Antimicrobial and antioxidant activities of Cinnamomum cassia essential oil and its application in food preservation

This study was designed to investigate chemical and antioxidant properties, as well as the antimicrobial and antibiofilm behaviour of Cinnamomum cassia essential oil (CCEO). MALDI-TOF MS Biotyper mass spectrometry was applied to evaluate the biofilms of Stenotrophonomonas maltophilia and Bacillus subtilis, while the antibiofilm ability of CCEO was assessed on wooden and glass surfaces. The antimicrobial activity by disc diffusion method, microdilution method, and vapour phase for two biofilm-producing bacteria and three Penicillium spp. were used. Antimicrobial and antibiofilm properties were assessed using the agar microdilution protocol. The vapour phase of Penicillium citrinum, P. crustosum, P. expansum, S. maltophilia, and B. subtilis on bread, carrot, potato, sweet potato, and apple in situ was studied. Specific molecular variations related to the biofilm formation and genetic analogies were evaluated with MSP spectra dendrograms of S. maltophilia and B. subtilis profiles were grown on different days. The results of disc diffusion and broth diffusion methods showed that CCEO was strongly effective against all tested microorganisms and the vapour phase method was effective and active against all Penicillium spp., but not strongly effective against bacteria in food preservation of food matrices.  * Corresponding author: Miroslava Kačániová, Department of Fruit Science, Viticulture and Enology, Faculty of Horticulture and Landscape Engineering, Slovak University of Agriculture, Tr. A. Hlinku 2, 94976, Nitra, Slovakia; Department of Bioenergetics, Food Analysis and Microbiology, Institute of Food Technology and Nutrition, University of Rzeszow, Cwiklinskiej 1, 35-601, Rzeszow, Poland, e-mail: miroslava.kacaniova@gmail.com, tel: +421-641-4715 Lucia Galovičová: Department of Fruit Science, Viticulture and Enology, Faculty of Horticulture and Landscape Engineering, Slovak University of Agriculture, Tr. A. Hlinku 2, 94976, Nitra, Slovakia, e-mail: l.galovicova95@gmail.com Veronika Valková: Department of Fruit Science, Viticulture and Enology, Faculty of Horticulture and Landscape Engineering, Slovak University of Agriculture, Tr. A. Hlinku 2, 94976, Nitra, Slovakia, e-mail: veronika.valkova@uniag.sk Eva Tvrdá: Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, 94976, Nitra, Slovakia, e-mail: eva.tvrda@uniag.sk Margarita Terentjeva: Institute of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Latvia University of Life Sciences and Technologies, K. Helmaņa iela 8, LV-3004, Jelgava, Latvia, e-mail: margarita.terentjeva@llu.lv Jana Žiarovská: Department of Plant Genetics and Breeding, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture, Tr. A. Hlinku 2, 94976, Nitra, Slovakia, e-mail: jana.ziarovska@uniag.sk Simona Kunová: Department of Food Hygiene and Safety, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, 94976, Nitra, Slovakia, e-mail: simona.kunova@uniag.sk  Tatsiana Savitskaya: Research Institute for Physical Chemical Problems, Belarusian State University, Leningradskaya str. 14, 220030, Minsk, Belarus, e-mail: savitskayaTA@bsu.by Dmitrij Grinshpan: Research Institute for Physical Chemical Problems, Belarusian State University, Leningradskaya str. 14, 220030, Minsk, Belarus, e-mail: Grinshpan@bsu.by Jana Štefániková: AgroBioTech Research Centre, Slovak University of Agriculture, Tr. A. Hlinku 2, 94976, Nitra, Slovakia, e-mail: jana.stefanikova@uniag.sk Soňa Felsöciová: Department of Microbiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, 94976, Nitra, Slovakia, e-mail: sona.felsociova@uniag.sk Nenad Vukovic: Department of Chemistry, Faculty of Science, University of Kragujevac, P. O. Box 12, Kragujevac, Serbia, e-mail: nvchem@yahoo.com * Corresponding author: Przemysław Łukasz Kowalczewski, Department of Food Technology of Plant Origin, Poznań University of Life Sciences, 31 Wojska Polskiego St., 60-624, Poznań, Poland, e-mail: przemyslaw.kowalczewski@up.poznan.pl, tel: +48-61-848-7297 Open Chemistry 2021; 19: 214–227 Open Access. © 2021 Miroslava Kačániová et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License.


