Combined Effect of Vacuum Packaging, Fennel and Savory Essential Oil Treatment on the Quality of Chicken Thighs

microorganisms

Article Combined Eﬀect of Vacuum Packaging, Fennel and Savory Essential Oil Treatment on the Quality of Chicken Thighs

Miroslava Kaˇcániová 1,2,* , Martin Mellen 3, Nenad L. Vukovic 4, Maciej Kluz 2, Czeslaw Puchalski 2, Peter Hašˇcík 5 and Simona Kunová 6

1 Department of Microbiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, 949 76 Nitra, Slovak Republic 2 Department of Bioenergy Technology and Food Analysis Faculty of Biology and Agriculture, University of Rzeszow, 35-601 Rzeszow, Poland; [email protected] (M.K.); [email protected] (C.P.) 3 Klas Holding, s.r.o., 984 01 Luˇcenec,Slovakia; [email protected] 4 Department of Chemistry, Faculty of Science, University of Kragujevac, P.O. Box 60, 34000 Kragujevac, Serbia; [email protected] 5 Department of Technology and Quality of Animal Products, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, 949 76 Nitra, Slovak Republic; [email protected] 6 Department of Food Hygiene and Safety, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, 949 76 Nitra, Slovak Republic; [email protected] * Correspondence: [email protected]

Received: 8 April 2019; Accepted: 9 May 2019; Published: 15 May 2019

Abstract: The aim of the present work was to evaluate the microbiological quality of chicken thighs after treatment by fennel (Foeniculum vulgare) and savory (Satureja hortensis) essential oil, stored under vacuum packaging (VP) at 4 0.5 C for a period of 16 days. The following treatments of ± ◦ chicken thighs were used: Air-packaging control samples (APCS), vacuum-packaging control samples (VPC), vacuum-packaging (VP) control samples with rapeseed oil (VPRO), VP (vacuum-packaging) with fennel essential oil at concentrations 0.2% v/w (VP + F), and VP with savory essential oil at concentration 0.2% v/w (VP + S). The quality assessment of APCS, VPC, VPRO, VP + F and VP + S products was established by microbiological analysis. The microbiological parameters as the total viable counts of bacteria of the Enterobacteriaceae family, lactic acid bacteria (LAB), and Pseudomonas spp. were detected. Bacterial species were identified with the MALDI-TOF MS Biotyper. The combination of essential oils and vacuum packaging had a significant effect (p < 0.05) on the reduction of total viable counts (TVC) compared with control group without vacuum packaging and the untreated control group. Though 15 genera and 46 species were isolated with scores higher than 2.3 from the chicken samples.

Keywords: Foeniculum vulgare; Satureja hortensis; chicken thigh; microﬂora

1. Introduction The number of reported cases of food - associated infections has increased rapidly so it is very important to focus on food safety from both consumers and food industry point of view [1]. One of very popular food commodity in recent decades in many countries is poultry meat, which is a highly perishable food providing an almost perfect medium for microbial growth, including both spoilage and pathogenic microorganisms [2,3]. One of the possibilities to prolong the shelf-life of the meat is application of natural preservatives, including oil extracts of herbs and spices [4]. The oil s are natural products extracted from plants and can be used as natural additives for food because of

Microorganisms 2019, 7, 134; doi:10.3390/microorganisms7050134 www.mdpi.com/journal/microorganisms Microorganisms 2019, 7, 134 2 of 10 their antibacterial, anti-fungal, antioxidant and anti-carcinogenic properties. [5–7]. Essential oils have been proven to show an inhibitory effect against a wide range of food spoiling microbes; however, the level of this effect depends on their concentration, method of testing, and the active constituents presented [8,9]. The antimicrobial activity of essential oils is the consequence of the presence of small terpenoids and phenolic compounds (thymol, carvacrol, eugenol) [10–12]. Foeniculum vulgare Mill. (fennel) is a biennial medicinal plant belonging to the family Apiaceae. The essential oil of fennel is used as flavoring agents in food products. The main components of fennel essential oil are trans-anethole, fenchone, and estragole [13–15]; among them, trans-anethole exhibits both antioxidants and antimicrobial activities [16]. In addition, Patra et al. [17] reported that anethole and its isomers are responsible for the antimicrobial activities of fennel oil. Satureja hortensis L. (summer savory) is a well-known aromatic and medicinal plant. Some parts of the Satureja hortensis plant such as leaves, flowers, and stem are frequently used for tea or as an additive in commercial spice mixtures for a lot of food and give the food the aroma and flavour [18–21]. Thymol, p-cymene, γ-terpinene, and carvacrol are the main components of savory oil, exhibiting strong biological and antimicrobial activity [22–25]. In this study, we aimed to investigate the effect of (Foeniculum vulgare) and savory (Satureja hortensis) essential oils on the shelf-life extension of fresh chicken thighs stored under vacuum packaging at 4 0.5 C for a period of 16 days. ± ◦ 2. Material and Methods

