Agnieszka Joanna Brodowska, Krzysztof ´Smigielski, Agnieszka Nowak, Katarzyna Brodowska, Rik Catthoor, and Agata Czy˙zowska

The overall objective of this study was to develop a decontamination method against microorganisms in cardamom (Elettaria cardamomum (L.) Maton) seeds using ozone as a decontaminating agent. Ozone treatment was conducted 3 times, at 24-h intervals, and the parameters of the process were determined assuring the least possible losses of biologically active substances (essential oils and polyphenols): ozone concentration 160 to 165.0 g/m3 ; flow rate 0.1 L/min; pressure 0.5 atm; time 30 min. After each step of decontamination, the microbiological profile of the cardamom seeds was studied, and the contaminating microflora was identified. Next to the microbiological profile, the total polyphenol content (TPC), composition of essential oils, free radical-scavenging capacity, total antioxidant capacity, ferric-reducing antioxidant power (FRAP), and LC–MS polyphenol analysis were determined. This study shows that extract from cardamom seeds after ozone treatment is characterized by a better radical scavenging activity (IC50 = 24.18 ± 0.04 mg/mL) than the control sample (IC50 = 31.94 ± 0.05 mg/mL). The extract from cardamom seeds after ozone treatment showed an improved FRAP activity as well (613.64 ± 49.79 mmol TE/g compared to 480.29 ± 30.91 mmol TE/g of control sample). The TPC and the total antioxidant capacity were negatively affected, respectively, 41.2% and 16.2%, compared to the control sample.

Abstract:

Keywords: antioxidant activity, cardamom seeds, decontamination, polyphenols, ozone

In essence, the decontamination methods used so far, largely pointed to: losses of biologically active compounds (polyphenols, essential oils); a negative impact on the sensory evaluation of products; and a need for the use of radiation sterilization. Proposed decontamination method is an alternative to existing ones due to elimination of these cons. It is universal for all form of spices (whole, powdered), and does not influence on the composition as well as amount of essential oils. The purpose of the study was to eliminate mentioned drawbacks through the development of an innovative technology using ozone as the decontaminating agent of plant materials and to obtain a product of a high microbiological purity and unchangeable composition.

Practical application:

Introduction The maintenance of high-quality products, especially the ones of plant origin, is an important topic in the food industry. The results of microbiological purity analysis are a significant factor in the evaluation of usefulness of plant materials for the production process (Khadre and others 2001). Unfortunately, many plants, including spices and herbs are grown and harvested under poor sanitary conditions and in warm and humid regions, which are the main reasons for their microbiological contamination (Mckee 1995; Banerjee and Sarkar 2003). Therefore, the main purpose of the food research is to find an innovative decontamination technology, which meets consumer demands and makes available fresh and safe products (Khadre and others 2001). MS 20140531 Submitted 4/1/2014, Accepted 7/7/2014. Authors Agnieszka ´ Joanna Brodowska, Smigielski and Brodowska are with Inst. of General Food Chemistry, Faculty of Biotechnology and Food Sciences, Lodz Univ. of Technology, Lodz, ˙ Poland. Authors Nowak and CzyZowska are with Inst. of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences, Lodz Univ. of Technology, Lodz, Poland. Author Catthoor is with Dept. of Sustainable Organic Chemistry and Technology, Ghent Univ., Coupure links 653, B-9000, Gent, Belgium. Direct inquiries to author Agnieszka Joanna Brodowska (E-mail: [email protected]). R  C 2014 Institute of Food Technologists

doi: 10.1111/1750-3841.12591 Further reproduction without permission is prohibited

There are currently several decontamination methods, which have been considered in terms of their safety and effectiveness. Many of the existing physical methods (carbon dioxide under pressure, microwaves, ionizing radiation, infrared radiation, extrusion, and steam treatment) have some limitations. Those include the loss of essential oils, changes in the chemical composition of plant materials, and unfavorable effects on their consistency. Chemical methods used for decontamination of spices and herbs utilize methyl bromide, formaldehyde, ethyl alcohol, or ethylene oxide. All of them significantly affect the amount of essential oils in plant materials (Chusri and others 2012; Brodowska and others 2014). In contrast, ozone, a strong oxidant and potent disinfecting agent, has already numerous applications in the food industry (Guzel-Seydim and others 2004). It has been shown that different microorganisms can display various vulnerabilities to ozone. Bacteria are more sensitive than fungi, and Gram-positive bacteria are more sensitive than Gram-negative ones. Obviously, bacterial spores are more resistant than vegetative cells (Kunicka - Styczy´nska and S´ migielski 2011). The study conducted by Akbas and Ozdemir (2008) demonstrated that the application of gaseous ozone to dried figs effectively reduced Escherichia coli, Bacillus cereus, and B. cereus Vol. 00, Nr. 0, 2014 r Journal of Food Science C1

C: Food Chemistry

The Impact of Ozone Treatment on Changes in Biologically Active Substances of Cardamom Seeds

Impact of ozone treatment on . . .

