there was a statistical grouping of samples according to their Raman spectra. PCA was used to reduce the dimensionality of multivariate biological data while preserving most of the variances, this fact were demonstrated previously by Lu et al 39. PCA results allowed the selection of those bands with the highest statistical weight for sample grouping.

401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417

Figure 2a) shows the sample grouping for spores in control samples and spores in samples exposed to BITC, both in vapor phase and in liquid phase (direct contact). The explained variance achieved for the first 2 PCs was satisfactory (92%). PCA figure shows that spores from control samples, were significantly different of spores from treated-samples. The fact that the control samples were all grouped together, both from vapour phase and liquid phase, showed that although the growth conditions were different the spores composition was similar. In the case of spores from treated samples, two different sample grouping were observed (BITC1-treated and BITC2-treated samples). Spores from treated samples in liquid phase were group all together and also together with part of spores from treated samples in vapour phase (BITC1-treated samples). These samples were widely different from control samples in PC1. As it has been observed in the visual analysis of the Raman spectra spores from treated samples in vapour phase (BITC1-treated and BITC2-treated), spores showed two different spectra profile patterns. The second group of spores from samples treated in the vapour phase (BITC2-treated samples) was placed in the PCA between control samples and BITC1-treated samples, which meant that the differences with control spores were smaller than in BITC1-treated samples.

418 419 420 421 422

The bands with a high score in spores from control samples were 1583 cm-1, 1448 cm-1, 1454 cm-1 and 2932 cm-1; whereas the bands with high scores spores from BITC-treated samples were 300-600 cm-1, 617 cm-1 and 987 cm-1. The biggest difference of the scores obtained for spores from BITC1-treated samples when compared to the scores of the BITC2-treated samples is the presence of 2932 cm-1 and the absence of 987 cm-1.

423 424

The results show that while the effect of BITC on spores is very similar among the different spores when samples are exposed to BITC in liquid medium (direct contact), the spores can be



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425 426 427

affected at different levels in vapour phase. When BITC acts in vapour phase, some spores will be affected at the same level than in direct contact, but some of them can suffer from a lower affection.

428 429 430 431

Figure 2b) shows the sample grouping for hyphae in control samples and in samples exposed to BITC, both in vapor phase and in liquid phase (direct contact). The pattern observed was very similar to the pattern observed for spores. In this case, the explained variance for the first two PCs was also satisfactory (83%).

432 433 434 435 436 437 438

As happened for spores, hyphae from control samples were very different from hyphae from treated samples, even though in this case more dispersion among control samples was observed. The PCA also showed two different groups in hyphae from treated samples, one group that enclosed hyphae from treated samples in the liquid phase and part of hyphae from samples treated in the vapour phase (BITC1-treated samples), that showed the biggest differences with control samples; and a second group (BITC2treated samples) consisted of part of hyphae from treated samples in the vapour phase.

439 440 441 442 443

According to PCA figure, the separation between BITC1-treated and BITC2-treated samples was due to bands linked to PC2. BITC2-treated samples were linked to bands 300-600 cm-1 and 987 cm-1 and BITC2-treated samples to 780 cm-1, 1410 cm-1, 1657 cm-1 and 2932 cm-1. All these results are in concordance with the previously discussion based on Raman results.

444 445

4. Conclusion

446 447 448 449

Raman imaging spectroscopy has proved to be a great tool for the study of A. ochraceus samples, providing information about its molecular composition and morphology, this technique allows a deep knowledge of the effects that BITC antimicrobial agent has on its spores and hyphae.

450 451 452 453 454 455

This work showed that BITC was accumulated in the mould cells where it caused alterations in essential cell components such as saccharides, amino acids, proteins, lipids or enzymes, thus impacting several cellular functions such as respiration, metabolism or cell cycle,. All these metabolic changes will affect the production of OTA, what makes BITC a great antifungal agent a very attractive packaging to guaranty the food safety.

456 457 458 459

This work demonstrates that BITC behaves as antimicrobial agent in direct contact (liquid medium) as well as in vapour phase. Therefore, positive results are expected for the use of BITC as antimicrobial agent in active stickers incorporated in food packaging.

460 461

5. Acknowledgments


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This work was supported by University of Zaragoza (PIFUZ-2012-B-CIE-001) within the scope of 2012/0254 REPSOL lubricantes y especialidades (Rylesa) and the project RYC-2012-11856 (Ramon y Cajal programme). Thanks are also given to Gobierno de Aragón and Fondo Social Europeo for financial help of Group GUIA, T-10.

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6. References

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Figure 1:

567 568 569 570 571

Normalized Raman imaging spectra so that in the average spectrum of all the maps for each same sample (n=5-8). Antifungal activity in liquid medium: (a) non-treated and BITC-treated spores and (b) non-treated and BITC-treated hyphae. Antifungal activity in vapour phase: (c) non-treated, BITC1-treated and BITC2-treated spores and (d) nontreated, BITC1-treated and BITC2-treated hyphae.

572

Figure 2:

573 574 575

Principal components analysis obtained with Raman spectra of spores (a) and hyphae (b) in different assays: vapour condition, non-treated (■) and BITC-treated (●) and in a liquid condition; non-treated (♦) and BITC-treated (▲).

576 577

Table 1: Raman spectroscopy bands of A. ochraceus and possible assignments according to literature. Band (cm-1) 300600 451 617 670 747 779 780 782 929 987 1002 1061 1127 1171 1227 1245 1294 1309 1337 1360 1392 1393 1437 1448 1454 1577 1583 1657 2932

Possible assignment

Ref.

(COC) Exocyclic deformations of saccharides, glycoside ring deformation Ring torsion of phenyl groups C-C twisting (protein) DNA dT(N-H out of plane bending) Fe (IV)=O Phosphate mode of DNA/ RNA DNA-RNA ʋ(C-C), stretching-probably in amino acids; Proline and Valine Phosphate (Pi) Amino acids C-C stretch random conformation skeletal Fatty acid and ʋ(C-N) (protein)] C-H bend Tyrosine (protein) (1220-1280 cm-1)T, A(DNA)/amide III (protein) and -CH bend (lipids) [Amide III] Methylene twisting CH3/CH2 twisting or bending mode of lipid Polynucleotide chain, CH2/CH3 wagging and twisting mode in nucleic acid and Trp δ(CH2)n of alkyl chains and Tryptophan C-N stretching, in quinoid ring-benzoid [O-C-O sym. Str and CH rocking] CH2 deformation (lipid) acyl chains CH bending mode of proteins and lipids Phospholipids Bound and free NADH Nucleic acid ring stretching vibration (especially G and A) and C=C bending mode of Phe C=C (lipids), n(C=C)cis (phospholipids) and triglycerides C-H stretching, CH2 and CH3 groups (proteins, lipids, nucleic acids carbohydrates)

578 579


40-42 33,43 33,44 33,45-48 49 50 42,43,45,48,51 33 33 52,53 33,47,54,55 33,56 20,33,45 33,45,47 45 33 33 47,54,57 33,47,48,51 33,40 58 59 60,61 33,47,51,55,59 56 33,62 33,54,57,63 64 39,41,57,59,63,65


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Raman Imaging Spectroscopy as a Tool To Investigate the Cell

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