Green pepper essential oil as a biopreservative agent for fish-based products: Antimicrobial and antivirulence activities against Pseudomonas aeruginosa KM01

LWT - Food Science and Technology 108 (2019) 6–13

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Green pepper essential oil as a biopreservative agent for fish-based products: Antimicrobial and antivirulence activities against Pseudomonas aeruginosa KM01

T

Kamila Myszkaa,∗, Anna Olejnika, Małgorzata Majcherb, Natalia Sobieszczańskaa, Anna Grygierb, Jolanta Powierska-Czarnyc, Magdalena Rudzińskab a b c

Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, Poznan, PL-60-627, Poland Department of Food Chemistry and Instrumental Analysis, Poznan University of Life Sciences, Wojska Polskiego 31, Poznan, PL-60-624, Poland Institute of Forensic Genetics, Al. Mickiewicza 3/4, Bydgoszcz, PL-85-071, Poland

A R T I C LE I N FO

A B S T R A C T

Keywords: Pseudomonas aeruginosa Bacterial virulence Fish-based products Green pepper essential oil β-caryophyllene

Pseudomonas aeruginosa is a common contaminant in fish-based products. Given the resistance of P. aeruginosa to synthetic preservatives, there is a need for novel tools to suppress the growth and virulence of this pathogen. This study investigated the potential of β-caryophyllene-rich green pepper essential oil (EO) as a biopreservative agent against the wild type KM01 strain of P. aeruginosa. Green pepper EO at selected concentration was nontoxic to FHs 74 Int and CCD 841 CoN human cell lines, as determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay. The sub-minimal inhibitory concentrations (MICs) of green pepper EO and β-caryophyllene were 120 and 25 nl ml−1, respectively; exposure of P. aeruginosa KM01 to these concentrations increased unsaturated fatty acids (FAs) (especially C16:1 cis-9 and C18:2 cis-9,12) and decreased saturated and hydroxylated FAs (especially 16:0, 3-OH-12:0, and 2-OH-16:0) in the bacterial cell membrane. An absence of anteiso-C15:0 synthesis was also observed in membrane FA profiles. The results of modeling and in situ experiments revealed that green pepper EO and β-caryophyllene inhibited the growth of KM01 (by 89%–95%) and attenuated bacterial virulence properties such as pyocyanin production (by 80%–89%) and elastase (by 40%–72%) and alkaline protease (by 28%–91%) activities.

1. Introduction Seafoods, especially fish and fish-based products, are one of the fastest-growing sources of food worldwide as well as a major part of revenue in many developing and developed countries (Tidwell & Allan, 2001). Being a rich source of proteins (containing all essential amino acids), unsaturated fatty acids, vitamins (D, A, and B), and minerals (calcium, iodine, zinc, and selenium), seafoods are nutritionally important, and thus their global consumption hit a record high over the last decade (Barik, 2017). However, the perishable nature of seafoods and their improper storage conditions decrease their safety and shelflife considerably (Wu, Pu, & Sun, 2019). Despite the implementation of quality management programs in the food industry, fish-based products continue to pose a threat by being responsible for foodborne-disease outbreaks, associated mostly with psychrotolerant bacterial pathogens (Elbashir et al., 2018). About one-fourth of the world's food supply and 30% of landed fish are lost due to microbial deterioration (Ghaly, Dave,

∗

Budge, & Brooks, 2010). Microbial spoilage begins within 12 h of catching a fish and proceeds rapidly (AMEC, 2003). Psychrotolerant Pseudomonas spp. (including P. aeruginosa) is one of the bacteria most frequently isolated from spoiled seafoods and contributes to the loss of product quality due to slime formation and the generation of off flavors and odors (Benie et al., 2017). P. aeruginosa may account for as much as 90% of microorganisms in spoiled fish and can cause gastrointestinal problems, particularly in immunocompromised and debilitated individuals. The prevalence of P. aeruginosa infection (including of the digestive system) is 11.5% in Europe and 17% in developing countries (Benie et al., 2017). For this reason, effective methods of preservation of seafoods against Pseudomonas spp. are required in order to maintain the safety and extend the shelf-life of such products. Chemical preservatives have been applied to fish-based products; for example, sodium acetate or butylated hydroxyanisole (BHA) can extend the shelf life of sliced salmon during refrigerated storage by a

Corresponding author. E-mail address: [email protected] (K. Myszka).

https://doi.org/10.1016/j.lwt.2019.03.047 Received 31 October 2018; Received in revised form 11 March 2019; Accepted 15 March 2019 Available online 20 March 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

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apparatus for 3 h with 300 ml deionized water. The resultant EO product was dried over anhydrous sodium sulfate and stored at 4 °C until further filtration. A GC-MS analysis of the EO was performed using a Hewlett-Packard HP 7890A GC apparatus coupled to a 5975C mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a Supelcowax-10 column (30 m × 0.25 mm × 0.5 μm). The operating conditions for GC-MS were as follows: helium flow, 0.8 ml/min; initial oven temperature, 40 °C (2 min) raised to 240 °C at a rate of 8 °C min−1 and held isothermally for 6 min. Retention indices were calculated for all peaks using a homologous series of C7–C24 n-alkanes. Mass spectra were recorded in electron impact mode (70 eV) in a scan range of m/z 33–350.