2.1. Preparation of Essential Oils for Testing and Their Chemical Composition The dried medicinal plants for essential oil isolation were donated by established growers. Essential oils were distilled in the large-scale distillation apparatus speciﬁcally designed for aromatic and medicinal plants. The apparatus consisted of the main distillatory apparatus, a steam condenser, a steam boiler, and an apparatus for the improvement of used water. There were two types of equipment—an HV-3000 with a height of 5250 mm, a width of 2180 mm, and a container for 200–250 kg of dried plant material and an HV-300 with a height of 3400 mm, a width of 1300 mm, and a container for 40–50 kg of dried plant material. An analysis of the essential oils was carried out with Hewlett-Packard 5890/5970 GC-MSD system. A chemical composition of the essential oils of Foeniculum vulgare Mill. was: t-anethol (32.8%), and of essential oil Satureja hortensis were carvacrol (45.2%), γ-terpinene (30.5%), α-terpinene (3.2%), and p-cimene (2.3%).

2.2. Preparation of Samples Rapeseed oil with any microorganisms was used as a solvent for concentration of essential oils. Rapeseed oil has good properties, and it is safe for human health. A vacuum packaging machine—type VB-6 (RM Gastro, Czech Republic)—was used for packaging. Fennel and savory essential oils (Calendula, Nova Lubovna, Slovakia) were transferred onto chicken breast with a micropipette to completely cover the surface of both sides of the meat. 0.2% v/w of fennel (Foeniculum vulgare) and savory (Satureja hortensis) essential oils were used for treatment. Both essential oils were prepared for a concentration of 0.2 % v/w in rapeseed oil (Brassica napus) purchased in a Slovak market. Fresh chicken thigh meat (cca. 300 g, skinless and boneless ﬁllet) was purchased from a local poultry processing factory within 1 h after slaughter and transferred to the laboratory of Slovak University of Agriculture, Nitra, Slovakia, in insulated polystyrene boxes on ice. The chicken meat was kept at 4 ◦C until the testing was initiated. In each group were three samples for microbiological analysis. Each experiment was replicated twice. The fresh chicken thigh samples were prepared as follows: Microorganisms 2019, 7, 134 3 of 10

Air-packaged control samples (APCS): Fresh chicken thigh meat was packed into polyethylene bags (bags for food packaging with low permeability, as well as gas and vapor protection) and stored aerobically at 4 ◦C. Vacuum-packaged control samples (VPC): Fresh chicken thigh meat was packed into polyethylene bags and stored at 4 ◦C anaerobically in the vacuum. Vacuum-packed control samples (VP) with rapeseed oil (VPRO): Fresh chicken thigh meat was treated with rapeseed oil for 1 min, packed in the polyethylene bags, and stored at 4 ◦C anaerobically in the vacuum. VP with fennel oil 0.20% v/w (VP + F): Fresh chicken thigh meat was treated with fennel oil for 1 min, packed to polyethylene bags, and stored at 4 ◦C anaerobically in the vacuum. VP with savory oil 0.20% v/w, (VP + S): Fresh chicken thigh meat was treated with savory oil for 1 min, packed to polyethylene bags, and stored at 4 ◦C anaerobically in the vacuum; Each sample was packaged immediately after treatment by using a vacuum packaging machine—type VB-6 (RM Gastro, Czech Republic)—and kept at 4 C 0.5 C for 16 days. ◦ ± ◦ 2.3. Microbiological Analysis 10 g of chicken thigh was sampled with sterile scalpels and forceps and immediately transferred into a sterile stomacher bag containing 90 mL of 0.1% buffered peptone water (BPW, pH 7.0, Oxoid, Basingstoke, UK); the sample was then homogenized for 60 s in a stomacher at room temperature. Sampling was carried out at on day 0, 4, 8, 12, and 16 of the experiment For each sample, appropriate serial decimal dilutions were prepared in 0.1% BPW solution. The amount of 0.1 mL of serial dilutions of prepared samples was spread on the surface of dry media. Total viable counts (TVC) were counted on a Plate Count Agar (PCA, Merck, Darmstadt, Germany) after incubation for 3 days at 30 ◦C, aerobically. The number of pseudomonads were determined on a Cephaloridine Fucidin Cetrimide agar (Oxoid, supplemented with SR 103, Basingstoke, UK) after incubation at 25 ◦C for 2 days, aerobically. For the detection of Enterobacteriaceae, 15 mL of molten (45 ◦C) Violet Red Bile Glucose Agar (Oxoid) was inoculated with 1.0 mL of the sample. Incubation was carried out at 37 ◦C for 24 h, aerobically. The number of lactic acid bacteria (LAB) were determined on a Man Rogosa Sharpe agar (Oxoid) after incubation at 25 ◦C for 5 days, anaerobically. Then, the agars were evaluated for bacterial growth, and 8 colonies (depending on the different morphological characteristics of colonies) per plate were selected for further confirmation with the MALDI-TOF MS Biotyper (Bruker Daltonics, Germany).

