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Combined antimicrobial effect of oregano essential oil and caprylic acidin minced beef
Radka Hulankova ⁎, Gabriela Borilova, Iva SteinhauserovaUniversity of Veterinary and Pharmaceutical Sciences Brno, Faculty of VeterinaryHygiene and Ecology, Department ofMeat Hygiene and Technology, Palackeho tr. 1/3, 612 42 Brno, Czech RepublicCEITEC — Central European Institute of Technology, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho tr. 1/3, 612 42 Brno, Czech Republic
⁎ Corresponding author at: University of Veterinary andFaculty of Veterinary Hygiene and Ecology, Department ofPalackeho tr. 1/3, 612 42 Brno, Czech Republic. Tel.: +42
Oregano essential oil (OEO) and caprylic acid (CA) are highly aromatic natural antimicrobials with limited indi-vidual application in food. We proved their combined additive effect when used in meat. Application of 0.5% CAand 0.2% OEO (v/w) with 0.1% of citric acid in vacuum packed minced beef inoculated with Listeriamonocytogenes at a concentration of 5 log cells/g reduced counts of lactic acid bacteria by 1.5 log CFU/g andcounts of psychrotrophic bacteria and L. monocytogenes by more than 2.5 log CFU/g at the end of storage at3 °C for 10 days. In sensory evaluation the samples with OEO showed during the whole experiment statisticallybetter scores than control, whereas the samples treated with CA showed worse colour attributes.
Raw meat can be contaminated by many pathogenic microorgan-isms. Although the majority of them are unable to multiply during re-frigerated storage, Listeria monocytogenes is well known for its abilityto grow at low temperatures. In order to inhibit growth of pathogensand to extend the shelf-life, antimicrobial substances can be added infood. Over the past years, research in the area of microbiological safe-ty and shelf-life of food has focused on natural antimicrobials as a re-sult of concern of consumers regarding synthetic additives.
Caprylic acid (octanoic acid; CA) is a saturated medium-chain fattyacid, occurring naturally in coconut oil, palm kernel oil and milk ofruminants as well as human breast milk (Hlongwane, Delves, Wan, &Ayorinde, 2001; Jensen, 2002; Jensen, Ferris, Lammi-Keefe, & Henderson,1990; Park, Juarez, Ramos, & Haenlein, 2007; Santos, Villarino, Zosa, &Dayrit, 2011). According to the Joint FAO/WHO Expert Committee onFood Additives caprylic acid is considered as safe when used as a flavour(JECFA, 1999). In the USA, caprylic acid is approved for surface applicationon RTE meat and meat products (USDA-FSIS, 2012). Caprylic acid isknown to have antibacterial properties against a wide range of bothgram-positive and gram-negative pathogens (Boyen et al., 2008;Burnett et al., 2007; Jang & Rhee, 2009; Kinderlerer & Lund, 1992;Nair, Vasudevan, Hoagland, & Venkitanarayanan, 2004; Nobmann,Smith, Dunne, Henehan, & Bourke, 2009; Skrivanova, Marounek,Benda, & Brezina, 2006; Sprong, Hulstein, & Van der Meer, 2001).
Pharmaceutical Sciences Brno,Meat Hygiene and Technology,0 541562750.
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Essential oils – volatile, aromatic oily liquids obtained from plantmaterial – are regarded as prime candidates for use as natural anti-microbials in food. Their antimicrobial properties have been longrecognized (Burt, 2004). Oregano essential oil (OEO) obtained fromOriganum vulgare L. has been successfully used many times for inhibitionofmicroorganisms both in vitro (Hammer, Carson, & Riley, 1999; Peñalveret al., 2005; Souza, Stamford, & Lima, 2006) and in meat, includingL. monocytogenes (Skandamis & Nychas, 2001; Tsigarida, Skandamis, &Nychas, 2000).
