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Potential application of essential oils as antimicrobial preservatives in cheese
Accepted Manuscript Potential application of preservatives in cheese
essential
oils
as
antimicrobial
Nasim Khorshidian, Mojtaba Yousefi, Elham Khanniri, Amir Mohammad Mortazavian PII: DOI: Reference:
S1466-8564(17)30321-1 doi:10.1016/j.ifset.2017.09.020 INNFOO 1858
To appear in:
Innovative Food Science and Emerging Technologies
Received date: Revised date: Accepted date:
16 March 2017 15 August 2017 28 September 2017
Please cite this article as: Nasim Khorshidian, Mojtaba Yousefi, Elham Khanniri, Amir Mohammad Mortazavian , Potential application of essential oils as antimicrobial preservatives in cheese. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Innfoo(2017), doi:10.1016/ j.ifset.2017.09.020
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ACCEPTED MANUSCRIPT
Potential application of essential oils as antimicrobial preservatives in cheese
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Nasim Khorshidian1, 2, Mojtaba Yousefi1, 2, Elham Khanniri1, Amir Mohammad Mortazavian3*
Student Research Committee, Department of Food Technology, Faculty of Nutrition Sciences and Food Technology/National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
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Food Safety Research Center (Salt), School of Nutrition and Food Sciences, Semnan University of Medical Sciences, Semnan, Iran
3
Department of Food Technology, Faculty of Nutrition Sciences and Food Technology/National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Corresponding authors:
Amir M. Mortazavian (ph.D)
Ph: + 98-912-7114977
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Fax: +98 21 22360657 E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract Increasing demand of consumers to use food products without preservatives or natural preservatives as possible has compelled the food industries for utilization of preservatives with herbal and microbial origins instead of artificial preservative in their production. Essential oils
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are volatile, natural plant-derived substances that are used in medicine, food flavoring and food preservation. These diverse compounds represent considerable potential antioxidant, antibacterial
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and antifungal activities via various mechanisms. This review represents an overview on the
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impact of essential oils and their constituents as natural antimicrobials versus common pathogenic and spoilage microorganisms in cheese along with the related mechanisms of actions.
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Industrial Relevance: Natural preservatives have proven popularity such that interest continues
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in substituting artificial additives with natural. Therefore, production of safe food without or with low amounts of synthetic preservatives is one of the most important challenges in the food
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industry. This review, introduces the potential application of essential oils as natural
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antimicrobial agents for reduction of common spoilage and pathogenic bacteria as well as molds and yeasts in cheese-making industry.
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Keywords: Cheese; Essential oil; Spoilage; Antimicrobial; Foodborne pathogen
ACCEPTED MANUSCRIPT 1. Introduction Food safety is one of the main issues in the food industry and apart from spoilage of foodstuff, there is always concern about the outbreak of foodborne illnesses among food manufacturers, regulatory agencies, researchers and consumers. Therefore, producing safer food is one of the
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most important priorities in the food industry (Friedman, Henika, & Mandrell, 2002; Mohamed,
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Zaky, Kassem, Abbas, Salem, & Said-Al Ahl, 2013; Smith-Palmer, Stewart, & Fyfe, 2001).
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Different methods, including thermal processing, lowering of water activity, various methods of
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packaging, irradiation, high pressure, high-intensity pulsed electric field processing and the addition of chemical preservatives have been utilized to produce safer foodstuffs (Čvek, Markov,
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Frece, Dragičević, Majica, & Delaš, 2010; Delves-Broughton, 2005; Jeong, et al., 2014; Lee,
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Yeo, Park, Kang, & Hahm, 2010; López, Sánchez, Batlle, & Nerín, 2005). Food-grade and GRAS food additives have been normally used by the food industry for some time; however,
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synthetic preservatives are now being replaced by natural preservatives as consumers seek foods
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for a healthier lifestyle (Zantar, Yedri, Mrabet, Laglaoui, Bakkali, & Zerrouk, 2014). Although a specific category for natural additives has not been defined, there are various natural
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antioxidants, antimicrobials, sweeteners and coloring agents that are derived from animals, plants
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and microorganisms (Carocho, Morales, & Ferreira, 2015). Bacteriocins, natamycin, reuterin from microbial sources; lysozyme, lactoperoxidase, lactoferrin from animal sources; polyphenols and essential oils from plant sources can be named as examples of natural preservatives (Carocho, et al., 2015). Essential oils (EO) that are lipophilic liquids, extracted from divers plants containing different natural, biologically active components have antimicrobial and antioxidant properties (Amatiste, et al., 2014; Hamedi, Razavi-Rohani, & Gandomi, 2014; Yousefi Asli, Khorshidian, Mortazavian, & Hosseini, 2017). Antimicrobial properties of
ACCEPTED MANUSCRIPT essential oils against a wide range of microorganisms have been reported in various studies (Čvek, et al., 2010; Delves-Broughton, 2005; Jeong, et al., 2014; Kotzekidou, Giannakidis, & Boulamatsis, 2008; Lee, et al., 2010; Smith-Palmer, et al., 2001; Yahyazadeh, Omidbaigi, Zare, & Taheri, 2008). Due to the hydrophobicity of essential oils’ components, they easily pass
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through the bacterial cell membrane interfering with molecular transport mechanisms leading to
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cell inactivation (Burt, 2004; Goñi, López, Sánchez, Gómez-Lus, Becerril, & Nerín, 2009).
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Essential oils commonly exhibit greater inhibitory properties against Gram-positive than Gramnegative bacteria due to the lipopolysaccharide barrier in the outer membrane of Gram-negative
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bacteria (Techathuvanan, Reyes, David, & Davidson, 2014; Tehrani & Sadeghi, 2015). As
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mentioned before, essential oils have antibacterial and antifungal activities against different microorganisms and as no review has been published in this context, the aim of this paper was to
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review the antimicrobial properties of essential oils derived from different plant sources used in
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different types of cheeses.