2.4. Mass Spectrometry Identification of Isolates The qualitative analysis of microbial isolates was performed with MALDI-TOF Mass Spectrometry (Bruker Daltonics, Germany). Isolates from the agar were transferred into 300 µL of distilled water. Then, a quantity of 900 µL of ethanol was added, and the tubes with bacterial suspension in water were centrifuged for 2 min at 14,000 npm. The supernatant was discarded, and the pellet was centrifuged repeatedly. After the remaining ethanol was removed, the pellet was allowed to dry. An amount of 10 µL of 70% formic acid was mixed with the pellet, and a 10 µL of acetonitrile was added. Tubes were centrifuged for 2 min at 14,000 npm, and 1 µL of the supernatant was used for MALDI identification. Once dry, every spot was overlaid with 1 µL of the α-Cyano-4-hydroxycinnamic acid (HCCA) matrix and left to dry at room temperature before analysis. Generated spectra were analyzed on a MALDI-TOF Microflex LT (Bruker Daltonics) instrument using Flex Control 3.4 software and Biotyper Realtime Classification 3.1 with BC-specific software. Criteria for reliable identification were a score of 2.0 at ≥ species level and 1.7 at genus level. ≥ 2.5. Statistical Analysis The statistical processing of the data obtained from each evaluation was implemented by means with Statgraphics Plus version 5.1 (AV Trading, Umex, Dresden, Germany). For each replication, the The antimicrobial activity of essential oil has been known for many centuries [26]. The total viable count (TVC) values for the tested groups of chicken thigh are shown in Figure 1. The initial TVC value of chicken thigh was 3.43 ± 0.33 log cfu/g on day 0. A similar result was observed by Ismail et al. [27], who reported mean TVC populations of 3.32–5.77 log cfu/g for various raw and processed chicken products. We observed the highest TVC, 4.58 ± 0.48 log cfu/g, in APCS on day eight, and the lowest TVC, 3.09 ± 2.39 log cfu/g, was found in VPC samples after day 16 of storage at 4 ± 0.5 °C. In samples treated with essential oils, the lowest TVC, 3.43 ± 0.33 log cfu/g, was found on day 0. For VP Microorganisms 2019, 7, 134 4 of 10 + F samples, the highest TVC, 4.57 ± 0.30 log cfu/g, was found on day 16; for VP + S samples, the highest TVC, 4.42 ± 0.41 log cfu/g, was found on day 1. Statistically significant differences (p < 0.05) weremean found was calculated,between AP andCS and the dataVPC, wereAPCS log and transformed. VPRO, APCS The and Student’s VP + F, VPCt-test and was VP performed + S, VPC and for VPstatistical + F, and analysis. VPRO and Diff VPC.erences Rapeseeds were evaluated oil treatments as significant did not if affectp < 0.05; the ptotal< 0.01; viablep < count0.001. (TVC). Our results were in agreement with the findings of Cosby et al. [28], Branen and Davidson [29], and Belfiore3. Results [30], and who Discussion did not find an effect of a disodium ethylenediametetra-acetate (EDTA) and nisin (NIS)The combination antimicrobial and activitystorage ofunder essential modified oil has atmosphere been known packaging for many (MAP) centuries or [vacuum26]. The totalpackaging viable (VP)count on (TVC) the TVC values in their for thestudy. tested Dawson groups et al. of [31] chicken reported thigh a arereduction shown in in growth Figure1 of. Theaerobic initial bacteria TVC byvalue 1–1.5 of chicken log cfu/g thigh in ground was 3.43 chicken0.33 log meat cfu after/g on 14 day days 0. A of similar storage result under was modified observed atmosphere by Ismail et ± packaging.al. [27], who The reported data of meanthe study TVC Adiguzel populations et al. of [32] 3.32–5.77 clearly indicated log cfu/g forthat various the essential raw and oil of processed Satureja hortensischicken products.plant has Westrong observed antimicrobial the highest activity TVC, against 4.58 0.48 both log bacteria cfu/g, inan APCSd fungi on, daysince eight, 25 bacteria, and the ± eightlowest fungi, TVC, and 3.09 one2.39 yeast log species cfu/g, were was foundinhibited. in VPC In the samples study afterby Mahboubi day 16 of and storage Kazempour at 4 0.5 [33]C. ± ± ◦ thymol,In samples p-cymene, treated with γ-terpinene essential, and oils, carvacrol the lowest were TVC, the 3.43 main0.33 components log cfu/g, was of S. found hortensis on day oil 0., and For ± thymolVP + F samples,played an the important highest TVC, role 4.57in pro0.30viding log antimicrobial cfu/g, was found activity. on day Many 16; forauthors VP + Sreported samples, that the ± fennelhighest essential TVC, 4.42 oil has0.41 good log antimicrobial cfu/g, was found properties on day [14,34] 1. Statistically, while Miquel significant et al. [35] diff founderences that (p fennel< 0.05) ± essentialwere found oils between exhibited APCS very andlow VPC,antimicrobial APCS and activity. VPRO, Our APCS results and VPof the+ F, TVC VPC are and in VPagreement+ S, VPC with and whatVP + hasF, and been VPRO previo andusly VPC. reported Rapeseeds and have oil treatments shown that did the not treatment affect the was total successful viable count for VP+F. (TVC).