The antimicrobial effect of both CA andOEO is aimed at the cellmem-brane, where caprylic acid probably disrupts the electron transport chainand oxidative phosphorylation (Desbois & Smith, 2010), whereas themajor antimicrobial components of OEO, carvacrol and thymol, increasecell membrane permeability (Burt, 2004).
The aim of this study on the combined effect of CA and OEO was toevaluate the possibility of lowering the individual concentrations ofthese compounds to a sensory acceptable level while maintaining theoverall antimicrobial effect. Since these compounds are highly aromatic,their individual application in food is therefore limited, especially ashigher concentrations of essential oils are usually needed in food (includ-ing meat) to obtain the same antimicrobial effect as in vitro (Barbosa etal., 2009).
2. Material and methods
2.1. Antimicrobial substances
The antimicrobials used in this studywere oregano essential oil (OEO)obtained from O. vulgare L. (Nobilis Tilia, Czech Republic), originatingfrom Spain and containing mainly carvacrol (72%), p-cymene (7.6%) and
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γ-terpinene (5.7%); caprylic acid (CA; ≥98%, Sigma-Aldrich, USA) andcitric acid (anhydrous, p.a., Lach-Ner, Czech Republic). Both CA and OEOwere used undiluted whereas citric acid was first dissolved in distilledwater. Concentrations of OEO and CA in the mixtures were based ontheir minimum inhibitory concentrations (MICs) determined for thesame L. monocytogenes strains in vitro (Hulankova & Borilova, 2011). Ap-proximately 2 × MIC of CA together with approximately 4 × MIC of OEOwas determined as the highest sensory acceptable concentration in meat(Table 1).
2.2. Experimental design
Beef inside rounds (3 pieces) were purchased from a local meatretailer and immediately transported to the laboratory and pre-pared for testing on the day of purchase. Meat was ground in ameat grinder (3 mm grinder plate) and inoculated with a mixtureof six L. monocytogenes strains (Table 2). Suspensions of the strainswere prepared from cells in the stationary phase using theMcFarland turbidity scale and further diluted. The strains werepresented in the inoculum in equal proportion. Meat was inoculatedwith 5 log cells/g (inoculumwas added in volume 10 ml/kg). After inoc-ulation and homogenisation in a laboratory knife mill (2000 rpm, 10 s),sterile distilled water (control) or the antimicrobials were added (v/w)and the mixture was homogenised again (2000 rpm, 20 s). Aliquots of50 g of the mixture were vacuum packed (99.0% of vacuum) usingVAC-STAR S-223 GX packing machine (VAC-STAR AG, Switzerland)and AMILEN foil bags (VF Verpackungen GmbH, Germany) (PA/PE 20/60 μm, oxygen permeability 50 cm3·m−2 · 24 h · 1 atm at 23 °C 75%r.h.). Thin bags (approx. 5 mm in diameter) were formed and storedat 3 ± 1 °C for 10 days. The analyses were performed after 0, 3, 6, and10 days of storage. At each sampling time, six bags of each groupwere taken for sensory evaluation, microbiological analysis, and colourand pH measurements, performed in this order. Four more bags wereused for sensory evaluation after cooking. In total, 200 samples wereanalysed.
2.3. Microbiological analysis
From each bag, 10 g of minced beef was aseptically weighted in astomacher bag and homogenised for 2 min with 90 ml of sterile buff-ered peptone water (Oxoid, UK, code CM1049). Serial dilutions insterile saline were made from the slurries and used for enumerationof lactic acid bacteria and psychrotrophic microorganisms accordingto standardized methods ISO 15214:1998 and ISO 17410:2001, respec-tively. Lactic acid bacteriawere enumerated onManRogosa Sharpe agar(MRS, Oxoid, UK, code CM1153) overlaid by the same medium and in-cubated at 30 °C for 3 days. Numbers of psychrotrophic microorgan-isms were determined by spread plating onto Standard Plate CountAgar (APHA, Oxoid, UK, code CM0463), incubation at 7 °C for 10 days.Numbers of L. monocytogenes were determined according to ISO11290–2:1998; after 1 h at 20 °C for resuscitation, the slurries wereserially diluted in sterile buffered peptone water, spread plated ontoALOA (Merck, Germany, code 100427) and the plates were incubatedat 37 °C for 48 h.