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2. Spoilage and pathogenic microorganisms in cheese
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Cheese is one of the most popular dairy products which is a rich source of essential nutrients such as proteins, vitamins, minerals, short-chain fatty acids and certain trans-fatty acids that can
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be regarded as a part of healthy diet (López-Expósito, Amigo, & Recio, 2012). Cheese spoilage can be caused by both bacteria and fungi. However, the type of the spoilage varies depending on the type of cheese. Common spoilage molds in cheeses belong to the genera Penicillium, Aspergillus, Cladosporium, Mucor, Fusarium, Monilia and Alternaria (Johnson, 2002). Yeasts frequently found in spoiled cheese include Candida spp., Yarrowia lipolytica, Pichia spp., Kluyveromyces marxianus, Geothricum candidum and Debaryomyces hansenii (Filtenborg, Frisvad, & Thrane, 1996; Fleet, 1990; Johnson, 2002; Kosse, Seiler, Amann, Ludwig, & Scherer,
ACCEPTED MANUSCRIPT 1997). The growth of mold is associated with undesirable taints and odors, liquefaction of the curd and in some cases, production of mycotoxins. In fresh cheeses such as cottage cheese with a higher pH compared to other types of cheeses, Gram-negative psychrotrophic species, such as Pseudomonas and some coliforms can cause spoilage. The most important psychrotrophic
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species are Pseudomonas spp., Alcaligenes spp., Achromobacter spp. and Flavobacterium spp.
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Other bacteria involved in cheese spoilage are Enterobacteriaceae, Bacillus spp., Clostridium
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butyricum, Clostridium tyrobutyricum and Clostridium sporogenes which cause blowing (Robinson, Tamime, & Wszolek, 2002).
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Although cheese is a safe food, occasionally recalls and outbreaks do occur (Kousta,
Listeria
monocytogenes,
Salmonella
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Mataragas, Skandamis, & Drosinos, 2010). The most serious outbreaks have been caused by and enteropathogenic
Escherichia coli
(EPEC).
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Enterohaemorrhagic strains of E. coli (EHEC), such as E. coli O157:H7 may cause serious
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infections and fatalities in the very young and the elderly. E. coli O157:H7 is considered to be a potentially high-risk pathogen in cheese due to its high tolerability to low pH for a long period of
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time and its association with unpasteurized milk. In this regard, soft cheeses made from raw milk
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are high-risk products (Reitsma & Henning, 1996). L. monocytogenes is a psychrotrophic and fairly heat-tolerant pathogen that can be present
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in raw milk. It may enter the cheese process if hygienic conditions are not maintained in the processing. In surface-ripened cheeses, the pH of the cheese is increased from around 5 to 6-7 because of desired surface mold growth in these types of cheeses. High pH along with high moisture content and temperature of the ripening rooms (8-12°C) provide suitable conditions for rapid growth of L. monocytogenes (Carpentier & Cerf, 2011; Gould, Mungai, & Barton Behravesh, 2014; Melo, Andrew, & Faleiro, 2015).
ACCEPTED MANUSCRIPT Salmonella can be found in cheeses made from raw milk and do not survive pasteurization (Villarruel-L, et al., 2016). However, they may also enter cheese as post-pasteurization contaminants if the process is not properly controlled. If acid production during manufacture is slow, Salmonella may grow during cheese making and have been shown to survive for more than
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60 days in some cheeses (Fernandes, 2009). Staphylococcus aureus is able to tolerate salt and
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moderate acidity and can multiply during cheese manufacture and ripening in soft cheeses. It
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may survive for long periods even in hard cheeses and if high enough populations are developed, enterotoxin may be produced which persists for many months even after the cells have lost their
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viability (Fernandes, 2009).
3. Essential oils and mechanisms of their antimicrobial activity
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Essential oils are natural aromatic and volatile liquids which might be derived from different
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parts of the plants, including flowers, roots, barks, leaves, seeds, peel, fruits, wood and the whole plants (Bozin, Mimica-Dukic, Simin, & Anackov, 2006; Hammer, Carson, & Riley, 1999;
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Hyldgaard, Mygind, & Meyer, 2012). The International Organization for Standardization (ISO)
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has defined essential oils as ‘product obtained from a natural raw material of plant origin, by steam distillation, by mechanical processes from the epicarp of citrus fruits, or by dry distillation,
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after separation of the aqueous phase if any by physical processes’, going on to specify that ‘the essential oil can undergo physical treatments, which do not result in any significant change in its composition’ (ISO/DIS9235, 2013). Essential oils are a mixture of various compounds characterized by aromatic smell, generally liquid colorless to slightly yellowish color and insoluble in water, but soluble in organic solvents (Nazzaro, Fratianni, De Martino, Coppola, & De Feo, 2013). There are almost 3000 different essential oils with approximately 300 used commercially in the flavoring and fragrances market (Burt, 2004). Their composition varies
ACCEPTED MANUSCRIPT depending on the type of the plant species, geographic origin of the plant, climate conditions, soil composition, vegetative cycle stage and the part of the plant that was used for EO extraction (Angioni, Barra, Coroneo, Dessi, & Cabras, 2006; Masotti, Juteau, Bessière, & Viano, 2003). They are usually secreted as secondary metabolites having roles in pollination or defense
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mechanisms against bacteria and fungi (Tajkarimi, Ibrahim, & Cliver, 2010). Essential oils are
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extracted by means of water or steam distillation, maceration and supercritical fluid extraction
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(Burt, 2004; Edris, 2007; Shannon, Milillo, Johnson, & Ricke, 2011). Although EOs are used primarily as flavoring agents in the food industry, due to possessing antimicrobial activity, they
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can be incorporated into food products in order to extend the shelf-life. However, the
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prerequisites of this application are knowledge about EOs properties, minimum inhibitory concentrations (MIC), the target microorganisms, the mode of action and the probable
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interactions with food matrix as well as sensory quality of the food. The antimicrobial activity of
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EOs can be completely associated with their active constituents (Hyldgaard, et al., 2012). About 90-95% of the whole EO comprises volatile components consisting monoterpenes and
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sesquiterpene hydrocarbons and their oxygenated derivatives besides aliphatic aldehydes,
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alcohols and esters. The non-volatile portion constitutes about 5-10% of the whole EO that includes mainly hydrocarbons, fatty acids, sterols, carotenoids, waxes, cumarines and flavonoids
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(Luque De Castro, Jiménez-Carmona, & Fernández-Pérez, 1999). The most active antimicrobial compounds present in EOs can be divided into four groups based on the chemical structure: terpens (e.g., p-cymene, limonene), terpenoids (e.g., thymol, carvacrol), phenylpropenes (e.g., eugenol, vanillin) and other compounds such as allicin or isothiocyanates (Hyldgaard, et al., 2012).