few days (Sallam, 2007). However, despite their antioxidative and antimicrobial properties, a number of recent studies have raised concerns about the safety of synthetic preservatives, especially regarding their endocrine-disrupting activity (Karpuzoglu, Holladay, & Gogal, 2013). It was demonstrated that parabens can induce oxidative stress, which has been implicated in the pathology of several human diseases (Giustarini, Dalle-Donne, Budge, & Brooks, 2009); for instance, a clear link has been reported between oxidative stress and DNA damage in mammalian Vero cells exposed to BHA (Pérez Martín et al., 2010). Oxidative DNA damage caused by reactive oxygen species can lead to a decline in cellular functions and represents the first step of mutagenesis, carcinogenesis, and aging (Valko et al., 2007). Due to the growing concerns regarding the safety of most synthetic seafood preservatives, alternative approaches to preservation using natural compounds have been investigated (Kykkidou, Giatrakou, Papavergou, Kontominas, & Savvaidis, 2009; Mastromatteo, Conte, & Del Nobile, 2010). Several studies have demonstrated the potent antioxidant and antimicrobial activities of essential oils (EOs) towards foodborne pathogens, and EOs derived from green pepper fruit have be evaluated in the food industry for their preservative potential (Nikolić et al., 2015). Based on the antimicrobial activities of monoterpene and sesquiterpene hydrocarbons, it is presumed that green pepper EOs target P. aeruginosa cells (Nikolić et al., 2015). Moreover, pepper EOs are placed in the Generally Recognized As Safe (GRAS) category by the U.S. Code of Federal Regulations and their documented high oral lethal dose (LD50) values in rats (> 5 g/kg) strengthen the possibly of utilizing pepper EOs as preservatives of food items (Tisserand & Young, 2014). Pepper fed to rats at doses 5–20 times the normal human intake did not have any adverse effects on growth, food efficiency ratio, blood cell counts, organ weights, serum aminotransferase and phosphatase activities, nitrogen balance, or fat content (Bhat & Chandrasekhara, 1986). Epidemiological studies have also suggested that daily consumption of green pepper EOs can reduce the risk of cancer and cardiovascular disease (Srinivasan, 2007). Manosroi, Dhumtanom, and Manosroi (2006) revealed the chemopreventive activity of EOs against human mouth epidermal carcinoma cells and mouse leukemia cells, with respective lethal concentration (LC50) values of 0.215 and 0.201 mg/ml. Biopreservation with green pepper EO can be highly effective in the case of raw and processed fish, which require the application of natural extracts/EOs from herbs or spices for flavor improvement. However, to date the beneficial effects of green pepper EOs in the food industry have not been systematically characterized. Therefore, in this study we investigated the antibacterial and antivirulence activity of green pepper EO against the wild-type food-associated P. aeruginosa KM01 strain. We first analyzed the chemical composition of green pepper EO by gas chromatography combined with mass spectrometry (GC-MS). To establish the sub-minimum inhibitory concentration (MIC) of green pepper EO and β-caryophyllene, we used the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess their cytotoxicity to normal human small intestine FHs 74 Int and colon CCD 841 CoN cells with the macrodilution assay. The antimicrobial effects of the test agents were evaluated by quantification of fatty acid (FA) methyl esters of P. aeruginosa KM01, and their antivirulence activities were verified by analyzing bacterial elastase and protease activities and pyocyanin production.

2.2. MTT cell proliferation assay The normal human small intestine FHs 74 Int and colon CCD 841 CoN cell lines (American Type Culture Collection [ATCC] CCL-241 and CRL-179, respectively) were supplied by LGC Standards (Łomianki, Poland). FHs 74 Int cells were grown in Hybri-Care Medium ATCC 46-X (ATCC, Manassas, VA, USA) supplemented with 30 ng ml−1 epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA) and 100 ml l−1 fetal bovine serum (Gibco, Grand Island, NY, USA). CCD 841 CoN cells were cultured in the ATCC-formulated Eagle's Minimum Essential Medium containing fetal bovine serum at a final concentration of 10%. FHs 74 Int and CCD 841 CoN cell cultures were maintained at 37 °C in a humidified atmosphere with 5% CO2. In order to reduce the risk of cell transformation, cells were used in experiments at a low passage number. The cells were grown in 96-well plates at an initial density of 2.0·104 cells cm−2. The 24-h cultures were treated with green pepper EO and β-caryophyllene at concentrations ranging from 7.81 to 500 and 3.13–200 nl ml−1, respectively. The samples were then incubated for 48 h under standard culture conditions. The effect of the green pepper EO and β-caryophyllene on cell viability was evaluated with the MTT (Sigma-Aldrich) assay (Mosmann, 1983). Briefly, FHs 74 Int and CCD 841 CoN cells treated with green pepper EO and β-caryophyllene were incubated with MTT solution (0.5 mg ml−1) at 37 °C for 3 h. Acidic isopropanol was added to each well at the end of the incubation period, and the culture plate was agitated on a microplate shaker for 20 min to dissolve the formazan crystals. The absorbance was measured at 570 and 690 nm (as a reference) on an M200 Infinite microplate reader (Tecan Group, Männedorf, Switzerland). Cell viability is expressed as % absorbance relative to untreated control cells. The 50% and 90% inhibitory concentrations (IC50 and IC90, respectively) were calculated based on the assay results. 2.3. Bacterial strain P. aeruginosa KM01 was isolated from commercial raw salmon (Salmo salar). The strain was confirmed by 16S rRNA gene restriction length polymorphism analysis and sequencing. Cells were preserved at −80 °C in cryovials (Medical Wire & Equipment, Corsham, UK). 2.4. Culture conditions P. aeruginosa KM01 was cultured in tryptic soy broth (TSB) (BD Biosciences, Franklin Lakes, NJ, USA) enriched with serial dilutions of green pepper EO (7.8–500 nl ml−1) and β-caryophyllene (3.13–200 nl ml−1) (Sigma-Aldrich) in dimethyl sulfoxide (SigmaAldrich) at 4 °C ± 1 °C for 72 h. The pH of the culture medium was 5.0.