C: Food Chemistry

spores. Ozonated and nonozonated dried figs showed no significant changes in color, pH, and flavor. To our knowledge, there are no published data on the impact of ozone treatment on changes in biologically active substances of spices. Therefore, the objective of this study was to investigate ozone decontamination of cardamom (Elettaria cardamomum (L.) Maton) seeds emphasizing the least possible losses of biologically active substances such as essential oils and polyphenols.

identified to the species level using API tests. API 20NE identified Gram-negative non-EE, API 20E identified EE and nonfermenting Gram-negative bacteria. API 50 CHB identified Bacillus species. Fungal colonies were later microscopically visualized and identified by morphological traits.

Chemicals 2-2-Diphenyl-1-picrylhydrazyl (DPPH); Folin-Ciocalteu’s reagent (2 N); (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid (Trolox); (+)-catechin; and 2,4,6-tri(2-pyridyl)s-triazine (TPTZ) were purchased from Sigma-Aldrich (St. Louis, Miss., U.S.A.). Plate count agar (PCA), DG18 medium, and bullion agar were purchased from Merck (Darmstadt, Germany). The oxidase test kit was obtained from Merck. API tests were purchased from BioM´erieux (Poland). Unless indicated otherwise, all chemicals were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland).

Gas chromatography–mass spectrometry (GC–MS) GC–MS analyses were carried out using a Trace GC Ultra gas chromatograph connected with a DSQ II mass spectrometer (Thermo Electron Corp., Waltham, Ma., U.S.A.). Chromatographic separations were performed on Rtx-1 nonpolar capillary column (30 m × 0.32 mm; 0.25 μm film thickness). The temperature program for Rtx-1: 60 to 300 °C at 4 °C/min. The injector (SSL) temperature was 280 °C, and transfer line temperature 200 °C. He was used as the carrier gas, flow rate 1 mL/min, split ratio 1:20. The identification of the essential oil components was based on a comparison of their retention indices (RI), mass spectra (NIST and Wiley libraries), and literature data (Kov´ats 1958; Adams 1995).

Isolation of essential oil Ground cardamom (E. cardamomum (L.) Maton) (40.0 g) was immersed in 800.0 mL water in a round-bottom flask. The essential oil was obtained by 3-h hydrodistillation of cardamom in an Material and Methods apparatus constructed in the Inst. of General Food Chemistry. This Plant material apparatus assures working without odor release, assures very good Cardamom (E. cardamomum (L.) Maton) seeds were purchased separation of phases, and allows control of the essential oil comfrom a local store in Lodz, Poland. They had been imported from position and the volatile phase, in a continuous way (S´ migielski and others 2009). India.

Ozone treatment The procedure of ozone treatment was performed in a roundbottom glass reactor at 20 to 25 °C. Ozone, previously generated from an oxygen bottle by a laboratory Ozone Generator BMT 803 N (BMT Messtechnik, Berlin, Germany), was transferred to research samples (40.0 g each). Samples were treated with ozone 3 times, at 24-h intervals under conditions as follows: ozone concentration 160 to 165.0 g/m3 ; flow rate 0.1 L/min; pressure 0.5 atm; time 30 min. The ozone concentration in the reactor was controlled by a digital Ozone Analyzer BMT 964 (BMT Messtechnik). Then, the spice samples were transferred to sterile packaging without any direct ozone contact with researcher. Moreover, for keeping additive protection it was installed ozone sensor (Eco Sensors Model A-21ZX, Newark, N.J., U.S.A.) in the laboratory.

Preparation of extracts Cardamom seed extracts (before and after ozone treatment) were obtained by mixing 3.0 g of the material, which had been ground before, with 30.0 mL pure methanol. This mixture was centrifuged (Labofuge 300, ThermoScientific, Waltham, Ma., U.S.A.) at 2500 rpm for 5 min at room temperature. The supernatants were recovered and stored in the refrigerator until analysis.