ACCEPTED MANUSCRIPT The mechanism of action of EOs against microorganisms has not yet been elucidated entirely and cannot be attributed to a single mechanism. There are several locations in the microorganisms which are supposed to be the sites of the action for EOs (Nazzaro, et al., 2013). The fundamental mechanisms of EOs antimicrobial activity are shown in Figure 1. The
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antimicrobial activity is pertained to the hydrophilic or lipophilic character of the EOs’
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components, the microorganism type and its cell wall structure. It is assumed that EOs are more
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effective against Gram-positive bacteria compared to Gram-negative ones (Cimanga, et al., 2002; Hyldgaard, et al., 2012; Smith-Palmer, Stewart, & Fyfe, 1998). The cell wall of Gram-positive
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bacteria consists of peptidoglycan (90-95%) along with teichoic acid and proteins linked to it.
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Since the major parts of EOs are hydrophobic, they interact with the cell membrane and easily pass through it to the cytoplasm. The cell wall structure in Gram-negative bacteria is more
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complex comprised of a monolayer of peptidoglycan surrounded by an outer membrane
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consisting of proteins and lipopolysaccharide (LPS). This outer cell boundary is charged, so it has a hydrophilic nature; however, hydrophobic compounds can pass through this barrier
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(Nazzaro, et al., 2013; Nikaido, 1994; Vaara, 1992).
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The lipophilic nature of the hydrocarbon skeleton and hydrophilic nature of functional groups of EOs play substantial roles in the antimicrobial effects of these compounds. The
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greatest antimicrobial activity in EOs is found with phenolic compounds, following in order by aldehydes, ketones, alcohols, ethers and hydrocarbons (Kalemba & Kunicka, 2003). The activity of phenols is attributed to the acidic characteristic of the hydroxyl group. These compounds change the cell permeability, interfere with the enzymes involved in energy production and interrupt the protein motive force that eventually leads to the cell death (Basim, Yegen, & Zeller,
ACCEPTED MANUSCRIPT 2000). The shape of the bacteria also can be determinative of the activity of EOs and it has been stated that rod-shaped cells are more susceptible than coccoid-shape (Nazzaro, et al., 2013).
4. Application of EOs in various types of cheese
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Table 1 summarizes selected publications on the antimicrobial activity of different
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essential oils applied in cheeses. The major compounds of each essential oil used in cheeses are
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shown in Table 2.
Effect of essential oil from Ocimum gratissimum (200, 400, 600, 800 or 1000 mg/L) was
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assessed against six fungi including Aspergillus flavus, Aspergillus tamarii, Fusarium poae,
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Fusarium verticillioides, Penicillium citrinum and Penicillium griseofulvum isolated from a traditional cheese named wagashi. Wagashi is a soft, brine-pickled cheese made from whole
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cow’s milk by adding the juice of crushed stems of Bryophylum and no rennet or lactic starter is
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used. It is almost fried before consumption. It was noted that mycelia growth was reduced by increasing the essential oil level. A significant fungistatic activity against all the examined
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species was observed with MIC values ranged from 800-1000 mg/L. Penicillium species and F.
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poae were the most susceptible fungi to the essential oil. O. gratissimum essential oil efficacy was attributed to its major compounds, including thymol, γ-terpinene and p-cymene (Philippe,
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Souaïbou, Guy, et al., 2012). Similarly, antifungal activity of O. gratissimum essential oil was attributed to thymol. It was demonstrated that thymol interacts with cell envelope to impair vesicles and cell membranes as well as ergosterol synthesis which has a role in membrane integrity and fluidity (Ahmad, et al., 2011; Hyldgaard, et al., 2012). In vitro antifungal activity of Cinnamomum zeylanicum and O. gratissimum (200, 400, 600, 800 or 1000 mg/L) were explored against six molds including Aspergillus terreus, Aspergillus ustus, Aspergillus niger, Aspergillus aculeatus, Penicillium brevicompactum and
ACCEPTED MANUSCRIPT Scopulariopsis brevicaulis isolated from wagashi cheese. Results showed that O. gratissimum had fungistatic activity against all species with MIC values in the range of 400-1000 mg/mL and fungicidal activity against A. terreus (MFC of 1000 mg/mL) and S. brevicaulis (MFC of 600 mg/mL) which were the most susceptible strains. C. zeylanicum showed only inhibitory effect on
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A. terreus, S. brevicaulis and P. brevicompactum. A. aculeatus was the most resistant strain to
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this essential oil. It was announced that O. gratissimum was more active against all the tested
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strains due to its prominent constituent, thymol (Philippe, Souaïbou, Paulin, Issaka, & Dominique, 2012).
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Essential oils (Pimenta racemosa, Syzygium aromaticum, Cinnamomum verum and Thymus
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vulgaris) were added to full-fat and low-fat cheeses at the levels of 0.1, 0.5 and 1% to evaluate their influence on L. monocytogenes and Salmonella Enteritidis growth at 4 and 10°C,
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respectively during 14 days of storage. It was concluded that at 1%, clove and cinnamon
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essential oils were the most effective oils regarding reduction of L. monocytogenes count in lowfat cheese within three days compared to the bay and thyme essential oils which reduced L.