2. Materials and methods 2.1. Isolation of green pepper EO and identification of constituent compounds

2.5. Estimation of MIC and selection of sub-MIC Isolation of green pepper EO and identification of constituent compounds were carried out according to our previous study (Myszka, Schmidt, Majcher, Juzwa, & Czaczyk, 2017). Briefly, ground green pepper fruits (50 g) were subjected to hydrodistillation in a Clevenger

The MICs of green pepper EO and β-caryophyllene against P. aeruginosa KM01 were determined according to Clinical and Laboratory Standards Institute guidelines (CLSI, 2006). A 6-h culture of P. 7

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2.7.3. Elastase activity The elastase activity of P. aeruginosa KM01 was measured with a microtiter-based assay based on the cleavage of the elastase-specific chromogenic peptide substrate N-succinyl-Ala-Ala-Ala-p-anilide (Sigma-Aldrich) as previously described (Galdino et al., 2017). The reaction mixture (100 μl) containing the cell-free culture supernatant, chromogenic substrate at 1 mM, and buffer (50 mM Tris-HCl, 10 mM CaCl2, 1 mM ZnCl2, and 150 mM NaCl [pH 8.0]) was incubated for 2 h at 37 °C in a 96-well microplate. A405 of the samples was measured with a microplate reader. The results are expressed in U l−1. The percentage inhibition of elastase activity was calculated with the formula (2):

aeruginosa KM01 was used as the inoculum with different dilutions of green pepper EO and β-caryophyllene (Sigma-Aldrich). The turbidity of the culture was adjusted with sterile saline to the equivalent of the McFarland 0.5 standard. P. aeruginosa KM01 was cultured for 72 h at 4 °C. The concentration of green pepper EO or β-caryophyllene resulting in visible growth inhibition was determined as the MIC. Sterile medium without and with EO and β-caryophyllene supplementation served as controls. Sub-MICs of green pepper EO and β-caryophyllene were determined for each experiment. 2.6. Determination of cellular FA methyl ester (FAME) profiles Cellular FAME profiles were determined as previously described (Whittaker et al., 2005). Briefly, P. aeruginosa KM01 cells were collected by centrifugation (3000×g, 10 min) after adjusting the absorbance at 540 nm (A540) to 1.5. A 1-ml volume of 3.75 N NaOH (1:1, methanol:distilled water) was added to each sample to saponify the FAs. The samples were then heated in a boiling water bath for 5 min, vortexed, heated for an additional 30 min in a boiling water bath, and then cooled. Next, 2 ml of 3.75 N HCl (1:1.18; methanol:6 N HCl) were added for the methylation of FAs. The samples were heated for 10 min at 80 °C. After cooling, FAMEs were isolated by adding 1.25 ml of a 1:1 hexane:methyl tert-butyl ether mixture. The samples were gently vortexed, and the lower phase was removed by pipetting before adding 3.0 ml of 0.3 N NaOH to the organic phase followed by vortexing for additional 5 min. The organic phase was collected for GC analysis of FAMEs, which was performed using a Trace 1300 systems (Thermo Fisher Scientific, Waltham, MA, USA) with a flame ionization detector. Hydrogen was used as the carrier gas at a flow rate of 1 ml/min. A 30 m × 0.25 mm × 0.25 μm column (HP-5MS; Agilent Technologies) was used to separate the FAs. The operating conditions were as follows: initial temperature, 150 °C increased to 185 °C at a rate of 4 °C min−1 and then to 200 °C at 2 °C min−1. The detector temperature was 250 °C. The injection was performed in a splitless manner. Chromeleon Chromatography Studio software (Thermo Fisher Scientific) was used for data collection. The abundance of individual FAs is shown as a percentage of total detected FAs.

100 − [C/D * 100]

where C denotes OD405 of samples containing the supernatant of P. aeruginosa KM01 grown in TSB medium supplemented with test substance; D denotes OD405 of samples containing the supernatant of P. aeruginosa KM01 grown in unsupplemented TSB medium. 2.7.4. Protease activity Proteolytic activity was determined by measuring the release of αamino groups with the trinitrobenzenesulfonic acid (TNBS) method (Marchand et al., 2009), which is based on the reaction of free amino groups with TNBS reagent (Sigma-Aldrich) at pH 9.2 in the dark. The results are expressed in U l−1. The percentage inhibition of protease activity was calculated with the formula (3): 100 − [E/F * 100]

(3)

where E denotes OD420 of samples containing the supernatant of P. aeruginosa KM01 grown in TSB medium supplemented with test substance; F denotes OD420 of samples containing the supernatant of P. aeruginosa KM01 grown in TSB medium. 2.8. In situ experiments evaluating the antivirulence properties of green pepper EO and β-caryophyllene against P. aeruginosa KM01 2.8.1. Salmon sample preparation, marination, and packaging One night before the experiment, frozen 25 g samples of commercial raw salmon were retrieved from the freezer and thawed overnight in a refrigerator (4°C–5°C). The thawed samples were then inoculated with 2 ml of P. aeruginosa KM01 culture standardized to an initial level of approximately 103 CFUg−1. The samples were then air-dried at 22 °C for 15 min (to allow bacterial attachment) in the biosafety cabinet (Thermo Scientific, U.S.A.) prior to treatment with marinade and packaging. The product marinade was composed of 95% olive oil and 5% vinegar (components are expressed as a percentage of total weight). All ingredients were obtained from local manufacturers. The 5 ml portions of marinade were enriched with different concentrations of green pepper EO and β-caryophyllene; marinade without test substances served as control samples. The prepared marinade was then poured onto the salmon samples. The pH of marinades was 5.5. The samples were packed in sterile 420 μm amorphous polyethylene terephthalate/polyethylene trays that were wrapped in polyvinyl chloride stretch films (Kraina Foils Packaging, Stanica Poland). All the samples were stored at 4 °C ± 1 °C for 72 h in order to simulate the conditions in the refrigerated chamber during the shelf life of raw salmon sold in shops (72 h).