Introduction
The genus Cinnamomum (Laureaceae family) consists of 250 species of wooden plants native to China, Southeast Asia, and Australia [1]. Trees and shrubs of the genus may be found in rainforests located within a wide range of altitudes, but are rare in latitudes with a typical seasonal climate [2]. Damp and well-drained locations are preferable for plant growth [2].
Essential oils (EOs) of Cinnamomum cassia, Cinnamomum zeylanicum, and Cinnamomum camphora are widely recognized for their various applications in the medicine and food industries [3,6]. The exact composition of the cinnamon EOs depends on the geographical origin and processing procedure. Cinnamon EOs have been used in medical remedies for centuries and their positive effects on the treatment of respiratory, gastrointestinal cardiovascular, and urinary disorders are well-described. EOs possess aphrodisiac, antihelminthic, antibacterial, insecticidal, antioxidant, antimutagenic activities, and tonic properties [3,6]. EOs from the leaves of Cinnamomum osmophloeum exhibit strong activity against bacteria, termites, mosquitoes, mildew, and other biological agents [4]. According to Verspohl et al. [5], the EO of C. cassia has expressed antidiabetic effects via the insulin-enhancing activity in vitro [6].
Cinnamon is a common ingredient in seasonings, sauces, bakery, confectionery, and drinks; the Food and Drug Administration has recognized cinnamon as a safe food additive [7].
Strong antifungal activity of cinnamon EOs was attributed to cinnamaldehyde abundance in the EO composition with up to 76.34% of the total of EO compounds. Antimicrobial activities against molds, e.g. Rhizopus nigricans, Aspergillus flavus, and Penicillum expansum, and bacteria, e.g. Staphylococcus aureus and foodborne pathogens, were studied previously [7][8][9][10].
Cinnamomum EOs may exhibit a significant antibiofilm activity: the EO from the trunk bark of C. burmannii interfered with planktonic cell growth and inhibited development of S. aureus and P. aeruginosa biofilms [11]. Trans-cinnamaldehyde has been shown to delay the formation of E. coli biofilm in urinary catheters [12].
The main objectives of this study were to study the chemical characteristics and antioxidant properties of Cinamomum cassia essential oil, the antibiofilm and molecular profile of biofilm formation of C. cassia, and the antimicrobial effectivity of C. cassia essential oil in vitro as well as in situ.

Essential oil
Cinnamomun casia essential oil (CCEO) was obtained from Hanus, a.s. (Nitra, Slovakia). In our study, chemical characterization was performed and antioxidant activity of the essential oil was measured as well. Thereafter, the antimicrobial, antibiofilm activity, and molecular profile of biofilm were evaluated.

Chemical composition of the essential oil
Gas chromatographic-mass spectrometric analysis (GC Agilent 7890B and MS Agilent 5977A, Agilent Technologies Inc., Santa Clara, CA, USA) of CCEO was done as reported previously [13,14]. Prior to the analysis, a CCEO sample was diluted in hexane (HPLC ≥97%, Sigma Aldrich GmbH, Darmstad, Germany) to a concentration of 10 µL/mL. One microlitre of the diluted sample was injected into the inlet (250°C) operated in split mode 1:10. The separation was achieved using a HP-5 ms capillary column (30 m × 0.25 mm × 0.25 µm film; Agilent Technologies). The oven temperature program was set to 50°C for the first 5 min, and subsequently increased to 240°C at the rate of 3°C/min, where it was kept constant for 2 min. Helium was used as a carrier gas at a constant flow (1.2 mL/min). The mass detector parameters were as follows: ionization energy of the filament -70 eV, transfer line temperature -250°C, MS source temperature -230°C, quadrupole temperature -150°C. The mass spectrometer was programmed under electron impact (EI) in a full scan mode at m/z 40-350 with a scanning rate of 2.4 scans/s. The identification of compounds was carried out by comparing mass spectra (over 80% match) with a commercial database NIST ® 2017, and Wiley library, retention times of reference standards (D-limonene, β-myrcene, and γ-terpinene; Sigma-Aldrich GmbH) comparison of data on the occurrence in CCEO with the literature [15][16][17][18][19][20][21]. The relative content of the identified compounds was calculated by dividing the individual peak area by the total area of all peaks. Each sample was measured in triplicate. The results were expressed as the means of three injections ± standard errors (SE).