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monocytogenes to less than 1 log10 cfu/mL during 10 days. In the case of full-fat cheese, only
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clove oil (at 1%) was effective in reducing L. monocytogenes to less than 1 log10 cfu/mL and at 0.1%, inhibiting the growth in low-fat and full-fat cheese. Clove oil at 0.5% in low-fat cheese,
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reduced S. Enteritidis to ≤ 1 log10 cfu/g, but recovered to 4.7 log10 cfu/g at day 14 (Smith-Palmer, et al., 2001). In a study by Govaris et al. (2011), antibacterial effects of oregano (0.1 or 0.2 mL/100 g) and thyme (0.1 mL/100 g) essential oils against L. monocytogenes and E. coli O157:H7 in feta cheese stored under modified atmosphere packaging (50% CO2 and 50% N2) at 4°C were determined. The results revealed that at 0.1 mL/100 g of oregano essential oil, L. monocytogenes and E. coli O157:H7 could survive up to 18 and 22 days of storage, respectively,
ACCEPTED MANUSCRIPT while at the level of 0.2 mL/100 g, the counts of survivors were up to 14 and 16 days. A similar trend as found with the oregano essential oil study was reported in the case of thyme essential oil at 0.1 mL/100 g. Moreover, both essential oils possessed a greater inhibitory effect toward L. monocytogenes compared to E. coli O157:H7. These results were consistent with other studies
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which evaluated the antimicrobial efficiency of oregano against E. coli O157:H7 and L.
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monocytogenes (Elgayyar, Draughon, Golden, & Mount, 2001; Gutierrez, Barry-Ryan, &
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Bourke, 2008; Santurio, De Jesus, Zanette, Schlemmer, Fraton, & Fries, 2014). In studies conducted by Burt and Reinders (2003), Sikkema et al. (1995) and De Sousa et al. (2012), cells
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of E. coli O157:H7 exposed to oregano essential oil were inactivated due to cell lysis.
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In a study by Amatiste et al. (2014), the antimicrobial activity of T. vulgaris L. and Origanum vulgare L. essential oils against S. aureus in fresh sheep cheese was assessed. The
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determined MIC and MBC of the essential oils were 4 and 8 µL/mL, respectively. In addition, it
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was observed the aforementioned essential oils had no effect on S. aureus count in cheese
EOs with cheese matrix.
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samples during 7 days of storage which was attributed to the interaction of active components of
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In another study, the effects of T. vulgaris essential oil toward S. aureus, L. monocytogenes and cheese starter cultures Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris
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were examined through MIC determination and measurement of viability in a Coalho cheesebased broth medium and a coalho cheese model. Coalho cheese is a semi-hard, a medium to high-moisture cheese typical of the northeast region of Brazil. It has a mild acidic flavor and fat content in the range of 35-60% in dry matter. The lower MIC for starter culture (1.25 µL/mL) compared to S. aureus and L. monocytogenes (2.5 µL/mL) was an indicative of a higher sensitivity of mesophilic starter culture. Determination of cell viability in coalho cheese-based
ACCEPTED MANUSCRIPT broth medium at various concentrations of EO (1.25, 2.5 or 5 µL/mL) showed no effect on S. aureus at 1.25 µL/mL, but at 2.5 µL/mL, both pathogenic bacteria and starter culture counts were reduced. At the level of 5 µL/mL, the counts of pathogenic bacteria decreased sharply and starter cultures were inhibited. Determination of cell viability in coalho cheese model showed
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that at 1.25 µL/mL, the essential oil had no impact on the counts of S. aureus and L.
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monocytogenes and the counts of Lacococcus ssp. were almost the same as the initial count.
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Essential oil at 2.5 µL/mL decreased S. aureus, L. monocytogenes and Lactococcus ssp. varying from 0.3 to 1 log cfu/g after 72 h of exposure. It was deduced that Lactococcus ssp. were more
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susceptible to essential oil and the selection of essential oil doses should be performed
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cautiously. Besides, a food matrix with higher fat and protein would impair the efficacy of antimicrobial components in essential oils (de Carvalho, et al., 2015).
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In a work performed by Zantar et al. (2014), essential oils of T. vulgari and Origanum
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compactum (0.05 and 0.1%) were added to goat cheese and their effects on some pathogenic and spoilage microorganisms were studied. Assessing antibacterial activity by disk diffusion method
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showed that the highest antibacterial activity for T. vulgari against S. aureus and the lowest
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activity for O. compactum toward Yersinia enterocolitica. Determination of antifungal activity by agar diffusion method at the concentrations of 1% and 3% indicated that O. compactum
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thoroughly inhibited the growth of A. flavus and Fusarium solani. EOs were added to goat cheese (0.05 and 0.1%) and it was observed that in samples fortified with 0.1% O. compactum, coliforms were not present from the first day of storage and from day 1 in the case of T. vulgari. In shelf-life estimation of cheese by monitoring the growth of molds and yeasts, it was announced that EOs extended the shelf-life of cheeses and O. compactum was more efficient in
ACCEPTED MANUSCRIPT this respect. Sensory evaluation of cheeses showed that samples containing T. vulgari were more acceptable by the panel group. De Souza et al. (2016) measured inhibitory effects of Origanum vulgare L. (0.6, 1.25, 2.5 or 5 µL/mL) on S. aureus, L. monocytogenes, L. lactis ssp. lactis and L. lactis ssp. cremoris in
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coalho cheese broth and slurry. MIC of essential oil against S. aureus and L. monocytogenes
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were 2.5 µL/mL and 0.6 µL/mL for mesophilic lactic acid bacteria. During 24 h, 0.6 µL/mL
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essential oil had no inhibitory effect toward both pathogenic bacteria in cheese broth whilst viable cell counts of starter culture decreased 1 log cfu/mL. A reduction of 1.8 and 1.5 log
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cfu/mL were observed at 1.25 µL/mL in L. monocytogenes and S. aureus, respectively after 24 h.
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The above-mentioned concentration showed the bactericidal effect on the lactic acid bacteria by decreasing 4 log cfu/mL of cell counts. O. vulgare at 2.5 µL/mL decreased S. aureus and L.
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monocytogenes by 2 and 2.5 log cfu/mL after 24 h and a sharp decrease (4 log cfu/mL) in the
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counts of starter culture after 1 h, occurred. The essential oil at 5 µL/mL showed a bactericidal effect against both pathogenic bacteria after 24 h. In cheese slurries, 0.6 µL/g essential oil had no
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impact on pathogenic and lactic acid bacteria counts. At 1.25 µL/g, the viable cell counts of both
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pathogenic bacteria and Lactococcus spp. decreased 1.5 and 2 log cfu/g, respectively after 72 h. Exposure to 2.5 µL/g essential oil caused a reduction of 2.5 and 2.8 log cfu/g in S. aureus and L.