2.7. Antivirulence properties of green pepper EO and β-caryophyllene against P. aeruginosa KM01 2.7.1. Preparation of cell-free supernatant P. aeruginosa KM01 cultures were grown with and without green pepper EO and β-caryophyllene supplementation at 4 °C ± 1 °C for 72 h. Cell densities were adjusted to an absorbance of 1.0 at a wavelength of 540 nm (OD540). Cultures were then centrifuged at 4000g for 15 min and the supernatants were passed through a 0.22 μm filter to obtain cell-free supernatants for the analysis of virulence factors. Sterile medium and medium containing green pepper EO and β-caryophyllene only served as the controls. 2.7.2. Determination of pyocyanin content Pyocyanin content was quantified as previously described (Huerta, Mihalik, Crixell, & Vattem, 2008). Briefly, P. aeruginosa KM01 was grown in the presence of green pepper EO and β-caryophyllene. Chloroform (Sigma-Aldrich) was added to the cell-free culture supernatant and the optical density of the chloroform layer was measured at a wavelength of 690 nm (OD690). The percentage inhibition of pyocyanin production was calculated with the following formula: 100 − [A/B * 100]

(2)

2.8.2. In-situ antimicrobial and antivirulence assays Verification of P. aeruginosa KM01 growth on cold-stored marinated salmons was carried out every 24 h. For this purpose, the products were aseptically opened and placed in a sterile polyethylene bag (Merck, Darmstadt, Germany). Next, 225 ml of 0.1% sterile peptone water (BD Biosciences) was added to achieve a 1:10 dilution. The samples were

(1)

where A denotes OD690 of the chloroform layer containing the supernatant of P. aeruginosa KM01 grown in TSB medium supplemented with test substance; B denotes OD690 of the supernatant of P. aeruginosa KM01 grown in unsupplemented TSB medium. 8

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caryophyllene (18.8%), limonene (17.5%), and β-myrcene (12.85%) were the most important compounds in green pepper EO, followed by γcarene (9.94%), β-pinene (7.95%), α-phellandrene (7.68%), and αpinene (6.92%). None of the remaining 12 volatile components was present at a concentration greater than 3% (Table 1).

homogenized for 2 min with a Pulsifier (Microgen Bioproducts, Camberley, UK). The suspension was serially diluted and 0.1-ml aliquots were surface-spread in duplicate on Cephaloridine Fucidin Cetrimide agar (Oxoid, Basingstoke, UK) plates that were incubated at 4 °C ± 1 °C for 72 h. The bacterial growth experiments were performed at least three times. Salmon samples treated with a marinate that was not supplemented with the test substances served as controls. The antimicrobial activity of the test substances was calculated with the following formula (4): 100 − [G/H * 100]

3.2. Effects of green pepper EO and β-caryophyllene on the viability and metabolic activity of FHs 74 Int and CCD 841 CoN cells We evaluated the cytotoxicity of green pepper EO and β-caryophyllene in normal human small intestine FHs 74 Int and colon CCD 841 CoN cells treated for 72-h with increasing concentrations of each substance up to 500 and 200 nl ml−1, respectively. The median and lethal inhibitory concentrations (IC50 and IC90, respectively) determined with the MTT assay are shown in Table 2. The IC50 values of green pepper EO in FHs 74 Int and CCD 841 CoN cells were 198 and 180 nl ml−1, respectively; the IC90 values were 338 and 326 nl ml−1, respectively. β-caryophyllene was not toxic to either cell line (IC50 > 200 nl ml−1) (Table 2).

(4)

where G denotes P. aeruginosa KM01 counts in salmon samples with the marinate containing the test substances; H represents P. aeruginosa KM01 counts in the reference samples. No Pseudomonas spp. was detected in the control samples of raw salmon. The marinated salmon samples were also assayed for virulence properties. Pyocyanin production and elastase and protease activities were evaluated as described above. Samples with the marinate without green pepper EO and β-caryophyllene supplementation served as controls.

3.3. MIC To evaluate the antimicrobial effects of green pepper EO and βcaryophyllene against P. aeruginosa KM01, we analyzed microbial growth by measuring the optical density. In this experiment, the concentrations of green pepper EO and β-caryophyllene that were not toxic to FHs 74 Int and CCD 841 CoN cells were screened for their antibacterial activity. Concentrations below the MIC of green pepper EO and β-caryophyllene (120 and 25 nl ml−1, respectively) were used in subsequent experiments, since they were shown to partly inhibit P. aeruginosa KM01 growth (Table 3).