Determination of total polyphenol content (TPC) Estimation of the TPC in the extracts was done following the Folin–Ciocalteu procedure (Kahkonen and others 1999) taking the modification of Singleton and Rossi (1965) into account. The absorbance was recorded at 760 nm using a Hewlett Packard 8453 spectrophotometer (Waldbronn, Germany). A standard curve was Microbiological analysis prepared using different concentrations (0.02 to 75 μg/mL) of The samples of cardamom were prepared according to ISO catechin. The results were expressed as mg catechin equivalent 6887–4. Total mesophilic bacteria count (TMC) and total bacterial (CE) per 100 g extract (CE/100 g). spore count (TSC) were determined on PCA medium (incubation at 30 °C, aerobically). For the determination of the amount of LC–MSn analysis of polyphenols bacterial spores, a thermal shock (80 °C, 10 min.) was provided Samples of E. cardamomum (L.) Maton extracts were 10-fold at the initial suspension. The Enterobacteriaceae count (EE) was concentrated by rotary evaporator (IKA RV10C S99; Staufen, determined on VRBG agar followed by incubation at 30 °C for Germany), dissolved in 1 mL of pure methanol and filtered 24 h. The total count of fungi (TFC) included isolation, culturing through a 0.22-μm membrane prior to analysis. This was inon Czapek-Dox agar, and incubation at 25 °C for 7 d. The colonies jected onto the high-performance liquid chromatograph (HPLC) were counted as colony forming units (CFU) per gram of sample. column. The HPLC was coupled on-line with an MS LTQ Velos The results were presented as mean ± standard deviation (SD). mass spectrometer (ThermoScientific). Chromatographic separaThe occurrence of the expected bacteria was confirmed on tion was achieved with a column operated at 45 °C. The mobile the basis of Gram-staining, oxidase and catalase tests, glucose phase consisted of solvent A (1 mL formic acid in 1 L deionized metabolism, endospore formation, motility, using standard pro- water) and solvent B (95% acetonitrile). The column used was a cedures (Banerjee and Sarkar 2003). Oxidation or fermentation Hypersil Gold 150 × 2.1, particle size 1.9 μm (ThermoScientific). of glucose was examined in Hugh Leifson (HL) medium afThe elution began with 96% to 85% A for 8 min, continued ter incubation for 72 h at 30 °C. Predominant bacteria were with 85% to 82% A for 12 min, from 82% to 60% A for 40 min, C2 Journal of Food Science r Vol. 00, Nr. 0, 2014

from 60% to 50% A for 4 min, the same for 3 min, from 50% to 96% A for 2 min, followed by washing and re-equilibration of the column. Mass spectra were recorded within 85 min. The injection volume was 10 μL. The flow rate was set at 220 μL/min. Electrospray ionization mass spectrometry was performed using a LTQ Velos mass spectrometer (ThermoScientific) equipped with an ESI interface and controlled by Excalibur software. Mass spectra were acquired in negative mode over the range m/z 120 to 1000. The I spray voltage was 4 kV. The sheath gas flow rate was 25 and auxiliary gas flow rate was 10. The desolvation temperature was 280 °C, and the source temperature was 350 °C. Peak identification was performed by comparison of the retention time and mass spectra of standards.

Antioxidant activity: free radical-scavenging activity Free radical-scavenging capacity of the methanolic extracts was determined spectrophotometrically, following the modified procedure described by Hatano and others (1988). The absorbance was measured after 30 min, using a spectrophotometer (Hewlett Packard 8453) at 517 nm. The scavenging activity was measured as the decrease in absorbance of the samples compared with DPPH standard solution. Lower absorbance of the reaction mixture indicated higher free radical-scavenging activity. The results were expressed as mean inhibiting concentration (IC50 ) (mg/mL); this parameter is defined as the concentration of substrate necessary to scavenge 50% DPPH free radicals. The determination was carried out in triplicate, and the results were expressed as mean values ± SD. Determination of total antioxidant capacity by the DPPH method The DPPH assay was done according to the method of BrandWilliams and others (1995). The absorbance was recorded using a Hewlett Packard 8453 spectrophotometer at 517 nm and quantified using Trolox (25.0 to 250.0 mg/mL) as a standard. The results were expressed as mg Trolox equivalent (TE)/100 g extract. Ferric-reducing antioxidant power assay (FRAP) The FRAP of the essential oils and extracts of cardamom seeds was tested using the assay of Oyaizu (1986), with some modifications. The FRAP assay measures the change in absorbance at 593 nm owing to the formation of a blue-colored FeII -tripyridyltriazine compound from the colorless oxidized FeIII form by the action of electron-donating antioxidants. A standard curve was prepared using different concentrations (25.0 to 250.0 mg/mL) of Trolox. The results were expressed as mmol TE/g extract. Statistical analysis All determinations were carried out in triplicate. Mean values with ±SD were reported for each case. Significance differences for multiple comparisons were considered by Mann–Whitney’s test (Statistica 10.) at the P < 0.05 level. Instrumentation Ozone decontamination: Ozone Generator BMT 803 N (BMT Messtechnik); digital Ozone Analyzer BMT 964 (BMT Messtechnik); ozone sensor (Eco Sensors Model A-21ZX); BMT software (Berlin, Germany). GC–MS analysis: Trace GC ultragas chromatograph connected with a DSQ II mass spectrometer (Thermo Electron Corp.); column—Rtx-1. Antioxidant activity