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monocytogenes after 72 h and a sharp decrease in Lactococcus spp. counts after 24 h. It was also noted that in order to decrease the bacteria count the same as the broth, higher concentrations were needed (De Souza, et al., 2016). Higher protein and fat in food compared to simulated condition could interact with essential oil constituents being unavailable to act on cell membrane (Burt, 2004). Furthermore, higher nutrients in cheese would provide a suitable condition for repairing the damaged cells of bacteria (Burt, 2004; Gill, Delaquis, Russo, & Holley, 2002).
ACCEPTED MANUSCRIPT In an experiment, zein-based edible film was prepared by incorporation of Zataria multiflora Bioss essential oil (1, 2, 3 or 4% w/v) in order to control the growth of S. Enteritidis, L. monocytogenes, E. coli and S. aureus in Iranian Feta cheese during 14 days of storage. The results showed that a sharp decrease in the bacterial count occurred in samples coated with
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fortified films (Ghasemi, Javadi, Moradi, & Khosravi-Darani, 2015). In another attempt, Z.
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multiflora Boiss essential oil (50, 100, 200, 400, 600 or 1000 ppm) antifungal activity toward A.
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flavus ATCC 15546 was reported in the culture media and Iranian ultra-filtered white cheese. Radial growth and spore production analysis in agar medium demonstrated that fungal growth
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and spore production were reduced 21.3%, 42.7% by using 50 ppm, 79.4%5 and 21.7% in the
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case of 100 ppm and 75.8% and 92.5% by using 200 ppm essential oil. Z. multiflora Boiss essential oil showed a greater impact on spore production compared to mycelia growth. At the
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concentrations higher than 400 ppm, no fungal growth was observed and a dose of 1000 ppm had
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a fungicidal effect. Also, reduction of 22.6%, 82% and 90% in mycelial dry mass at the concentrations of 50, 100 and 150 ppm were reported while aflatoxin secretion was reduced
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31%, 97.6% and 99.4% at the same concentrations. Z. multiflora Boiss essential oil had an
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inhibitory effect on the radial fungal growth and aflatoxin production at all levels used in cheese samples, but fungal growth and aflatoxin production were not completely inhibited at any
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concentration (Gandomi, et al., 2009). Accordingly, Z. multiflora Boiss effect on growth and citrinin production by P. citrinum in culture media and mozzarella cheese was explored. MIC and MFC were reported as 200 and 400 ppm, respectively. In YES broth containing 100 and 200 ppm essential oil, citritin level was reduced 69 and 92%, respectively. Evaluation of the efficiency of EO activity (50, 100, 200, 400, 600, 800 or 1000 ppm) on fungal radial growth and citrinin production in mozzarella cheese depicted that radial growth and citrinin production were
ACCEPTED MANUSCRIPT inhibited at all levels of EO. However, P. citrinum was not completely inhibited by EO at 1000 ppm and an inhibition of 89.94% for fungi growth and 87% for citrinin production were reported. (Noori, et al., 2012) Eshaghi et al. (2014) added Z. multiflora Boiss essential oil at various amounts (0.05, 0.1,
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0.2 or 0.4%) to the milk for preparation of Gouda cheese and its influence on microbiological
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characteristics and formation of biogenic amines was studied during 90 days of ripening.
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According to the obtained results, increasing the essential oil level led to a decrease in tyramine and histamine amounts. At 0.05%, no significant reduction was observed in tyramine and
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histamine levels compared to the control samples, while 5, 22 and 44% reduction in tyramine
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and 14, 29 and 46% reduction in histamine were observed at the levels of 0.1, 0.2 and 0.4% at the end of storage. The counts of mesophilic lactobacilli decreased 0.5, 1.2 and 1.5 log cfu/g in
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cheese samples at 0.1, 0.2 and 0.4% on the day 15 of ripening. At 0.2 and 0.4% essential oil,
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Enterobacteriaceae reduced 0.68 and 0.75 log cfu/g at the end of ripening (90 days). In cheese samples containing 0.1, 0.2 and 0.4% essential oil, lactococci counts reduced 0.27, 0.58 and 1.3
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log cfu/g compared to the control group at the end of storage. At the same concentrations, the
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aerobic mesophilic counts were reduced 0.57, 0.77 and 1.57 log cfu/g and a reduction of 0.58, 0.84 and 2.2 log cfu/g in yeast counts were noticed. Regarding sensory evaluation, samples
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treated by 0.2% essential oils were the most preferable ones (Eshaghi Gorji, Noori, Nabizadeh Nodehi, Jahed Khaniki, Rastkari, & Alimohammadi, 2014). The impact of thyme and clove essential oil (1.5% v/v) incorporated into the whey protein isolate-based edible film applied for Kashar cheese on the growth of E. coli, L. monocytogenes and S. aureus during 60 days of storage was investigated. It was pointed out that thyme essential oil had a higher antimicrobial activity in comparison with clove essential oil. In cheese samples
ACCEPTED MANUSCRIPT coated with edible films containing thyme and clove essential oil, the number of E. coli were 5.86 and 6.12 log10 cfu/g on day 0 of storage which were decreased 2.25-4.49 and 1.57-4.91 log10 cfu/g on day 60. In the case of L. monocytogenes, the bacterial counts in thyme-coated cheese samples reduced from 5.66 on day 0 of storage to 4.40 log10 cfu/g on day 60. This suppression
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was from 5.93 to 4.81 log10 cfu/g from day 0 to 60 of storage in samples coated with clove-
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fortified edible films. S. aureus counts in samples coated with films containing thyme essential
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oil were 5.49 and 4.16 log10 cfu/g on day 0 and 60 of storage, respectively. In cheeses coated
respectively (Kavas, Kavas, & Saygili, 2015).
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with clove essential oil, S. aureus on day 0 and 60 of storage were 5.64 and 4.63 log10 cfu/g,
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The main components of essential oils in abovementioned studies include thymol and carvacrol. It is presumed that thymol would disrupt outer and inner cell membranes and interact
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with membrane proteins and intracellular targets (Hyldgaard, et al., 2012; Nazzaro, et al., 2013).