Microorganisms
Stenotrophomonas maltophilia and Bacillus subtilis were obtained from a milk producer, while Penicillium expansum, P. crustosum and P. citrinum were isolated from grapes. MALDI-TOF MS Biotyper (Brucker, Daltonic, Germany), 16S rRNA, and ITS sequencing were used for the identification.

Antimicrobial activity
The disc diffusion method was applied for detection of antimicrobial activity.

Minimum biofilm inhibitory concentration (MBIC)
MBIC against S. maltophilia was performed in microtitration plate [24]. Bacterial suspension preparation, incubation with CCEO, staining with crystal violet and acetic acid, and the evaluation of results were described previously [13,14,25]. MBIC was defined as the concentration of CCEO with absorbance less or equal to the negative control. The test was performed in triplicate.

Bread making process
The baking formula included 250 g wheat flour T650, 150 mL water, 2.5 g sucrose, 5 g salt, and yeast. The dough was fermented in a fermentation cabinet (MIWE cube; Pekass s.r.o., Plzeň; Czech Republic) at 32°C, with relative humidity 85% for 40 min. The loaves were baked at 180°C for 17 min with 160 mL of water, then at 210°C for 10 min in a steamy oven (MIWE cube). Freshly baked bread was left to rest at room temperature for 2 h.

Water activity and moisture content
Water activity (a w ) was assessed in cooled breadcrumbs (Lab Master a w Standard, Novasina; Switzerland) at 25 ± 0.3°C. The moisture content was detected by moisture analyser (Kern DBS 60-3, Kern & Sohn GmbH; Germany) at 120°C.

In situ antifungal analysis of bread
The bread was sliced (150 mm of thickness) and placed into 0.5 L sterile glass jars (Bormioli Rocco, Italy). A 5 µL of suspension of fungal spores (1 × 10 6 spores/mL) in sterile PBS with 0.5% Tween 80 with density of 1-1.2 McFarland was applied for bread inoculation. CCEO concentrations of 125, 250, and 500 µL/L (EO + ethyl acetate) were used for the impregnation of sterile paper discs (6 cm). The discs were inserted into the jar lid and jars were tightly closed. The sample material was incubated at 25 ± 1°C for 14 days in the dark and microbial colonies with visible mycelial growth were selected for confirmation [26].

Vapour phase of antimicrobial assay with vegetables and apple
An amount of 5 µL of inoculum was applied on 5 mm of thickness carrot slices, which were placed onto PDA (potato dextrose agar, Oxoid, Basingstoke, UK) agar. The dilution of CCEO in ethyl acetate (1:1) to a final volume of 125, 250, and 500 μL was used for the impregnation of a 55 mm sterile filter paper disc. After evaporation of ethyl acetate, the discs were placed between the lid and agar and incubated at 37°C for 18-24 h and at 25°C for 72 h for bacteria and fungi, respectively [14].

Biofilm development and molecular characteristics on different surfaces with MALDI-TOF MS biotyper
Pre-inoculated S. maltophilia and B. subtilis cultures were grown on a glass slide and a wooden toothpick with the addition of 1% CCEO. Sampling was done at 3, 5, 7, 9, 12, and 14 days of incubation on a shaker (170 rpm, 37°C) with a sterile cotton swab. The control samples were obtained from the planktonic cell suspension. MALDI-TOF MicroFlex analysis (Bruker Daltonics, Germany) with a standard global spectrum (MSP) produced by the MALDI Biotyper 3.0 software (Bruker Daltonics) was processed to the dendrogram method using Euclidean distances [27].

Statistical analysis
All experiments were performed in triplicate. Microsoft™ Excel ® was used for data analysis. MBIC50 and MBIC90 (concentration for 50 or 90% reduction of bacterial biofilm) were evaluated with logit analysis.
Ethical approval: The conducted research is not related to either human or animal use.

Chemical characterization of Cinnamomum cassia essential oil (CCEO)
Earlier reports have shown that CCEO obtained from different parts of plants, e.g. leaves and bark, differ in their composition [28,29]. Age of the trees, growing seasons or months, and sampled material (bark or xylem) were reported to affect the chemical composition of the CCEO [30][31][32][33]. Individual constituents of CCEO may be also affected by growing seasons or months for branches and leaves [32], as well as a natural variety of the sampled parts of the branches [33]. In our study, the main volatile compounds of CCEO were cinnamaldehyde (61.57%), trans-4methoxycinnamaldehyde (13.78%), cinnamyl acetate (5.35%), and o-hydroxy-cinnamic acid (4.12%) ( Table 1).