Table 1Concentrations of the antimicrobials used in this study.
Group Concentrations (v/w)
Control –
A 0.5% caprylic acid (CA)B 0.2% oregano essential oil (OEO)C 0.5% CA + 0.2% OEOD 0.5% CA + 0.2% OEO + 0.1% citric acid (w/w)
2.4. Physico-chemical analysis
Colour was instrumentally measured by the CIE L*a*b* systemusing a Minolta CM-5 spectrophotometer (Konica Minolta, Japan).The instrument was standardized against a white reference plate.Five measurements were taken from each sample over a measuringarea of 30 mm, 10° viewing angle and illuminant D65. CIE L*— lightness,a*— redness, b*— yellowness, C*— chroma and h*— huewere calculatedusing available software (Spectra Magic 3.61).
The pH values were measured with a calibrated portable pHmeter(pH610, Eutech Instruments) fitted with a combination Double Poreglass electrode (Hamilton, Switzerland).
2.5. Sensory evaluation
Odour and overall acceptability were evaluated immediately afteropening the package by thirteen trained assessors (staff members of theDepartment ofMeatHygiene andTechnology) using anine-point hedonicscale where 1 — dislike extremely, 5 — neutral, and 9 — like extremely.Odour was evaluated using both fresh and cooked samples. For the latter,the thin bags with meat tempered to room temperature were immersedin a water bath and cooked at 85 °C for 15 min.
2.6. Statistical analysis
The statistical analysis was performed using statistical softwareStatistica 7.1 (StatSoft Inc., USA) and level of significance 0.05. All theresults (microbiological data after reversion to logarithmic values) wereevaluated using a one-way ANOVA with post hoc Tukey HSD test.
3. Results
3.1. Antimicrobial activity
The combined antimicrobial effect of 0.5% CA and 0.2% OEO on L.monocytogenes, lactic acid bacteria and psychrotrophic microorgan-isms is shown in Fig. 1. Application of CA alone (2 × MIC in vitro)didn't lead to any significant inhibition of any group of microorgan-isms in comparison with control, whereas the application of OEOalone (4 × MIC in vitro) inhibited the growth of psychrotrophic mi-croorganisms (P b 0.001). The combined application of CA and OEOresulted in significantly lower populations of L. monocytogenes incomparison with control or individual applications of the antimicro-bials (P b 0.001) as the numbers of L. monocytogenes decreased dur-ing storage from 4.63 log CFU/g to 3.68 log CFU/g. When citric acidwas added, even more pronounced decrease from 4.70 log CFU/g to2.38 log CFU/g was noted. The combination of CA and OEO was lesseffective on population of lactic acid bacteria, when only the mixturewith citric acid led to significant inhibition in comparison with thecontrol group (P b 0.001). The psychrotrophic microorganisms wereinhibited by mixture of CA and OEO with or without citric acid to alarger extend, as the numbers were more than 2 log CFU/g lower inthe end of the storage then the numbers in the control group.
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Fig. 1. Effect of OEO, CA and their combination with or without citric acid on selectedmicro-organisms (A: L. monocytogenes, B: lactic acid bacteria, C: psychrotrophicmicroorganisms) inminced beef during storage at 3 °C for 10 days. (■) Control, (▲) 0.5% CA (v/w), (□) 0.2%OEO(v/w), (○) 0.5% CA and 0.2% OEO, (♦) 0.5% CA, 0.2% OEO and 0.1% citric acid (w/w).