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The interaction of thymol with membrane results in the release of K+ and ATP due to the change in the permeability of membrane (Walsh, Maillard, Russell, Catrenich, Charbonneau, & Bartolo,
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2003; Xu, Zhou, Ji, Pei, & Xu, 2008). Moreover, thymol integrates with polar head-groups
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located in the lipid bilayer that would cause alterations in the cell membrane (Di Pasqua, Betts, Hoskins, Edwards, Ercolini, & Mauriello, 2007; Turina, Nolan, Zygadlo, & Perillo, 2006). It was
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assumed that thymol could form a complex membrane or periplasmic proteins through hydrogen bonds and hydrophobic interactions (Juven, Kanner, Schved, & Weisslowicz, 1994). Di Pasqua et al. (2010) noted that thymol would damage the citrate metabolic pathway in addition to influencing the enzymes involved in ATP synthesis. Carvacrol is supposed to change the cell membrane structure as well as its fatty acids profile and therefore, increase the permeability and fluidity of the membrane. It has the capability of influencing the outer
ACCEPTED MANUSCRIPT membrane of Gram-negative bacteria and releasing LPS. It was also suggested that its hydroxyl group acts as a carrier for transportation of monovalent cations by carrying H+ into the cytoplasm and transporting K+ back out. Contrarily, Veldhuizen et al. (2006), announced that hydroxyl group in carvacrol was not fundamental in the revealing of antimicrobial activity, but instead, it
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was related to non-hydroxyl groups. Furthermore, it was observed that carvacrol could affect
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protein motive force and synthesis of flagellin thus, decreasing motility of bacteria. It was
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announced that components such as eugenol, carvacrol and cinnamaldehyde would prevent the membrane-bound ATPase activity in E. coli and L. monocytogenes (Sikkema, et al., 1995).
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Ribeiro et al. (2013) studied rosemary essential oil (20% v/v) as a way to control
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multidrug-resistant E. coli in coalho cheese. MIC of essential oil was 200 µL/mL which was added to cheese samples inoculated with 105 cfu/mL of test strain and stored at 8°C for 7 days. It
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was revealed that incorporation of essential oil caused a reduction of 2.3 log cycles in the
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bacterial population within the first 24 h of storage that was stable during the whole period of storage. In a similar study, the inhibitory effect of Eugenia caryophyllata Thumb leaves essential
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oil on contaminating microorganisms of coalho cheese was investigated. MIC and Minimum
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Cidal Concentration (MCC) of essential oil were in the range of 2.5-5 and 5-20 µg/mL, respectively. The lowest MIC levels were associated with Y. enterocolitica, Candida albicans,
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Candida parapsilosis and Candida krusei. Cidal concentration of E. caryophyllata essential oil against yeasts was 2-4 fold higher than MIC, while it was 4-8 fold higher for bacteria. This point showed the higher sensitivity of yeasts to essential oil in comparison with bacteria. No cidal effect was reported for Pseudomonas aeruginosa because of being Gram-negative and having low sensitivity toward essential oil. Clove essential oil was also added at 2.5, 5, 10 or 20 µg/g after whey drainage and salting of the cheese and its impact on mesophilic bacteria and fungi in
ACCEPTED MANUSCRIPT vacuum-packed cheese during 15 days of refrigerated storage was determined. Essential oil at 5, 10 or 20 µg/g significantly lowered the count of mesophilic bacteria compared to the control cheese samples. The essential oil at 2.5-20 µg/g showed a static effect on fungi count in comparison to the control samples. It was also implied that due to the oil loss during cheese
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pressing in addition to the presence of fat and protein in the cheese matrix and interaction with
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the essential oil components, antimicrobial effect of the essential oil was alleviated. Furthermore,
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because of a higher availability of nutrients in food matrix compared to laboratory media, repairing the bacterial damaged cells occurred faster (Trajano, Lima, de Souza, & Travassos,
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2010). The main constituent of E. caryophyllata essential oil is eugenol which is believed to
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change the membrane and fatty acid profile, influence the transport of ions and ATP as well as the inhibition of the enzymes such as ATPase, histidine decarboxylase, amylase and protease
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(Nazzaro, et al., 2013; Thoroski, Blank, & Biliaderis, 1989; Wendakoon & Morihiko, 1995).
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Jeong et al. (2014) examined antimicrobial activity of 8 essential oils against Penicillium spp. during cheese ripening. According to the antifungal activity assays, cinnamon (bark and
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leaf) had the highest activity and was selected for incorporation at 2, 5, 10 or 20% (v/v) into the
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cheese. It inhibited the growth of all fungi at levels more than 10% v/v. The cinnamon essential oils effect on cheese starters (FD-DVS ABT-5, KAZU 1 and FD-DVS FLORA-DANICA) was
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also determined. Lactobacilli in FD-DVS ABT-5 starter were not influenced by cinnamon essential oils at any concentration whereas cinnamon leaf essential oil (≥ 20%) and cinnamon bark oil (≥ 10%) inhibited the growth of lactobacilli in KAZU 1 and at ≥ 5%, both essential oils had an inhibitory effect against lactobacilli in FD-DVS FLORA-DANICA starter. The antifungal activity was ascribed to the major components including eugenol, cinnamaldehyde and linalool (Jeong, et al., 2014). Cinnamaldehyde represents three possible mechanisms of action at different
ACCEPTED MANUSCRIPT concentrations. At low levels, it prevents the enzymes involved in cytokine interactions, at sublethal levels, prohibits ATPase and at lethal concentrations disturbs cell membrane. The mode of action in fungi is impairing of cell division (Hyldgaard, et al., 2012; Nazzaro, et al., 2013). Moosavy et al. (2013) described the antibacterial effect of Mentha spicata essential oil (2
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or 2.5% v/w) on L. monocytogenes in traditional Lighvan cheese at different ripening
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temperature (4 or 14°C) and salt water concentrations (12 or 15%) during 60 days. Essential oil
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reduced the growth of L. monocytogenes significantly, but there was no significant difference between the two concentrations used. The number of L. monocytogenes was reduced with
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increasing the essential oil level, salt water percentage, ripening temperature and storage period.
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It was inferred that some environmental factors such as temperature and salt percentage would act as synergists and enhance the efficiency of the essential oil.