5 4.5 4 3.5 3 U/g 2.5

log CF 2 1.5 1 0.5 0 D0 D4 D8 D12 D16 Day of storage

Figure 1. Changes (log cfu/g) in population of Total Viable Count (TVC) in chicken thigh stored in air Figure(APCS, 1. );Changes stored under(log cfu/g) vacuum in population (VPC, ); storedof Total under Viable vacuum Count packaging(TVC) in chicken with rapeseed thigh stored oil (VPRO, in air (AN);P storedCS, ♦ under); stored vacuum under packaging vacuum (VPC,with Foeniculum ■); stored vulgare under 0.2% vacuum essential packaging oil (VP with+ F, rapeseed); and stored oil × (VPRO,under vacuum ▲); stored packaging under withvacuumSatureja packaging hortensis with0.2% Foeniculum essential oilvulgare (VP + 0.2%S, ).essential Each point oil (VP is the + meanF, ×); andof three stored samples under takenvacuum from packaging two replicate with experimentsSatureja hortensis (n = 30.2%2 =essential6). Error oil bars (VP show + S, ● SD.). Each point × is the mean of three samples taken from two replicate experiments (n = 3 × 2 = 6). Error bars show SD. Our results were in agreement with the findings of Cosby et al. [28], Branen and Davidson [29], and BelfioreThe processing [30], who and did storage not find of an meat effect prior of a disodiumto consumption ethylenediametetra-acetate can have a significant (EDTA) effect andon meat nisin quality(NIS) combination [36]. Members and of storage the Enterobacteriaceae under modified on atmosphere raw beef, lamb, packaging pork, and (MAP) poultry or vacuum products, packaging as well as(VP) on onoffal the [37] TVC, were in their identified study. in Dawson the previous et al. [study31] reported [38]. In aour reduction study Enterobacteriaceae in growth of aerobic as a hygiene bacteria indicatorby 1–1.5 log[39] cfu, counts/g in ground on day chicken 0 were meat 3.75 after± 0.46 14 log days cfu/g of storage(Figure under 2). On modified day 16 atmosphere of storage, Enterobacteriaceaepackaging. The data reached of the 4.87 study ± 0.04 Adiguzel log cfu/g et al. in [32 A]P clearlyCS sample indicateds. In the that case the essentialof VPC, oilthe of countSatureja of Enterobacteriaceaehortensis plant has was strong lowest antimicrobial at 3.75 ± 0.46 activity log cfu/g against on both day bacteria0 and highest and fungi, at 4.88 since ± 0.01 25 bacteria, log cfu/g eight on fungi, and one yeast species were inhibited. In the study by Mahboubi and Kazempour [33] thymol, p-cymene, γ-terpinene, and carvacrol were the main components of S. hortensis oil, and thymol played an important role in providing antimicrobial activity. Many authors reported that fennel essential oil has good antimicrobial properties [14,34], while Miquel et al. [35] found that fennel essential oils exhibited very low antimicrobial activity. Our results of the TVC are in agreement with what has been previously reported and have shown that the treatment was successful for VP+F. day eight. The counts of Enterobacteriaceae in VPRO was highest at 0.43 ± 1.06 log cfu/g on day 16 and lowest at 3.75 ± 0.46 log cfu/g on day 0. For VP + F, the lowest and highest Enterobacteriaceae counts were from 2.55 ± 1.99 log cfu/g on day 16 to 3.75 ± 0.64 log cfu/g on day 0. For VP + S, the lowest and highest counts were from 0.67 ± 1.03 log cfu/g on day eigh to 3.75 ± 0.64 log cfu/g on day 0. Statistically significant differences (p < 0.05) were found between all tested groups without VP + F, VP + S, APCS, and VPC. MicroorganismsThe essential2019, 7 ,oils 134 and their components are known because of their activity against a wide5 of 10 variety of microorganisms including gram-negative and gram-positive bacteria. There were found out thatThe gram processing - negative and bacteria storage ofare meat more prior resistant to consumption to the antagonistic can have effects a significant of essential effect oils on than meat gramquality-positive [36]. Members ones. It can of the beEnterobacteriaceae connected with on theraw structure beef, lamb, of the pork, outer and membrane, poultry products, where no as lipopolysaccharidewell as on offal [37 ],are were present. identified F. vulgare in the seed previous extracts study and [oil38]. are In rich our studyin transEnterobacteriaceae-anethole and otheras a compounds,hygiene indicator and they [39], have counts positive on day effect 0 were against 3.75 C.0.46 albicans log cfu, E./g coli (Figure, P. putida2). On, andday 16 other of storage, similar ± organismsEnterobacteriaceae [14]. Mihajilovreached-Krstev 4.87 et0.04 al. log[40] cfu reported/g in APCS that samples.E. coli O157:H7, In the caseS. typhimurium of VPC, the, S. count aureus of ± andEnterobacteriaceae L. monocytogeneswas are lowest inhibited at 3.75 with0.46 S. hortensis log cfu/g essential on day 0 oil. and Essential highest atoils 4.88 of S.0.01 hortensis log cfu show/g on ± ± wideday eight. spectrum The counts and inhibition of Enterobacteriaceae effect againstin VPRO growth was of highest the human at 0.43 and1.06 phytopatogenic log cfu/g on day and 16 food and ± spoilagelowest at bacteria, 3.75 0.46 fungi log and cfu /yeastsg on day species. 0. ± 6