2.9. Statistical analysis Data were analyzed using STATISTICA v.10.0 (StatSoft, Tulsa, OK, USA). Mean differences were evaluated by one-way analysis of variance and Tukey's parametric post hoc test. Results from three independent experiments were used for statistical analysis. P < 0.05 was considered significant. 3. Results

3.4. Effect of green pepper EO and β-caryophyllene on P. aeruginosa KM01 membrane FA composition

3.1. Composition of green pepper EO

The membrane FA composition of P. aeruginosa KM01 cultured in the TSB without the test substances was evaluated (Table 4). The membrane contained 11 FAs constituting three classes of compound: saturated (S)FAs, unsaturated (U)FAs, and hydroxylated (H)FAs. These classes refer to the ratios of (i) dodecanoic acid (C12:0), tetradecanoic acid (C14:0), hexadecanoic acid (C16:0), and heptadecanoic acid (C17:0); (ii) 9-cis-hexadecenoic acid (C16:1 cis-9), linoleic acid (C18:2 cis-9,12), oleic acid (C18:1 cis-9), and elaidic acid (C18:1 trans-9); and (iii) 2-hydroxydodecanoic acid (2-OH-C12:0), 3-hydroxydodecanoic acid (3-OH-C12:0), and 3-hydroxydecanoic acid (2-OH-C16:0). The main membrane FAs of P. aeruginosa KM01 grown in TSB medium without the test substances were C18:2 cis-9,12 (37.7%), followed by C16:1 cis-9 (17.7%), C16:0 (15.9%), and C18:1 trans-9 (11.1%). None of the remaining FAs were present at concentrations exceeding 10% (Table 4). Application of sub-MIC concentrations of green pepper EO and βcaryophyllene to the culture medium reduced the abundance of membrane FAs in P. aeruginosa KM01, especially 3-OH-C12:0, C16:0, 2-OHC16:0, C17:0, and C18:1 trans-9 in cells grown in the presence of green pepper EO; and 3-OH-C12:0, C14:0, C16:0, 2-OH-C16:0, C17:0, and C18:1 cis-9 in cells grown with β-caryophyllene. Green pepper EO increased the abundance of UFAs (from 69.8% to 84.9%) and decreased that of SFAs (from 24.8% to 12.6%) and HFAs (from 5.4% to 2.4%). The increase in UFAs was mainly due to an increased proportion of C16:1 cis-9 and C18:2 cis-9,12, which constituted 22.36% and 52.9%, respectively, of total membrane FAs. The decreases in SFAs and HFAs were mostly due to decreased proportions of C16:0, 3-OH-12:0, and 2OH-16:0. Similar changes were observed in P. aeruginosa KM01 exposed to sub-MIC concentrations of β-caryophyllene, including an increase in UFAs (89.4%) and decreases in SFAs (7.9%) and HFAs (2.8%). The former was mainly due to a high proportion of C16:1 cis-9 (37.7%) and

A total of 19 compounds in green pepper EO accounting for 100% of total EO were identified and quantitatively analyzed (Table 1). Monoterpene and sesquiterpene hydrocarbons were the predominant class of substances (68% and 31.1%, respectively), whereas oxygenated terpenoids (i.e., caryophyllene oxide) constituted only 0.9% of the total. βTable 1 Chemical composition [%] of dried green pepper oil extract. Compound

RI Wax

RI DB-5

Yield percentage (n-3)

α-pinene camphene β-pinene γ-carene β-myrcene α-phellandrene limonene γ-terpinene p-cymene α-terpinolene elemene α-cubebene linalool β-caryophyllene α-humulene β-bisabolene β-edusmene δ-cadinene caryophyllene oxide

939 953 980

1035 1080 1118 1148 1158 1170 1208 1249 1274 1279 1456 1472 1544 1594 1668 1736 1741 1748 1962

6.92 0.78 7.95 9.94 12.85 7.68 17.5 0.96 1.85 0.87 0.93 1.2 0.7 18.8 2.6 2.78 2.54 2.25 0.9

990 1006 1030 1072 1026 1083 1348 1100 1414 1449

1573

Values in bold type correspond to the most abundant constituents. % Composition: percentage composition calculated from the chromatogram obtained on the Supelcowax 10 column (normalized peak area %). RI Wax: retention index on Supelcowax 10 column. RI DB-5: retention index on DB-5 column. 9

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Table 2 IC50 and IC90 for green pepper EO and β-caryophyllene with respect to normal human small intestine FHs 74 Int and colon CCD 841 CoN cell lines. FHs 74 Int IC50 (nl ml Green pepper EO β-caryophyllene

CCD 841 CoN −1

)

IC90 (nl ml

198 ± 8.0 > 200

−1

338 ± 44.0 > 200

)

IC50 (nl ml−1)

IC90 (nl ml−1)

180 ± 2.0 > 200

326 ± 65.0 > 200

Table 3 Antibacterial activity of green pepper EO and β-caryophyllene against P. aeruginosa KM01 (nl ml−1).

Green pepper EO β-caryophyllene

MIC

sub-MIC

140 ± 1.0 30 ± 0.5

120 ± 0.5 25 ± 1.5

Table 4 Membrane fatty acid composition [%] of Pseudomonas aeruginosa KM01 treated with green pepper EO and β-caryophyllene. Fatty acid

C12:0 2-OH-C12:0 3-OH-C12:0 C14:0 Iso-C15:0 Anteiso-C15:0 C16:0 C16:1 cis-9 2-OH-C16:0 C17:0 C18:2 cis-9,12 C18:1 cis-9 C18:1 trans-9

Total composition [%] under different growth conditions TSB medium with green pepper EO