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Antimicrobial activity of some plants’ essential oils, including dill, caraway, coriander,
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basil and lemon balm against L. monocytogenes, S. aureus, Bacillus cereus, E. coli O157:H7 and Salmonella typhimurium was surveyed in cheese yogurt (Mohamed, et al., 2013). It was
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illustrated that caraway essential oil had the highest inhibitory effect followed by dill, coriander,
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basil and lemon balm. Therefore, caraway and dill were selected for further studies. The MIC of caraway and dill essential oils were 0.003 and 0.005 mL/mL, respectively. The aforementioned
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essential oils showed the most inhibitory activity against L. monocytogenes and S. typhimurium after 7 and 14 days in cheese yogurt samples. A reduction of 1.3 and 4 log cycles after 7 and 14 days in S. aureus counts were reported by caraway. In addition, it reduced E. coli O157:H7 counts 1.7 log cycles after 7 days and completely inhibited the growth of bacterium after 14 days of storage. Dill essential oil decreased S. aureus number by 0.9 and 1.5 log cycles after 7 and 14 days and inhibited E. coli O157:H7 by 1 log cycle after 7 days and no bacterium was detected
ACCEPTED MANUSCRIPT after 14 days of storage. B. cereus was inhibited thoroughly by caraway and dill essential oils after 7 and 14 days of storage, respectively. These two essential oils were not effective on the growth of lactic acid bacteria (L. delbrueckii ssp.bulgaricus and S. thermophilus) that was in agreement with the results obtained by Ehsani and Mahmoudi (2012) and Zantar et al. (2014),
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implying no influence of Pimpinella anisum, Allium ascalonicum, T. vulgari and Origanum
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essential oils on the growth and metabolic activity of lactic acid bacteria. In contrast, the
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antibacterial effect of Origanum vulgare toward mesophilic lactic acid bacteria was reported by De Souza et al. (2016). In addition, the inhibitory effect of Origanum vulgare var hirtum
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essential oil on lactic acid bacteria was assessed in vitro and in two types of cheeses. The
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essential oil (50, 100, 150 or 200 µg/g) was added to the milk containing S. thermophilus CRL 728 and CRL 813, L. delbruekii ssp. bulgaricus CRL 656 and CRL 468 and Lactococcus lactis
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ssp. lactis CRL 597. Evaluation of cell viability and biological activity showed no change in the
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growth, acidifying activity and fermentative activity of lactic acid bacteria even at the highest concentration of essential oil (Marcial, Gerez, De Kairuz, Araoz, Schuff, & De Valdez, 2016).
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Jay et al. (2005) also pointed out that in comparison with other Gram-positive bacteria, lactic
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acid bacteria were very resistant to EOs. The main component in M. spicata, caraway and dill essential oils is carvone. It would disrupt pH gradient and membrane potential of cells.
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Oosterhaven et al. (1995) announced that carvone reduced the growth of E. coli, S. thermophilus and L. lactis through interrupting the metabolic energy status of the cells. Helander et al. (1998) observed no effect of carvone on E. coli and S. typhimurium as well as the intracellular ATP pool. Fernandes et al. (2016) prepared microencapsulated form of rosemary essential oil and examined its antimicrobial effect in Minas frescal cheese during 15 days at 6°C. Addition of
ACCEPTED MANUSCRIPT 0.5% microencapsulated essential oil reduced the mesophilic bacterial count 1.36 log cycles after three days and 0.73 log cycles after 15 days of storage. In a study carried out by Sadeghi et al. (2016), essential oil of Mentha pulegium at various concentrations (7.5, 15 or 30 µL/mL) on the growth of L. monocytogenes inoculated at 103 cfu/mL in Iranian white-brined cheese during 60
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days was examined. The results showed that in control samples without essential oil up to day 7,
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the L. monocytogenes growth was enhanced, but it was decreased gradually during 60 days. In
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treated samples, the maximum growth lasted for 14 days following a log reduction in bacterium count in other days. The highest antibacterial effect was observed at the level of 0.03%, although
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0.015% essential oil was more acceptable from consumers’ point of view. The compounds
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responsible for the antibacterial effect were pulegone, piperitenone and 1, 8 cineole. Pimpinella anisum (750, 1500 or 3000 ppm) and Allium ascalonicum (500, 1000 or 2000
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ppm) were added to cheese milk and their influence on the growth of E. coli was studied in
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Iranian white-brined cheese during 60 days of ripening. The highest bacterial count reduction was observed at the highest essential oils concentration and A. ascalonicum was more effective
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in this respect. On the other hand, A. ascalonicum essential oil at 750 ppm was the most
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preferred sample by the panelists. The antimicrobial activity of P. anisum essential oil and A. ascalonicum were attributed to benzene derivatives and organo sulfide compounds, respectively;
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that constituted the major parts of the oils (Ehsani et al. 2012). Bunium persicum (black cumin) essential oil was studied in terms of in vitro antibacterial activity against S. typhimurium, E. coli O157:H7, S. aureus and L. monocytogenes as well as its effect at 1 and 2% (w/v) on E. coli O157:H7 and L. monocytogenes in Iranian white cheese. MIC and MBC for E. coli O157:H7, L. monocytogenes, S. typhimurium and S. aureus were 10, 5, 10, 1.25 µL/mL and 20, 10, 20 and 2.5 µL/mL, respectively. In cheese samples containing essential
ACCEPTED MANUSCRIPT oil, the number of E. coli O157:H7 and L. monocytogenes colonies decreased during storage period (45 days) and in this regard, a higher level of essential oil was more effective. It was noted that L. monocytogenes was more sensitive compared to E. coli O157:H7. The authors emphasized that although the higher dose of essential oil reduced the bacterial number in cheese
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samples significantly, but it was not enough to consider the cheese as safe and it was suggested
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that these natural preservatives should be accompanied with other preservation methods. Sensory
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evaluation of un-inoculated cheese samples containing black cumin EO revealed that fortified cheese with essential oil received higher scores than control samples in all sensory characteristics
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(flavor, texture, odor, color and general acceptability) and lower level (1%) of essential oil was
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more acceptable during 45 days of storage (Ehsani, Hashemi, Naghibi, Mohammadi, & Khalili Sadaghiani, 2016). In agreement with the study, Hassanein et al. (2013) reported that
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incorporation of black cumin essential oil (0.1 or 0.2%) in Domiati soft cheeses increased the
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acceptability of products compared to control samples during 30 days of storage. Cuminum cyminum L. essential oil (7.5, 15 or 30 µL/100 mL) was applied alone and in
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combination with a probiotic bacteria Lactobacillus acidophilus (0.5% w/v) in Iranian white-
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brined cheese in order to determine their antibacterial activity against S. aureus during 75 days of storage. It was deduced that essential oil and the probiotic bacteria had synergistic effects on
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decreasing the growth of S. aureus in cheese. The greatest inhibitory activity during 75 days of storage was found in samples containing 30 µL/100 mL essential oil plus 0.5% probiotic. However, regarding cheese acceptability, the cheese manufactured by the milk containing 15 µL/100 mL essential oil received the highest score by the panelists (Sadeghi, Akhondzadeh Basti, Noori, Khanjari, & Partovi, 2013).