2 log CFU/g

0 D0 D4 D8 D12 D16 -1 Day of storage

Figure 2. Changes (log cfu/g) in population of Enterobacteriacae in chicken thigh stored in air (APCS, ); Figurestored 2. under Changes vacuum (log (VPC,cfu/g) in); population stored under of vacuumEnterobacteriacae packaging in withchicken rapeseed thigh oilstored (VPRO, in airN );(A storedPCS, ♦under); stored vacuum under packaging vacuum with(VPC,Foeniculum ■); storedvulgare under 0.2%vacuum essential packaging oil (VP with+ F, rapeseed); stored oil under (VPRO, vacuum ▲); × storedpackaging under with vacuumSatureja packaging hortensis 0.2%with essentialFoeniculum oil (VPvulgare+ S, 0.2%). Eachessential point oil is the(VP mean + F, × of); stored three samples under vacuumtaken from packaging two replicate with Satureja experiments hortensis (n =0.2%3 essential2 = 6). Error oil (VP bars + showS, ●). Each SD. point is the mean of three × samples taken from two replicate experiments (n = 3 × 2 = 6). Error bars show SD. For VP + F, the lowest and highest Enterobacteriaceae counts were from 2.55 1.99 log cfu/g on day ± 16 toLactic 3.75 acid0.64 bacteria log cfu/g (LAB) on day, a 0.facultative For VP + S,anaerobe the lowest, are and recognized highest counts as an important were from competitor 0.67 1.03 logof ± ± othercfu/g spoilage on day eigh related to 3.75 microbial0.64 groups log cfu /underg on day VP/ 0.modified Statistically atmosphere significant packaging differences (MAP (p <) conditions0.05) were ± [38,41found]. betweenThe results all testedof Ntzimani groups [42] without indicate VP +thatF, VP LAB+ S,are APCS, an important and VPC. part of precooked chicken microfloraThe essential, irrespective oils and of theirthe packaging components conditions are known and because the antimicrobial of their activity treatment against acombination. wide variety Thisof microorganisms could contribute including to their rapid gram-negative growth between and gram-positive days 0 and two bacteria. of storage, There similar were to found LAB outgrowth that ingram beef - stored negative under bacteria MAP are at more5 °C [43] resistant. Dominant to the antagonisticLAB bacterial eff speciesects of essentialin pre-cooked oils than chicken gram-positive product storedones. Itincan air and be connected MAP are reported with the structureas being the of thesignificant outer membrane, part of natural where microflora no lipopolysaccharide of freeze-chilled are chickenspresent. F.with vulgare numberseed of extracts 6.1–7.5 and log oilcfu/g are [4 rich4,45 in]. trans-anethole and other compounds, and they have positiveThe einitialffect against numberC. of albicans LAB ,countsE. coli, P.