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Fig. 2. Effect of OEO, CA and their combination with or without citric acid on pH ofmeat. (■) Control, (▲) 0.5% CA (v/w), (□) 0.2% OEO (v/w), (○) 0.5% CA and 0.2%OEO, (♦) 0.5% CA, 0.2% OEO and 0.1% citric acid (w/w).
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3.2. The pH values and colour
The initial pH of the control group increased from 5.38 to no morethan 5.53 by the end of storage and the pH values of the treated sam-ples did not change noticeably during storage (Fig. 2). Although thepH of samples treated with CA or withmixture of CA and OEO showedslightly lower values, only at the group treated withmixture of CA, OEOand citric acid statistically lower pH values were observed (P b 0.001)in comparison with other groups, as the average pH at the end of stor-age was as low as 5.25 in this group.
The parameters of colour CIE L*, a*, b*, chroma and hue are shown inTable 3. Except for the first measure after vacuum packaging the valuesremained constant during storage. In lightness, there was a statistical
difference between all groups. Lightness was increasing in a row:control–OEO–CA–mix–mix with citric acid. Redness was significantlyhigher in control and samples treated with OEO than in other groups,while yellowness of the control samples was the lowest. The highestlevel of yellowness was measured at the two groups containing bothOEO and CA.
3.3. Sensory evaluation
The results of sensory analysis are shown in Fig. 3. Samples treatedwith CA only were assessed with the lowest attribute scores throughoutthe storage and judged as unacceptable. However, the other experimentalgroups (samples treated with OEO) exhibited better scores than the con-trol group and stayed above the neutral point (score 5) up to the sixth dayof storage. Odour of cooked samples treated with volatile OEO was morepronounced, but still evaluated as acceptable. In overall acceptability thesamples treatedwithmixtures of OEO andCAwere not statistically differ-ent (P = 0.91 and P = 0.97) than control, as the better scores in odourwere outweighed by worse appearance (colour).
4. Discussion
This study showed that the combined application of OEO and CA inmeat had a positive effect on its microbiological quality during vacu-um storage. We also proved the additive effect of these compoundswhen used in meat, which was successfully tested previously in anin vitro study (Hulankova & Borilova, 2011).
Addition of citric acid to the mixture of CA and OEO further in-creased its antimicrobial effect against L. monocytogenes (P b 0.001)and lactic acid bacteria (P = 0.01). Citric acid can act as an antimicro-bial and an acidulant, as it is well known that effectiveness of bothfatty acids and essential oils increases with decreasing pH (Boyen etal., 2008; Juven, Kanner, Schved, & Weisslowicz, 1994). When com-pared to control, combination of OEO, CA and citric acid (all in lowconcentrations) decreased the counts of psychrotrophic bacteria bymore than 2.5 log CFU/g at the end to storage and the same wastrue for L. monocytogenes, suggesting the possible use of the afore-mentioned mixture in control of this psychrotrophic pathogen. Al-though the attention is focused mainly on RTE meats and meatproducts, presence of L. monocytogenes in fresh beef can representan important hazard due to the possibility of cross contaminationthrough cooking utensils or consumption of inadequately cookedmeat (Barros et al., 2007). A decrease of L. monocytogenes in vacuumpacked beef by 2–3 log CFU/g at the end of storage was reported byTsigarida et al. (2000) after application of 0.8% (v/w) of OEO. In our
Table 3Effect of OEO, CA and their combination with or without citric acid on colour parameters (mean ± S.D.) of minced beef during storage at 3 °C for 10 days. A: 0.5% CA (v/w), B: 0.2%OEO (v/w), C: 0.5% CA and 0.2% OEO, D: 0.5% CA, 0.2% OEO and 0.1% citric acid (w/w).
a–eMeans with different superscripts within the same day and parameter differ significantly (P b 0.05).
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study, similar results were achieved with a mixture containing only0.2% of this highly aromatic essential oil.