assay: centrifuge (Labofuge 300, ThermoScientific); spectrophotometer Hewlett Packard 8453. LC–MSn analysis: the HPLC was coupled on-line with an MS LTQ Velos mass spectrometer (ThermoScientific); column—Hypersil Gold 150 × 2.1, particle size 1.9 μm (ThermoScientific); LTQ Velos mass spectrometer (ThermoScientific) equipped with an ESI interface and Excalibur software (ThermoScientific); rotary evaporator IKA RV10C S99.

Results and Discussion Microbiological analysis and changes during ozone treatment No color change was observed, the samples of cardamom seeds retained their original color during ozone treatment. Our observations are in agreement with the study investigated by Akbas and Ozdemir (2008), where dried figs retained their original color during ozone treatment. In addition, the same authors confirmed in another study that no color change was observed in kernels, shelled and ground pistachios samples treated with ozone (Akbas and Ozdemir 2006). The results of the microbial analyze of cardamom seed samples are summarized in Table 1. The results indicate a medium level of contamination. The TMC was in an unacceptable range (>104 CFU/g), but the total fungal and EE counts were at an acceptable level of contamination. The relatively low occurrence of EE indicates the hygienic quality of cardamom (Banerjee and Sarkar 2003). McKee (1995) described the study conducted by Garrido and others (1992), which dealt with the mold contamination of spices. According to that study, mold count was extremely high (105 CFU/g) in stick and ground cinnamons, capers, saffron, badian, cardamom, juniper, fennel, artemisia, bay leaf, mint, parsley, rock tea, and ground mustard seed. Several countries have laid out some specifications for microbial parameters in spices. However, India, from where cardamom seeds were derived, has not specified microbial standards of individual spices (http://www.indianspices.com). At present, there are no specified maximum contaminant levels of spices in the European Union. However, the microbiological contamination of spices should be as limited as possible to ensure food safety for consumers. Ozone concentration (160 to 165.0 g/m3 ) was used for 30 min at 24-h intervals to inactivate the contaminating microflora of the cardamom seeds. Our study showed that the contamination of seeds with mesophilic bacteria decreased during ozone treatment from 105 CFU/g to 103 CFU/g, while the total fungal count varied between 102 and 10 CFU/g. On the other hand, the members of EE decreased from 102 to 10 CFU/g. There were no statistically significant differences at P < 0.05 between the total mesophilic count in control samples and after the 1st ozone treatment samples. It means that ozone treatment was not sufficient to reduce mesophilic bacteria, which proves that they are not significantly affected by ozone (Kunicka - Styczy´nska and S´ migielski 2011). In addition, results indicated statistically significant differences between TMC in samples after the 1st and 2nd treatment. The 3rd treatment resulted in a 2.22 log reduction in TMC. The study confirmed that ozone was very effective in reducing the fungal count during the 1st treatment, a 1.15 log reduction was observed. In contrast, a 1.04 log reduction in EE was observed after the last ozone treatment. In both, the total fungal and EE counts, there were statistically significant differences noticed between control and ozonated samples. Vol. 00, Nr. 0, 2014 r Journal of Food Science C3

C: Food Chemistry

Impact of ozone treatment on . . .

Impact of ozone treatment on . . . Table 1–Occurrence of microbial contamination in cardamom (E. cardamomum (L.) Maton) seeds before and after the 1st, 2nd, and 3rd ozone treatments. Ozone treatment Before After

Total mesophilic bacteria count

C: Food Chemistry

control ozone 1 ozone 2 ozone 3

5.26 5.23 3.64 3.04

± 0.12a b, b, c ± 0.09 ± 0.02aa, bb ± 0.01aa, bb, cc

Total fungal count

Enterobacteriaceae count

2.15 ±0.04d < 1.00∗dd < 1.00∗dd < 1.00∗dd

2.04 ±0.03e < 1.00∗ee 1.00 ±0.00ee < 1.00∗ee

All results are given as log (CFU/g). The results obtained were expressed as mean ± SD with n = 3. ∗ not detected at the level 10 CFU/1 g. a Statistically no significant differences at a probability of P < 0.05. b Statistically no significant differences at a probability of P < 0.05. c Statistically no significant differences at a probability of P < 0.05. d Statistically no significant differences at a probability of P < 0.05. e Statistically no significant differences at a probability of P < 0.05. aa Statistically significant differences at a probability of P < 0.05. bb Statistically significant differences at a probability of P < 0.05. cc Statistically significant differences at a probability of P < 0.05. dd Statistically significant differences at a probability of P < 0.05. ee Statistically significant differences at a probability of P < 0.05.