(Figur putidae ,3) and was other 3.95 similar ± 0.18 organismslog cfu/g (day [14]. 0). Mihajilov-Krstev The number of et LABal. [40 in] AC reported ranged that fromE. coli3.45O157:H7, ± 0.46 logS. cfu/g typhimurium on day 16, S. to aureus 4.06 ±and 0.08L. log monocytogenes cfu/g on dayare four inhibited. In the case with VPCS. hortensis, the highestessential count oil. of Essential LAB 4.31 oils ± 0.29 of S. log hortensis cfu/g wasshow marked wide spectrum on day 16 and of storage inhibition, and eff theect lowes againstt 3.69growth ± 0.08 of log the humancfu/g on and day phytopatogenic 12. In VPRO, VP+F and, food and spoilageVP+S, the bacteria, highest fungiLAB c andounts yeasts of 3.95 species. ± 0.18 log cfu/g Lacticwere observed acid bacteria on day (LAB), 0, and a facultative the lowest anaerobe, comprised are 3.35 recognized ± 0.21 on asday an 12, important 3.39 ± 0.48 competitor on day 16 of, andother 3.28 spoilage ± 0.39 relatedlog cfu/g microbial on day groupseight for under each VP group,/modified respectively. atmosphere packaging (MAP) conditions [38, 41]. The results of Ntzimani [42] indicate that LAB are an important part of precooked chicken microflora, irrespective of the packaging conditions and the antimicrobial treatment combination. This could contribute to their rapid growth between days 0 and two of storage, similar to LAB growth in beef stored under MAP at 5 ◦C[43]. Dominant LAB bacterial species in pre-cooked chicken product stored in air and MAP are reported as being the significant part of natural microflora of freeze-chilled chickens with number of 6.1–7.5 log cfu/g [44,45]. Microorganisms 2019, 7, 134 6 of 10

The initial number of LAB counts (Figure3) was 3.95 0.18 log cfu/g (day 0). The number of LAB ± in AC ranged from 3.45 0.46 log cfu/g on day 16 to 4.06 0.08 log cfu/g on day four. In the case ± ± VPC, the highest count of LAB 4.31 0.29 log cfu/g was marked on day 16 of storage, and the lowest ± 3.69 0.08 log cfu/g on day 12. In VPRO, VP+F, and VP+S, the highest LAB counts of 3.95 0.18 log ± ± cfu/gStatistically were observed significant on day differences 0, and the lowest(p < 0.05) comprised were found 3.35 between0.21 on AC day and 12, VPRO, 3.39 0.48 AC onand day VP 16, + ± ± S,and VPC 3.28 and 0.39VP + log S, VPC cfu/g and on day VP eight+ F, and for VPRO each group, and VPC. respectively. ± 6