To find a compromise between effective doses of flavouring agentslike essential oils and sensory acceptability is a difficult task (Skandamis& Nychas, 2001). Although several studies reported better sensory prop-erties of minced beef or sheep meat treated with 0.8–1% of OEO in com-parison to control (Govaris, Solomakos, Pexara, & Chatzopoulou, 2010;Skandamis &Nychas, 2001; Tsigarida et al., 2000), other authors regardedthe concentrations of essential oils in meat close to 1% as unacceptable(Chouliara, Karatapanis, Savvaidis, & Kontominas, 2007; Solomakos,Govaris, Koidis, & Botsoglou, 2008). In our case, the assessors found theodour of meat treated with higher than 0.2% OEO as too strong and nolonger pleasant. The differences in sensory acceptance can be explainedby different composition of OEO resulting in different aroma intensity,as it's well known that the composition of essential oils is greatly variable(Burt, 2004). Geographical and individual differences in preferences of as-sessors could also play a role.
Samples treated with CA only showed very low scores in sensoryanalysis, as the odour was evaluated by assessors as unpleasant. Theodour of CA is usually described as ‘rancid’ (Santos et al., 2011). How-ever, Burnett et al. (2007) didn't find any differences in organolepticevaluation between RTE meats and poultry products treated with1% caprylic acid and the untreated controls. In our study, the mixturesof CA with OEO showed high odour scores as the less pleasant smell ofCA was overlaid by the aroma of OEO. Combination of antimicrobialsubstances led to a lowering of concentrations of these highly aromaticnatural antimicrobials on sensory acceptable level while maintainingthe overall antimicrobial effect.
Beside worse sensory properties of samples treated with CA, anegative impact on meat colour was observed, too. All the groupswhere CA was used showed higher values of lightness and yellownessthan the other groups and lower values of redness in the CIE L*a*b*system. The negative impact on meat colour also reflected in loweroverall acceptance scores. Direct application of caprylic acid ledprobably to partial denaturation of meat proteins and oxidation of
myoglobin, resulting in discolouration. Occurrence of colour changesin meat to brighter or brownish due to application of organic acidswith regard to decontamination of carcasses andmeat cuts has been stud-ied and discussed previously by many authors, e.g. in a review bySmulders andGreer (1998). At the point of sale, colour is themost impor-tant attribute of meat quality, since it is the first quality attribute seen bythe consumer (Troy & Kerry, 2010). What is more, the brown colour ofraw minced beef (as in premature browning) can lead to misinterpreta-tion of cooked internal colour and consumption of undercooked meat(Hunt, Sorheim, & Slinde, 1999).
5. Conclusions
We proved that a combination of medium-chain fatty acid (CA)and essential oil (OEO) can act as an effective antimicrobial agent inmeat. Given the results of our previous in vitro study (Hulankova &Borilova, 2011), the mixture also has a potential to be effective inmeat against gram-negative bacteria like Salmonella spp. or the familyEnterobacteriaceae generally, which could be beneficial to prove.However, the issue of discolouring remains to be solved. Moreover,it is worthwhile to note that natural antimicrobials like essentialoils in comparison with synthetic additives have no standard compo-sition; their composition can differ between harvesting seasons andbetween geographical sources (Burt, 2004), affecting their effective-ness. Interactions with other intrinsic and extrinsic factors like pH,fat level, storage temperature and amount of oxygen within the pack-aging should be always taken in account, too when using these natu-ral antimicrobials in food.
Acknowledgement
This study was financially supported by IGA VFU (Grant numberIGA 196/2009/FVHE).
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Fig. 3. Effect of OEO, CA and their combinationwith or without citric acid on sensory charac-teristics (A: odour of raw samples, B: odour of cooked samples, C: overall acceptability) ofminced beef during storage at 3 °C for 10 days. (■) Control, (▲) 0.5% CA (v/w), (□) 0.2%OEO (v/w), (○) 0.5% CA and 0.2% OEO, (♦) 0.5% CA, 0.2% OEO and 0.1% citric acid (w/w).
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