TSB medium with βcaryophyllene

TSB medium

3.8 ± 0.15 2.8 ± 0.11 ND ND ND ND ND 39.5 ± 0.15 ND ND 48.3 ± 0.12 ND 5.6 ± 0.22

10.8 ± 0.49 2.4 ± 0.11 ND 1.8 ± 0.08 ND ND ND 22.3 ± 0.01 ND ND 52.9 ± 0.02 9.7 ± 0.04 ND

4.5 ± 0.35 1.0 ± 0.08 1.4 ± 0.01 1.4 ± 0.01 2.3 ± 0.09 1.8 ± 0.07 14.1 ± 0.01 17.7 ± 0.01 3.0 ± 0.02 3.0 ± 0.02 35.4 ± 0.03 3.3 ± 0.02 11.1 ± 0.08

Fig. 1. The inhibitory potential of green pepper EO and its major constituent βcaryophyllene against pyocyanin biosynthesis by P. aeruginosa KM01. Also shown is the significance of the experimental data determined by ANOVA (p, F). Means with the same letters (a–b) do not differ significantly (p < 0.05).

C18:2 cis-9,12 (46.1%), whereas the latter were attributable to an absence of C14:0, C16:0, 3-OH-C12:0, and 2-OH-C16:0 in the membrane FA profile relative to the control. Additionally, P. aeruginosa KM01 responded to the toxic stress of exposure to a sub-MIC concentration of βcaryophyllene by suppressing the synthesis of 13-methyltetradecanoic acid (iso-C15:0) and 12-methyltetradecanoic acid (anteiso-C15:0) (Table 4).

Fig. 2. Inhibitory potential of green pepper EO and its major constituent βcaryophyllene against elastase activity of P. aeruginosa KM01. Also shown is the significance of the experimental data determined by ANOVA (p, F). Means with the same letters (a–b) do not differ significantly (p < 0.05).

3.5. Effect of green pepper EO and β-caryophyllene on the virulence of P. aeruginosa KM01

points in the presence of green pepper EO and β-caryophyllene, respectively (Fig. 3).

Given that sub-MIC concentrations of green pepper EO and β-caryophyllene altered the ratio of SFAs to UFAs and suppressed anteisoC15:0 production, we speculated that the test substances could also adversely affect the virulence properties of P. aeruginosa KM01. In particular, anteiso branched-chain FAs have been associated with bacterial resistance to abiotic stresses (Sun & O'Riordan, 2010). In this work, we evaluated the effects of green pepper EO and β-caryophyllene on pyocyanin biosynthesis and protease and elastase activities in P. aeruginosa KM01. At sub-MIC concentrations, green pepper EO and β-caryophyllene were highly effective in suppressing pyocyanin production (Fig. 1). At all examined time points, green pepper EO and β-caryophyllene reduced pyocyanin synthesis by over 70% (P < 0.05) and by 60% (P < 0.05), respectively. P. aeruginosa KM01 has high elastase activity, but this was suppressed by 70% and 60% following treatment with green pepper EO and β-caryophyllene, respectively (Fig. 2). Similarly, protease activity declined by over 50% and 30% at all experimental

3.6. Effect of green pepper EO and β-caryophyllene on P. aeruginosa KM01 growth and virulence in salmon samples To extend the results obtained in the in vitro experiments, we evaluated the true effects of green pepper EO and β-caryophyllene on the growth and virulence activity of exogenously provided P. aeruginosa KM01 on model salmon-based products during storage. The results summarized in Table 5 show the inhibitory effect of examined substances on P. aeruginosa KM01 growth. This inhibition was independent of the storage period and equaled 95% for green pepper EO and 90% for β-caryophyllene (P < 0.05) (Table 5). The green pepper EO added to the marinade also attenuated the virulence of P. aeruginosa KM01 on salmon meat, as evidenced by the decreases in pyocyanin production (80%) and protease (90%) and 10

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the antimicrobial activities of EOs applied to food products has not been investigated in detail. In the present study, we explored the antibacterial and antivirulence activities of green pepper EO and its major constituent β-caryophyllene against a food-associated Pseudomonas strain. Since the biological activity of EOs is typically attributed to their major compounds (Ribeiro-Santos, Andrade, Ramos de Melo, & Sanches-Silva, 2017), we first analyzed the chemical profile of green pepper EO extracted from dried, unripe Piper L. fruits from Malaysia. Unripe pepper fruit has been shown to aid in the digestion of food and has antioxidant activity (Srinivasan, 2007). We found that green pepper EO mainly contains monoterpene and sesquiterpene hydrocarbons, with β-caryophyllene, limonene, and β-myrcene being particularly abundant. These results are in agreement with previous studies reporting that β-caryophyllene and limonene are the major volatile constituents of green pepper EO (Nikolić et al., 2015; Orav, Stulova, Kailas, & Müürisepp, 2004). However, only β-caryophyllene shows potent antimicrobial activity, which was demonstrated against food-associated Pseudomonas fluorescens KM06 (Myszka et al., 2017). β-caryophyllene tends to accumulate in the bacterial membrane, which reduces membrane fluidity and permeability (Helander et al., 1998; Sikkema, de Bont, & Poolman, 1994). In contrast, limonene and β-myrcene cannot be incorporated into or alter the properties of bacterial membranes (Hąc-Wydro, Flasiński, & Romańczuk, 2017; Iurescia et al., 1999). Moreover, Pseudomonas spp. are β-myrcene-utilizing bacteria; the myrA, myrB, myrC, and myrD genes encoding alcohol dehydrogenase, aldehyde dehydrogenase, acyl-coenzyme (Co)A synthetase, and enoylCoA-hydratase participate in β-myrcene catabolism in the Pseudomonas M1 strain (Iurescia et al., 1999). Given the low toxicity of synthetic βcaryophyllene to humans (Sitarek et al., 2017), we selected this compound for examination of the antimicrobial activity of green pepper EO. Some EOs can influence mitochondrial enzyme activity and induce injury to eukaryotic cells, leading to their death (Raut & Karuppayil, 2014). They can also damage the mitochondrial membrane by causing their depolarization and increasing their permeability and fluidity (Döll-Boscardin et al., 2012). We therefore investigated the cytotoxicity of green pepper EO and β-caryophyllene on FHS 74 Int and CCD 841 CoN cells, since this is critical for its use as a food biopreservative. To the best of our knowledge, this is the first report evaluating the viability of FHS 74 Int and CCD 841 CoN cells exposed to different concentrations of β-caryophyllene-rich green pepper EO. Previous studies focused on investigating the cytotoxicity of only selected components of EOs, so that they could be applied as references to ensure the safe use of EOs in the food industry. In general, studies on the cytotoxicity of different EOs in cells of the gastrointestinal tract have been scarce (Raut & Karuppayil, 2014; Vigan, 2010). In this work, green pepper EO had a dose-dependent inhibitory effect on the growth of both cell lines, whereas synthetic β-caryophyllene was non-cytotoxic at the tested concentrations (IC50 > 200 nl ml−1). These results are in line with the observations of Sinha, Jothiramajayam, Ghosh, and Mukherjee (2014)