Antioxidant potential of CCEO
The DPPH radical inhibition value for CCEO was identified to be 42.04 ± 0.42%. The antioxidant properties of different parts of plant have been investigated with notable antioxidant properties reported [3]. Significant DPPH scavenging activity of C. cassia oil (92.4%) was detected previously [35].

Antimicrobial properties of CCEO
Strong antibacterial activity of CCEO was found against Stenotrophomonas maltophilia 27.33 ± 0.58 mm and Bacillus subtilis (20.33 ± 1.53 mm). A lesser degree of activity was reported against P. citrinum (13.53 ± 1.15 mm), P. crustosum (10.64 ± 0.58 mm), and P. expansum (10.33 ± 1.53 mm). Antibacterial activity of CCEO against bacteria, including food-borne pathogens such as Staphylococcus aureus, Listeria monocytogenes, Streptococcus oralis, S. anginosus, Escherichia coli, and B. subtilis, was reported [36][37][38]. Strong bacterial inhibitory effects in the present study support the findings on the antibacterial activities of CCEO. Antimicrobial activities were reported in other Cinnamomum plants, and Chinese cinnamon EO was reported to inhibit the growth of molds in foods with potential practical applications [39].

Minimum inhibitory concentration of CCEO
CCEO showed the highest activity against the S. maltophilia (MIC = 0.05 μL/mL) and against B. subtilis (MIC = 0.10 μL/mL). The high activity against P. citrinum (MFC = expansum (MFC = 0.20 μL/mL) was found which was in agreement with previous studies [36,38]. Our results showed that the best antimicrobial activity of CCEO was found against biofilm-producing strains of S. maltophilia and the worst antiicrobial activity was reported against microscopic filamentous fungus P. citrinum. The MIC extracts of C. cassia against S. aureus were in the range of 0.3-2.0 mg/mL [34]. Bud and bark extracts expressed higher antimicrobial activity against S. aureus and A. baumannii in comparison with the leaf extracts [34]. According to Manso et al. [40], the MIC and MFC of CCEO (100 and 200 ppm) were lower than 400 and 800 ppm recorded for oregano EO.

Moisture content and water activity
The moisture content of the bread was 42.23 ± 0.54% and water activity −0.9435 ± 0.005. Moisture content and water activity may alter the shelf-life of the bakery with elevated parameters and may enhance the microbial growth [41][42][43][44]. For white bread, the a w value was reported to be within the range of 0.94-0.97 [45], making the product more susceptible to microbial spoilage, especially molds. Intermediate moisture between 35 and 42% [46][47][48][49] is typical for bread, and that was in agreement with our data.
Aspergillus, Rhizopus, Penicillium, Mucor, Monilia, and Eurotium were common molds in bread [50]. P. expansum may withstand harsh environmental conditions which was a prerequisite of application of the mold in the experiment [51].

In situ antifungal activity of CCEO on vegetables
The highest antifungal activity of the CCEO on carrot was recorded against all tested fungi at a concentration of 125 µL/L, on potato at a concentration of 250 µL/L, and on sweet potato at a concentration of 500 µL/L ( Figure  2a-f). The growth of Aspergillus niger was inhibited using 500, 1,000, and 2,000 μL of CCEO/L of air; doses of 300, 800, and 1,500 μL of CCEO/L of air were reported as the MIC against P. expansum [52].

In situ antibacterial activity of CCEO on carrot, potato, and apple
A higher antibacterial effect of CCEO at 500 µL/plate was recorded against B. subtilis (Figure 3a-h) on the carrot, potato, and apple. Contrasting results for Citrus aurantium essential oils (CAEO) were obtained in the previous in situ research; there the high antimicrobial potential of CAEO against B. subtilis was found to be at 62.5 µL/plate of CAEO concentration. EO of the bark of C. cassia regulated proliferation of L. monocytogenes in meats without sensorial changes of the product. Specifically, CCEO has reduced the microbial growth under laboratory conditions in comparison with naturally contaminated samples [53].
Previous studies were focused on the antibacterial activity of cinnamon against meat food-borne pathogens, including Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus, Arcobacter butzeiri, and Arcobacter skirrowii [54][55][56]. In cheese, the antibacterial activity of the CEEO against L. monocytogenes, S. aureus, and Salmonella enterica was more profound at ∼23°C, indicating a possible application as a natural food preservative [57]. The results of in vitro studies showed that the same concentrations of EOs exhibited different antifungal activity when tested on fruits in vivo [58]. Host/antifungal/pathogen interaction and different extrinsic factors can lead to divergent results in in vitro and in vivo experiments. Alteration of site action [59] or structural changes due to hydrolysis, degradation, and polymerization [60] of fruits under in vivo condition may explain the differences in recorded results. Similar results have been previously reported [61,62].