Table 2–Detected bacterial species of cardamom (E. cardamomum ternaria, Cladosporium, and Virticillim) (Kunicka - Styczy´ nska and (L.) Maton) seeds before and after the 1st, and 3rd ozone treat- S´ migielski 2011; Cheng and others 2013). ment. Ozone treatment Before After 3rd

Species

Percentage distribution

A. hydrophila B. megaterium B. cereus B. pumilus A. hydrophila B. cereus

60 3 29 8 40 60

Bacteria isolated from the samples before ozone treatment included Aeromonas hydrophila, Bacillus megaterium, B. cereus, and Bacillus pumilus. After the last ozone treatment, only A. hydrophila and B. cereus were found. The detected species are presented in Table 2. Predominant bacteria included A. hydrophila, found in 60% of the samples analyzed (105 CFU/g) and B. cereus, occurring in 29% of the samples of cardamom seeds (104 CFU/g). The following occurrence of bacterial species was noticed: B. megaterium (3%) and B. pumilus (8%). However, after the 3rd ozone treatment the presence of bacterial species was as follows: B. cereus was found in 60% of the samples of cardamom (102 CFU/g) and A. hydrophila was detected in 40% of the samples tested (102 CFU/g). According to EFSA scientific opinion the infective dose of B. cereus is 105 to 106 CFU. Taking into account our results, before ozone treatment the contamination of cardamom seeds was at the level 5.2 × 104 CFU/g. After ozone treatment the contamination level of B. cereus was at the level 102 CFU. The food is maintained as safe if the level of contamination is 10 times lower than the infective dose. Thus, although B. cereus and A. hydrophila are harmful to humans and cause foodborne illness, after the last step of treatment they occurred at safe levels of contamination (http://www.efsa.eu.int). Before ozone treatment, 3 likely fungal species, including Cladosporium sp. (45%), Acremonium strictum (45%), and Rhizopus nigricans (10%), were found. According to the literature, the extent of spice contamination depends on numerous factors. It may have a primary character caused by microorganisms naturally present on plants. Not only the epiphytic microflora (species of the genera Pseudomonas, Enterobacter, Flavobacterium, Alcaligenes, Aerobacter, Chromobacterium, and Spirillium), but also secondary contamination with soil-, water-, or airborne microorganisms during harvesting, drying, transporting, and storage influence the microbial profile (species of Bacillus, Pseudomonas, Chromobacter, Aeromonas, Penicillium, Fusarium, AlC4 Journal of Food Science r Vol. 00, Nr. 0, 2014

Identification of essential oil The results obtained from the GC–MS analysis of the essential oil of the cardamom seeds isolated before and after the (3rd) ozone treatment are summarized in Table 3. The odor profile showed that the oil contains more than 60 constituents, out of which 32 were identified representing 98.05% and 98.25% of the total oil before and after ozone treatment, respectively. The constituents were identified by comparing the RIs of authentic samples and by comparing their mass spectra with those of standard libraries (NIST and Wiley) and the literature (Kov´ats 1958; Adams 1995). To correct minor variations in the injection volume n-alkane, C12 was used as an internal standard. Then the response from the analyte peak was compared to that standard. 1,8-Cineole, terpinyl acetate, linalool, sabinene, linalyl acetate, α-terpinol, myrcene, and geraniol were the major constituents occurring in oil before and after ozone treatment. If we compare the composition of essential oil before and after ozone treatment, we can see that the major constituent were terpinyl acetate (37.17%) and 1,8-cineole (37.84%) before and after ozone treatment, respectively. Referring to the study by Amma and others (2010), the characteristic flavor of cardamom was contributed by the esters, alcohols, and 1,8-cineole. The obtained results indicated that they are comparable. In addition, the statistical analysis confirmed a few significant differences between each constituent at the P < 0.05 level, which means that ozone treatment of cardamom seeds does not influence the chemical composition of essential oils much. Determination of TPC The total phenolic content (TPC) of the methanol extracts of cardamom was determined by the Folin–Ciocalteau method with some modifications. Catechin was used as the standard. The total phenolic content was expressed as mg CEs per 100 g of extract. Table 4 shows the results. A higher phenolic content was recorded in samples before ozone treatment (96.6 mg/100 g extract). The samples after the 3rd ozone treatment were characterized by 41.2% loss compared to a sample before treatment (56.8 mg/100 g extract). These results were in agreement with those reported by Amma and others (2010) and Deepa and others (2013), despite the fact that they used gallic acid as a standard. The reaction mechanism between ozone and polyphenols is very complex and still is not well understood (Diaz

Impact of ozone treatment on . . . Table 3–Comparison of the composition of essential oil of cardamom (E. cardamomum (L.) Maton) seeds obtained before and after the 3rd ozone treatment.