3 log CFU/g 2

0 D0 D4 D8 D12 D16 Day of storage

Figure 3. Changes (log cfu/g) in population of lactic acid bacteria (LAB) in chicken thigh stored in air Figure(APCS, 3. );Changes stored under(log cfu/g) vacuum in population (VPC, ); storedof lactic under acid vacuumbacteria packaging(LAB) in chicken with rapeseed thigh stored oil (VPRO, in air (AN);P storedCS, ♦ under); stored vacuum under packaging vacuum with (VPC,Foeniculum ■); stored vulgare under0.2% vacuum essential packaging oil (VP + withF, ); rapeseed stored under oil × (VPRO,vacuum ▲ packaging); stored withunderSatureja vacuum hortensis packaging0.2% essentialwith Foeniculum oil (VP + vulgareS, ). Each0.2% pointessential is the oil mean (VP of+ F, three ×); storedsamples under taken vacuum from two packaging replicate with experiments Satureja hortensis (n = 3 0.2%2 = 6). essential Error bars oil (VP show + S, SD. ●). Each point is the × mean of three samples taken from two replicate experiments (n = 3 × 2 = 6). Error bars show SD. Statistically significant differences (p < 0.05) were found between AC and VPRO, AC and VP + S, VPCDifferent and VP + essentialS, VPC andoils, VP such+ F,as andoregano VPRO, were and applied VPC. to control the growth of LAB. Oregano oil with aDi concentrationfferent essential of 1% oils, was such more as oregano,effective werethan the applied concentration to control of the 0.1% growth in regard of LAB.s to reducing Oregano theoil with populations a concentration of LAB, ofwhile 1% thewas combination more effective of than(modified the concentration atmosphere packaging) of 0.1% in regardsMAP and to oreganoreducing oil the at populationsa concentration of LAB, 1% had while the the greatest combination effect, as of described (modified by atmosphere Chouliara packaging)et al. [2]. Kivanc MAP etand al. oregano[46] reported oil at that a concentration oregano and 1%its hadessential the greatestoil inhibited effect, the as growth described of Lactobacillus by Chouliara plantarum et al. [2,]. butKivanc oregano et al. spice [46] reported stimulated that Lactobacillus oregano and plantarum its essential acid production.oil inhibited Lactobacillus the growth plantarumof Lactobacillus in a liquidplantarum medium, but oregano stimulated spice its stimulated growth andLactobacillus acid production plantarum of cuminacid production. at concentrationLactobacillus 0.5, 1.0 plantarum and 2.0 %in ( aw liquid/w). Lactobacillus medium stimulated plantarum its with growth acid andproduction acid production is inhibited of cumin with atessential concentration oil from 0.5, cumin 1.0 and in h2.0igh % concentration (w/w). Lactobacillus (300 and plantarum 600 ppm).with In acid our production study, the iscount inhibited of Pseudomonas with essential spp. oil was from found cumin only in ohighn day concentration 0, with a number (300 and of 3.43 600 log ppm). cfu/g In in our all study, samples the.count of Pseudomonas spp. was found only on day 0,Together, with a number 15 genera of 3.43 were log cfu identified/g in all, samples.and they were Aeromonas, Aromatoleum, Buttiauxella, ClostridiumTogether,, Enterobacter 15 genera, wereHafnia identified,, Lactobacillus and, theyLysinobacillus were Aeromonas, Pantotea, Aromatoleum, Pseudomonas, Buttiauxella, Rahnella, , RaoultellaClostridium, Serratia, Enterobacter, Staphylococcus, Hafnia, Lactobacillus, and Yersina, Lysinobacillus. In our study, Pantotea, only microorganisms, Pseudomonas, Rahnella identified, Raoultella with a , scoreSerratia of, Staphylococcusmore than two, andwereYersina mentioned.. In our A study, total onlyof 239 microorganisms colonies were identifiedidentified with with a this score score of more for bacterialthan two species were mentioned. identification. A total From of 239fifteen colonies genera were, 46 identifiedspecies were with isolated. this score Within for bacterial the Aeromonas species genusidentification., A. veronii From was fifteen the most genera, frequently 46 species isolated were, isolated.while Clostridium Within the novyiAeromonas (4%) was genus isolated, A. veronii less frequentlywas the most (Table frequently 1). Among isolated, LAB, while onlyClostridium Lactobacillus novyi spp.(4%) were was isolated isolated with less frequently12 species. (Table Genus1). PseudomonasAmong LAB, was only representedLactobacillus spp. with were9 species isolated, and with P. 12 flurorescens species. Genus (80%),Pseudomonas P. gessardiiwas (80 represented%), and P. proteoliticawith 9 species, (80%) and wereP. flurorescens among the(80%), mostP. abundant. gessardii (80%), Enterobacteriac and P. proteoliticaeae were(80%) comprised were amongof Buttiauxella the most, Enterobacterabundant. Enterobacteriaceae, Hafnia, Rahnellawere, Raoultella comprised, and of SerratiaButtiauxella, with, Enterobacter Rahnella aquatis, Hafnia ,(48Rahnella%) and, Raoultella Serratia , plymuthicaand Serratia (,48 with%) beingRahnella the aquatis most (48%)abundant and Serratia (Table plymuthica1). The similar(48%) results being the of mostthe chicken abundant meat (Table after1). treatment with essential oils were found in a study by Kačániová et al. [47].