Antibiofilm properties of CCEO
MBIC50 and MBIC90 (minimal biofilm inhibition concentration) values were 3.71 and 5.36 μL/mL for B. subtilis, and 4.94 and 6.21 μL/mL for S. maltophilia, respectively. Commercially available Cinnamomum zeylanicum EO was effective in the inhibition of the biomass and viable counts of P. aeruginosa in biofilm at concentrations of 0.12-1.92 mg/mL. Biomass was completely inhibited at 1.92 mg/mL and the significant reduction of viable cells was recorded [63]. C. zeylanicum EO exhibited effect on 41.7 and 33.3% of P. aeruginosa and S. aureus biofilms [64].

Biofilm formation and molecular profile on surface following treatment with CCEO
The development of B. subtilis biofilm is shown in Figure 4. Spectra of the growing biofilms were presented in pairs for analysis of development on different surfaces. Significant differences in spectra between the glass and planktonic spectrum were found on the 3rd day of experiment. Furthermore, significant differences between both experimental and control groups were identified on 5th day (Figure 4b). Differences between experimental and control groups were recorded during the 7-14th days of experiment (Figure 4c-f). The development stages of spectra of B. subtilis and S. maltophilia were similar to previously reported aftertreatment with different essential oils [13,14].
A dendrogram was constructed to make a grouping pattern of B. subtilis for analysed experimental groups ( Figure 5). Two main clusters were generated at level 0.94 which were divided into six subclusters. No specific grouping was there, but most of glass samples together with control were grouped in the wider main cluster. The highest similarity of subclusters was seen for samples on the 3rd, 12th, and 14th day of experiment. All control groups were included in the same cluster, which showed larger MSP distances when compared. The separation of the experimental group in the cluster was due to changes in the biofilm structure after the CCEO application.
MALDI-TOF mass spectra of S. maltophilia biofilms are shown in Figure 6. Spectra (a-f) were paired according to the day of biofilm development. Similarities between experimental and control samples were observed during the 3rd and 7th days of experiment (Figure 6a-c). Inhibition of pattern of experimental spectra was identified at the 9th up to the 12 days of the experiment (Figures 6d and e), after which CCEO interrupted the expansion of S. maltophilia biofilm. Spectra of the 14th day of experiment indicated complete degradation of the biofilm (Figure 6f).
The dendrogram of S. maltophilia consisted of three main clusters with the highest similarity of MSP found for control and wood samples on the 12th and 14th days of the experiment. The constructed dendrogram (Figure 7) shows the highest diversity for KSM samples on the 5th and 9th, and for SMD samples on the 3rd day of experiment. The other samples expressed relatively different spectra.

Conclusions
CCEO showed a satisfactory biological activity and strong inhibitory effect on microorganisms with significant influence in the food models. The main component of the CCEO was cinnamaldehyde which is known for strong inhibitory antimicrobial and antifungal action. The antibiofilm of CCEO was found to have the strongest antibiofilm action and was reported during the 7-14 days of experiments. Evaluation of CCEO revealed antioxidant and antimicrobial properties of CCEO. The vapour phase method is to be used for studies of inhibitory activities of bacteria and molds in the food model. According to the obtained results, CCEO could be suitable to reduce the damage caused by the fungi and biofilm-forming bacteria. EOs were obtained from edible plants and are safe for humans and environment. However, in order to be used as the organic alternative to chemical fungicides, deeper investigations about the absence of whatever form of toxicity are needed. Conflict of interest: Przemysław Łukasz Kowalczewski, who is the co-author of this article, is a current Editorial Board member of Open Chemistry. This fact did not affect the peer-review process. The authors declare no other conflict of interest.
Data availability statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.