Nr.

Compounds

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

α-Thujene α-Pinene Sabinene β-Pinene Myrcene β-Cymene 1,8-Cineole Linalool oxide cis-Sabinene hydrate Linalool trans-p-Menth-2-en-1-ol α-Terpineol 4-Terpinenol α-Terpinol Linalyl acetate Geraniol Geranyl vinyl ether Terpinyl acetate Neryl acetate Geraniol acetate Nerolidol acetate Isopulegone Terpinyl propionate Limonene oxide Isopulegyl acetate β-Selinene Torreyol (S)-(+)-Carvone acetate trans-Nerolidol Nerolidol Pterine-6-carboxylic acid 2-Myristynoyl pantetheine Total

After ozone treatment

%

RI

%

RI

0.10 1.09∗ 3.05∗ 0.52 1.09 0.28 36.50 0.71 0.05 5.42∗ 0.17 0.11 1.01 2.56 2.75 1.21 0.44 37.17 0.36 0.88 0.11 0.13 0.08 0.51 0.11 0.19 0.20 0.26 0.85 0.09 0.01 0.02 98.05

926 930 966 969 986 1016 1021 1056 1083 1087 1108 1153 1164 1176 1241 1245 1270 1336 1351 1368 1388 1415 1423 1442 1466 1481 1508 1543 1552 1572 1789 2301

0.10 1.22∗ 3.24∗ 0.53 1.01 0.31 37.84 0.67 0.06 5.24∗ 0.16 0.11 0.98 2.47 2.76 0.99 0.46 36.00 0.37 0.90 0.09 0.15 0.09 0.63 0.13 0.17 0.22 0.35 0.86 0.10 0.02 0.02 98.25

925 930 966 969 986 1016 1022 1057 1076 1087 1108 1153 1164 1176 1242 1246 1270 1337 1351 1368 1388 1414 1423 1442 1464 1482 1508 1540 1551 1572 1789 2303

The results obtained were expressed as mean ± SD with n = 3 according to Mann–Whitney’s test. ∗ Values are significantly different at P < 0.05.

Table 4–Determination of total polyphenol content (TPC) of LC–MSn analysis of polyphenols methanolic extract from cardamom (E. cardamomum (L.) Maton) The phenolic compounds present in cardamom extracts before seeds before and after the 3rd ozone treatment (mg CE/100 g of and after the 3rd ozone treatment were identified by LC–MS. A extract).

total of 30 polyphenols were detected. Preliminary identification Before ozone treatment After the 3rd ozone treatment

TPC (mg CE/100 g of extract) of the phenolic compounds was carried out by comparing retention times and masses with those of 15 authentic standards. For the 96.6 ± 0.5 56.8 ± 4.6 remaining 15 compounds, for which no standards were available,

The results obtained were expressed as mean ± SD with n = 3 according to Mann–Whitney’s test. Values are significantly different at P < 0.05. Total phenolic content expressed as mg of catechin equivalent/100 g of sample.

and others 2005). The reduction in polyphenol content may be connected to the oxidation of phenolic compounds by gaseous ozone (Gurol and Vatistas 1987). Gould and Weber (1976) confirmed that the phenol–ozone system results in the creation of new molecules. Most probably, a carbon–carbon double bond can be attacked by ozone, giving rise to a new dicarbonyl compound. Subsequent oxidation of phenolic compounds may lead to a series of aldehyde-acids and diacids with 10 possible 6-carbon compounds. Moreover, dihydric phenols can be created as a result of hydroxylation of the benzene ring of phenol. Furthermore, Fernandez and others (2009) suggest that ozone–phenol reactions are faster than ozone–benzene reactions, but they are influenced by the numbers and positions of –OH groups present on the compound structure.

identification was based on accurate mass measurements of the pseudomolecular [M–H]− ions because polyphenols contain one or more hydroxyl and/or carboxylic acid groups, and mass spectra data were acquired in negative ionization mode (Duke and others 2003; Shan and others 2005). Additionally, the absorbance maxima UV λ max of the standards were used for the identification of these compounds in the investigated extracts of cardamom before and after ozone treatment. The results of accurate mass measurements matched the elemental composition of all the compounds analyzed (Table 5). The identified polyphenols, mostly phenolic acid derivatives, were gallic, caffeic, p-coumaric, hydroxycinnamic, and ellagic acid derivatives. The glycosylated forms of flavonoids, quercetin rutinoside, quercetin-3-coumarylo-galactoside, quercetin 3-Oglucuronide, and kaempferol pentoside were detected. With regard to the class of flavanols, the overlapping of the UV-vis spectrum of catechin with those of the catechin derivatives confirmed the presence of catechin compounds.