Table 1. Identification of bacteria isolated from chicken meat.

Species No. of Bacterial Colonies/No. of Samples Percentage % Aeromonas veronii 16/25 64.00 Aromatoleum alkani 2/25 8.00 Aromatoleum buckelii 6/25 24.00 Microorganisms 2019, 7, 134 7 of 10

The similar results of the chicken meat after treatment with essential oils were found in a study by Kaˇcániová et al. [47].

Table 1. Identiﬁcation of bacteria isolated from chicken meat.

Species No. of Bacterial Colonies/No. of Samples Percentage % Aeromonas veronii 16/25 64.00 Aromatoleum alkani 2/25 8.00 Aromatoleum buckelii 6/25 24.00 Aromatoleum evansii 9/25 36.00 Buttiauxella gaviniae 10/25 40.00 Buttiauxella izardii 4/25 16.00 Buttiauxella warmboldiae 6/25 24.00 Clostridium diﬃcile 2/25 8.00 Clostridium novyi 1/25 4.00 Enterobacter amnigenus 2/25 8.00 Enterobacter cloacae 4/25 16.00 Hafnia alvei 3/25 12.00 Lactobacillus amylophilus 2/25 8.00 Lactobacillus bifermentans 3/25 12.00 Lactobacillus casei 5/25 20.00 Lactobacillus fructivorans 3/25 12.00 Lactobacillus parabuchneri 4/25 16.00 Lactobacillus paracasei 6/25 24.00 Lactobacillus paraplantarum 5/25 20.00 Lactobacillus pentosus 15/25 60.00 Lactobacillus plantarum 2/25 8.00 Lactobacillus reuteri 5/25 20.00 Lactobacillus salivarius 5/25 20.00 Lactobacillus zeae 6/25 24.00 Lysinibacillus fusiformis 4/25 16.00 Pantoea ananatis 2/25 8.00 Pseudomonas anguilliseptica 3/5 60.00 Pseudomonas brenneri 2/5 40.00 Pseudomonas ﬂuorescens 4/5 80.00 Pseudomonas fragi 3/5 60.00 Pseudomonas gessardii 4/5 80.00 Pseudomonas lundensis 3/5 60.00 Pseudomonas proteolytica 4/5 80.00 Pseudomonas resinovorans 2/5 40.00 Pseudomonas taetrolens 6/25 24.00 Rahnella aquatis 12/25 48.00 Raoultella ornithinolytica 4/25 16.00 Seratia fonticola 5/25 20.00 Serratia liquefaciens 5/25 20.00 Serratia odorifera 9/25 36.00 Serratia plymuthica 12/25 48.00 Staphylococcus capitis 9/25 36.00 Staphylococcus caprae 5/25 20.00 Staphylococcus epidermis 6/25 24.00 Staphylococcus pasteuri 7/25 28.00 Yersinia enterocolitica 2/25 8.00

4. Conclusions The essential oils of Feniculum vulgare (fennel) and Satureja hortensis (savory) as natural food preservatives with potential antimicrobial activity in food industry were studied in our study. The use of fennel oil and savory oil was the most eﬀective against the growth of the total viable count of Microorganisms 2019, 7, 134 8 of 10 lactobacilli and Enterobactericeae in this study. Based on microbiological analyses, treatment with fennel and savory oil prolongs the shelf-life of chicken thighs, in comparison to control samples.

Author Contributions: M.K. and S.K. were responsible for the design of the study; M.K., M.M., S.K., M.K., C.P., and P.H. conducted the in vivo study and collected the samples. M.K. and N.L.V. performed the lab analysis. M.K., S.K., N.L.V., C.P., and M.K were responsible for writing and editing the manuscript. All authors have carefully revised and approved and final version of the manuscript. Funding: This research was funded by Slovak Research and Development Agency APVV-15-0544. Acknowledgments: The paper was supported by the project. The research leading to these results has received funding from the European Community under project no. 26220220180: Building Research Centre “AgroBioTech”. Conflicts of Interest: The authors declare no conflict of interest.

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