Fig. 3. Inhibitory potential of green pepper EO and its major constituent βcaryophyllene against protease biosynthesis by P. aeruginosa KM01. Also shown is the significance of the experimental data determined by ANOVA (p, F). Means with the same letters (a–b) do not differ significantly (p < 0.05).

Table 5 Growth inhibition [%] of Pseudomonas aeruginosa KM01 in salmon-based products during refrigerated storage. Also shown is the significance of the experimental data determined by ANOVA (p, F). Means with the same letter in column do not differ significantly (p < 0.05). Storage time [h]

24 48 72

Marinade component Green pepper EO [%] (p = 0.929; F = 0.07)

β-caryophyllene [%] (p = 0.906; F = 0.09)

95a ± 4.00 94a ± 1.73 95a ± 1.40

90a ± 4.15 89a ± 3.20 90a ± 1.45

elastase activities (70%). Similar antivirulence activity was exhibited by β-caryophyllene added to the salmon marinade. In this work, pyocyanin production and protease and elastase activities were decreased by 88%, 40%, and 30%, respectively (Table 6). 4. Discussion The acquired resistance of P. aeruginosa to synthetic food preservatives has highlighted the need for novel strategies to control the proliferation of bacteria in foodstuffs. Strategies that perturb bacterial homeostasis mechanisms—for example, by altering the cell membrane and/or extracellular enzyme production—can inhibit cell proliferation (Khan, Tango, Miskeen, Lee, & Oh, 2017); that is, bacteria remain in the lag phase or die before homeostasis can be restored. Thus, the safety and quality of foods can be ensured by targeting bacterial physiology (Burt, 2004). EOs have been demonstrated to exert such effects on foodborne pathogenic/spoilage microorganisms (Burt, 2004), although

Table 6 Inhibition of the virulence activities of Pseudomonas aeruginosa KM01 in salmon-based products during refrigerated storage. Also shown is the significance of the experimental data determined by ANOVA (p, F). Means with the same letter in column do not differ significantly (p < 0.05). Storage time [h]

Marinade component β-caryophyllene [%]

Green pepper EO [%] activity

24 48 72

pyocyanin (p = 0.875; F = 0.135)

elastase (p = 0967; F = 0.340)

protease (p = 0.966; F = 0.330)

pyocyanin (p = 0.744; F = 0.304)

elastase (p = 0.966; F = 0.033)

protease (p = 0.875; F = 0.135)

80a ± 3.8 78a ± 3.0 81a ± 2.9

72b ± 1.5 70b ± 2.0 70b ± 1.5

91c ± 2.1 91c ± 1.9 89c ± 2.0

88a ± 2.0 89a ± 0.5 88a ± 1.5

40b ± 3.5 42b ± 3.9 40b ± 4.7

30c ± 3.0 28c ± 3.0 31c ± 4.0

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virulence in in vitro experiments indicated that sub-MIC concentrations of green pepper EO and synthetic β-caryophyllene are effective antipathogenic agents. These results are in accordance with the finding that mandarin (Citrus reticulata) EO exerts anti-infective effects by suppressing the production of elastase in P. aeruginosa (Luciardi, Blázquez, Cartagena, Bardón, & Arena, 2016), and may thus be a suitable agent for reducing P. aeruginosa pathogenicity in food products. Also, exposure of P. aeruginosa to rosemary and cinnamon EOs showed a significant reduction in pyocyanin production and protease activity, as demonstrated by Araby and El-Tablawy (2016) and Kalia et al. (2015). Based on the results of in vitro assays, in this work the sub-MIC concentrations of green pepper EO and β-caryophyllene toward P. aeruginosa KM01 were further tested in in situ experiments. The antimicrobial and antivirulence effects were monitored against P. aeruginosa KM01 inoculated into marinated fish. In this work, pre-screening of the salmon samples revealed no pre-existing P. aeruginosa. In our study, the marinade was acidified with vinegar according to Negi (2012), who reported higher bioactivity of EOs upon acidic pH; at low pH, EOs behave in a more hydrophobic manner and their entry into cells is easier. Although vinegar has been used as a food preservative for millennia, it cannot limit the growth of acid-tolerant P. aeruginosa. The natural habitats of P. aeruginosa span the pH range of 4.5–9.5; P. aeruginosa synthesizes significant amounts of 2’ alanyl-phosphatidylglycerol (A-PG) in response to acidic conditions, which is necessary to maintain the membrane lipid homeostasis (Klein et al., 2009). In the in situ experiments, addition of the green pepper EO and βcaryophyllene managed the bacterial growth and suppressed production of all tested virulence factors. Similar bactericidal trends against common meat contaminants were revealed for juniper and winter savory EOs introduced to beef marinade (Vasilijević et al., 2019). Red wine marinade supplemented with an MIC concentration of EO remarkably decreased the counts of Enterobacteriaceae and lactic acid bacteria (Vasilijević et al., 2019). Because β-caryophyllene-rich green pepper EO exhibited notable antibacterial and antivirulence effects in marinated salmon, further investigation of its biopreservative properties should be carried out. Taking into account that micro- and nanoemulsions have been suggested as tools that transport active components of EOs to food and enhance the functional properties of treated products, their applications with β-caryophyllene-rich green pepper EO in the food industry should be considered, to increase and prolong the antibacterial and antivirulence activity of green pepper EO.