Vol. 00, Nr. 0, 2014 r Journal of Food Science C5

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Before ozone treatment

Impact of ozone treatment on . . . Table 5–LC-MS analysis of phenolic compounds identified in methanolic extracts from cardamom (E. cardamomum (L.) Maton) seeds before and after the 3rd ozone treatment. RT before RT after ozone ozone treatment treatment Peak (min) (min)

C: Food Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

2.15 3.23 3.66 4.20 8.28 9.49 10.16 11.58 12.18 13.23 14.26 16.24 16.74 17.16 18.43 21.76 22.80 23.10 25.13 35.19 38.78 39.14 44.80 47.86 52.15 53.70 54.34 58.40 63.61 68.80

2.15 3.23 3.66 4.20 8.28 9.50 10.12 11.50 12.18 13.23 14.26 16.24 16.67 16.99 18.52 22.29 22.45 23.10 25.01 35.44 37.66 39.37 44.90 47.59 52.03 53.44 54.34 58.26 63.61 68.77

max 218, 250, 258 259 235, 263 266 235, 262 236, 266, 292 256 262, 305 240, 295 243, 279, 305 309, 241 250, 267, 320, 353 256, 354 253, 353 244, 278, 225 244, 225 247 246 247, 288, 320, 353 278 250, 269 250, 270 243 251, 353 248, 270 248, 270, 314 252, 353 246, 222 355, 256 251, 353, 319

m−

MS–MS

377.5 341.2 225 181 292 202, 244 205 173, 111 299 137, 239, 179, 209 329 209, 167, 269, 181, 239 393 347 325 279 210 125 351.5 249, 333, 267 377.5 331, 341 609 301 609 301 477 301 703 657, 639 187 125 243 183, 225 187 125 595 445 419 555 393 685 639 403 153, 359, 385, 197, 331, 138, 247, 187, 173 329.5 314 241.6 179, 197 255.6 193, 211 329.5 314 266 97, 221, 193 343.5 328 593.5 277, 315, 413, 241, 233

Compound NI Sinapic acid Catechin derivatives Gallic acid derivative NI NI Stilbene derivative p-Coumaric acid derivative Gallic acid derivative Caffeic acid derivative Caffeic acid derivative Quercetin rutinoside Quercetin-3-coumarylo-galactoside Quercetin 3-O-glucuronide Catechin derivative Gallic acid derivative Stilbene derivative Gallic acid derivative Quercetin and hydroxycinnamic acid derivative NI Lariciresinol derivative Flavone glycoside Tentatively iridoid NI, flavonole derivative Stilbene derivative Hydroxycinnamic acid derivative NI, flavonole derivative Coumestrol Ellagic acid derivative Quercetin and hydroxycinnamic acid derivative

NI, not identified.

The LC–MS profile of methanolic extract of cardamom reported in the literature included gallic acid, catechin, eugenol, benzoic acid, benzoic acid derivatives (p-hydroxybenzoic acid), chlorogenic acid, p-coumaric acid, ferulic acid, cinnamic acid derivatives (o-hydroxycinnamic acid), quercetin glycosides, sinapic acid, rutin, vanillic acid, procatechuic acid, myricetin glycosides (myricitrin, myricetin-3-O-rutinoside), caffeic acid hexoside, and caffeic acid (Duke and others 2003). Comparing the polyphenols composition of the cardamom extracts before and after the 3rd ozone treatment revealed no differences in identified compounds by LC-MS analysis.

Antioxidant activity: free radical scavenging activity and total antioxidant capacity by DPPH method DPPH is a stable free radical with a characteristic absorption at 517 nm (purple color of solution). Results were expressed as mean inhibiting concentration (IC50 ) (mg/mL) of the DPPH. From the IC50 values (the concentration of the sample resulting in a 50% inhibition of the free radical), it was revealed that methanol extract of cardamom treated with ozone had a higher DPPH radical scavenging activity (24.18 ± 0.04 mg/mL) than samples not treated with ozone (31.94 ± 0.05 mg/mL) (Table 6). The results after ozone treatment are statistically different. A lower IC50 value of cardamom sample after (the 3rd) ozone treatment indicated the greater radical scavenging activity. Such conflicting results could be explained by the oxidation process, which occurred during ozone treatment. The ozone caused degradation of polyphenols, which were related to their total antioxidant capacity, thus statistically differences observed in TPC could be the reason C6 Journal of Food Science r Vol. 00, Nr. 0, 2014

Table 6–Total antioxidant capacity and free radical scavenging activity of methanolic extracts from cardamom (E. cardamomum (L.) Maton) seeds before and after the 3rd ozone treatment. Total antioxidant capacity (mg TE/100 g of extract) IC50 (mg/mL) Before ozone treatment After the 3rd ozone treatment

10.47 ± 0.67 8.77 ± 0.79

31.94 ± 0.05 24.18 ± 0.04

The results obtained were expressed as mean ± SD with n = 3 according to Mann–Whitney’s test. Values are significantly different at P < 0.05. Total antioxidant activity expressed as mg of Trolox equivalent /100 g of sample. IC50 values were expressed in mg/mL.

for the same differences in total antioxidant capacity (Shan and others 2005). The DPPH assay was done according to the method of BrandWilliams and others (1995). Trolox was used as a standard. The amount of total antioxidant capacity of the methanol extract of cardamom samples was expressed as TEs per 100 g of extract. The obtained results (Table 6) indicated higher total antioxidant capacity in cardamom samples before ozone treatment (10.47 mg/100 g extract) and a little lower (8.77 mg/100 g extract) after (the 3rd) ozone treatment.

FRAP Determination of the FRAP is a simple direct test of antioxidant capacity. In this study, the assay of reducing activity was based on the reduction of ferric to the ferrous form in the presence of reductants (antioxidants) in the tested samples. The antioxidant activity of essential oils and extracts before and after the 3rd ozone

Impact of ozone treatment on . . .

FRAP assay (mmol TE/g) Extract Essential oil

Before ozone treatment After the 3rd ozone treatment Before ozone treatment After the 3rd ozone treatment

480.29 613.64 72.41 55.03

± ± ± ±

30.91 49.79 4.31 1.73

The results obtained were expressed as mean ± SD with n = 3 according to Mann–Whitney’s test. Values are significantly different at P < 0.05. FRAP expressed as mg Trolox equivalent/g of sample.

treatment, determined by the FRAP method, is summarized in Table 7. Of the 2 essential oils (before and after the 3rd ozone treatment), the first showed stronger ferric ion reducing activity with 72.41 ± 4.31 mmol TE/g of essential oil. However, there was a significant difference between FRAP in both extracts. Cardamom extract isolated from the plant material after the 3rd ozone treatment exhibited a significant antioxidant potential with 613.64 ± 49.79 mmol TE/g extract. According to the study conducted by Deepa and others (2013) and our obtained results, cardamom extract after ozone treatment showed significant FRAP activity. In our study, essential oils of cardamom possessed lower ferric-reducing capacities than its extracts.

Conclusion Our results show that ozone treatment changes the antioxidant activity of cardamom (E. cardamomum (L.) Maton) seeds. Samples treated with ozone showed greater free radical scavenging activity than samples not treated with ozone. There were no significant differences between the composition of essential oils before and after ozone treatment. The study showed that ozone treatment allowed to obtain a microbiologically clean product with a similar composition of essential oils, although the results of antioxidant activity and polyphenols were not satisfactory under the used decontamination conditions. No change in color was observed in cardamom seeds samples treated with ozone. Therefore, ozone treatment of cardamom (E. cardamomum (L.) Maton) seeds still needs to be further optimized to become a promising method for maintaining biologically active substances during the decontamination process.

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Author contributions Agnieszka J. Brodowska drafted the manuscript, designed and conducted all of studies, and interpreted the results. Krzysztof S´ migielski drafted the concept of ozone decontamination, and discussed the results. Agnieszka Nowak helped with the microbiological analysis and interpretation of those results. Katarzyna Brodowska and Rik Catthoor conducted the antioxidant activity assays. Agata Czy˙zowska helped with the LC–MS analysis.

References Adams RP. 1995. Identification of essential oil components by gas chromatography/mass spectroscopy. Carol Stream, Ill.: Allured Publishing Co.

Supporting Information Additional Supporting information may be found in the online version of this article at publiser’s website: Fig. 1. Spectrum of GC-MS analysis of essential oil of cardamom seeds before ozone treatment Fig. 2. Spectrum of GC-MS analysis of essential oil of cardamom seeds after the 3rd ozone treatment

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Table 7–Ferric-reducing antioxidant power (FRAP) of the essen- Akbas MY, Ozdemir M. 2006. Effectiveness of ozone for inactivation of Escherichia coli and tial oils and methanolic extracts from cardamom (E. cardamoBacillus cereus in pistachios. Int J Food Sci Tech 41:513–9. mum (L.) Maton) seeds before and after the 3rd ozone treatment Akbas MY, Ozdemir M. 2008. Application of gaseous ozone to control populations of Escherichia coli, Bacillus cereus and Bacillus cereus spores in dried figs. Food Microbiol 25:386–91. (mmol TE/g).