and Raut and Karuppayil (2014), who also stated that low doses of EOs might be safe in humans. Sinha et al. (2014) revealed that the palmarosa, citronella, and lemongrass EOs at concentrations below 100 μg ml−1 do not modify the mitochondrial activity of lymphocytes and do not generate reactive oxygen species (ROS), considered as a critical factor responsible for cell transformation. Similarly, Dahham et al. (2015) evaluated the effect of β-caryophyllene from Aquilaria crassna EO on a panel of normal human cell lines and observed that it was safe to use in 3T3-L1 and RGC-5. The photomicrographic images displayed that β-caryophyllene did not affect the morphology of the normal cell lines (Dahham et al., 2015). In some cases, high concentrations of green pepper EO can impart undesirable sensory properties to the treated food and elicit allergic reactions (Hassoun & Çoban, 2017). Green pepper EO is characterized by a strong odor and flavor that can potentially leave a bad aftertaste, which could diminish its acceptance as a food ingredient (Myszka, Leja, & Majcher, 2019). We therefore examined the antibacterial activities of green pepper EO and β-caryophyllene at sub-MICs and found that they suppressed the growth of P. aeruginosa KM01 at concentrations of 120 and 25 nl ml−1, respectively; moreover, the FA composition of the bacterial cell membrane was altered by these treatments, with notable increases in UFAs including C16:1 cis-9 and C18:2 cis-9,12 and reductions in SFAs including C14:0, C16:0, 3-OH-C12:0, and 2-OH-C16:0 in the membrane. This is consistent with the observation that exposure of Listeria monocytogenes, Escherichia coli, and Salmonella enteritidis to EOs and their constituents increased UFAs and decreased SFAs in membrane FA profiles (Di Pasqua, Hoskins, Betts, & Mauriello, 2006; Siroli, Patrignani, Gardini, & Lanciotti, 2015). C18:2 abundance may be increased in bacteria in response to toxic stress caused by EOs as a selfdefense mechanism (Luz et al., 2014). In general, a higher percentage of UFAs increases membrane fluidity, while straight-chain SFAs are more densely packed and thus have the opposite effect (Wang, Zeng, Wang, Brennan, & Gong, 2018). A high SFA content in the cytoplasmic membrane of bacteria is essential for maintaining normal cellular functions, including cell division and diffusion (Wang et al., 2018). The present findings also show that P. aeruginosa KM01 responds effectively to the toxic stress induced by green pepper EO and β-caryophyllene by suppressing synthesis of iso- and anteiso-C15:0. The lack of anteisoC15:0, which has a much lower phase transition temperature (25.8 °C) than other FAs with the same number of carbon atoms (Rogiers, Kebede, van Loey, & Michiels, 2017) can explain the change in bacterial membrane fluidity—and consequently, growth inhibition—in P. aeruginosa KM01 in the presence of green pepper EO and β-caryophyllene. We also examined whether sub-MIC concentrations of green pepper EO and β-caryophyllene can diminish the virulence of foodborne P. aeruginosa KM01, as reflected by the production of pyocyanin and elastase and alkaline protease activities. Pyocyanin, the virulence factor of P. aeruginosa cells, plays a major role in chronic infections in humans (Oliver, Cantón, Campo, Baquero, & Blázquez, 2000). It is believed that the low molecular weight and zwitterionic properties of pyocyanin allow the toxin to easily permeate through cell membranes (Hall et al., 2016). Alkaline protease and elastase of P. aeruginosa inactivate human γ-interferon, a key factor for innate and adaptive immunity against bacterial infections. A lack of γ-interferon results in autoinflammatory and autoimmune diseases (Hoge, Pelzer, Rosenau, & Wilhelm, 2010). Both cytokines of P. aeruginosa are able to inhibit the function of neutrophils, especially interfering with their chemotaxis, which gives the bacterium an advantage in escaping phagocytes of the host defense system (Hoge et al., 2010). Moreover, elastase can destroy host cells/ tissues, impairing the permeability barrier and inhibiting protein production to promote cell death (Fata Moradali, Ghods, & Rehm, 2017). It was demonstrated that P. aeruginosa isolated from foods of animal origin harbor genes encoding exo-proteases; a high prevalence of mostly the lasB gene (89%) encoding elastase was detected in 204 foodborne isolates (Benie et al., 2017). In the present study, the observed suppression of P. aeruginosa KM01

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