ACCEPTED MANUSCRIPT An edible coating based on whey protein isolate and alginate fortified with 1.5 (v/v) ginger essential oil was applied in coating of Kashar cheese and its antimicrobial impact on S. aureus and E. coli O157:H7 was analyzed during 30 days of storage at 4°C. Bacteriostatic effect of the edible coating containing ginger essential oil against S. aureus was observed on the 7th
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day that the bacterial count was reduced to 2.48 log10 cfu/g while in the case of E. coli, this effect
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was detected from the 1st day of storage and the highest level was reported on the 30th day. The
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bactericidal effect was only considered in S. aureus on the 30th day without any effect on E. coli in this time (N. Kavas, Kavas, & Saygili, 2016). In another study by the same authors, an edible
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film based on egg white protein was developed and various levels of sage and lemon balm
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essential oils (0.5, 1 or 2% v/v) were incorporated in order to investigate their antimicrobial efficacy against E. coli O157:H7, L. monocytogenes, S. aureus and yeast and molds in Lor
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cheese during 30 days. It was indicated that sage essential oil had a higher antibacterial effect
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compared to balm essential oil at all concentrations whereas the latter possessed a greater antifungal activity. Edible films with essential oils (0.5%) exhibited a bacteriostatic effect against
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all microorganisms from the 1st day of storage. Sage essential oil had a bactericidal effects
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against L. monocytogenes, S. aureus and yeast-molds on day 15 and 30 of storage, respectively. At 1% (v/v), sage and lemon balm essential oil demonstrated a bacteriostatic effect against S.
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aureus, L. monocytogenes and E. coli from the 1st day of storage. On day 15, a bactericidal effects of sage essential oil against bacteria and antifungal activity were detected. With respect to lemon balm essential oil, no yeasts were observed in cheese samples and a bactericidal effect against L. monocytogenes was reported on day 30. Similarly, at 2% (v/v), sage and lemon balm essential oil demonstrated a bacteriostatic effect against S. aureus, L. monocytogenes and E. coli from the first day of storage. Sage essential oil in the edible film inhibited the growth of S.
ACCEPTED MANUSCRIPT aureus, L. monocytogenes and yeast and molds on day 7 and E. coli was inhibited on day 15. Lemon balm essential oil depicted bactericidal effect against S. aureus and L. monocytogenes on day 15 while in the case of E. coli, this effect was reported on day 30. The most resistant bacterium to essential oils was E. coli O157:H7, followed by S. aureus and L. monocytogenes
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(Kavas & Kavas, 2016). In edible films, greater levels of antimicrobial agents of essential oils
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would remain in the film and at the surface of the food which prevents the microorganisms for a
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longer period (Cagri, Ustunol, & Ryser, 2002; Coma, Martial-Gros, Garreau, Copinet, Salin, & Deschamps, 2002).
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In another similar study by Kavas and Kavas (2014), whey protein isolate-based edible
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films containing mint essential oil (1, 2, 3 or 4% v/v) were applied in Lor cheese and microbiological quality was assessed during 15 days of storage. Antimicrobial effect was
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augmented by increasing the mint essential oil concentration and storage duration. Mint oil at the
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level of 4% had bactericidal and antifungal activity and all of the microorganisms were prohibited. The highest antimicrobial activities at the level of 3% were for S. aureus (0.83 log10
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cfu/g with 5.9 log10 cfu/g decrease), yeast-molds (1.41 log10 cfu/g with 4.8 log10 cfu/g decrease),
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L. monocytogenes (2.25 log10 cfu/g with 4.53 log10 cfu/g reduction) and E. coli (2.89 log10 cfu/g with 4.01 decrease). The most resistant microorganism to essential oil was E. coli and the most
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sensitive was S. aureus. The bactericidal effect against S. aureus started from the day 15 at a dose of 2% and on day 7 at a dose of 3% essential oil. This effect at the level of 3% essential oil were recorded on day 10 and 15 of storage for yeast-molds and L. monocytogenes, respectively. Antibacterial effectiveness of tarragon, spearmint and pennyroyal essential oils individually and in combination with monolaurine was evaluated in culture medium and white cheese against L. monocytogenes. Determination of MIC in Mueller Hinton broth showed the
ACCEPTED MANUSCRIPT values of 50 ppm, 0.4, 2 and 0.2% for monolaurine, spearmint, tarragon and pennyroyal essential oils, respectively. Fractional inhibitory concentration indices (FIC) determined for combination of monolaurine along with spearmint, tarragon and pennyroyal essential oils were 0.75, 0.66 and 1, respectively. The results were indicative of an additive effect of these combinations. The
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results of antibacterial activity of essential oils (0.2, 0.4 or 0.6% v/v) individually or in
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combination with monolaurine (200 or 400 ppm) in soft cheese during 168 h revealed that by
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increasing the essential oil level, antilisterial activity was enhanced whilst in the case of monolaurine, no significant further reduction at the level of 400 ppm was reported compared to
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200 ppm. The highest inhibition was observed in a combination of 0.6% pennyroyal and 400
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ppm monolaurine. Addition of spearmint essential oil to milk caused a significant reduction in L. monocytogenes count and this reduction was amplified in the presence of monolaurine.
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Nevertheless, no difference was observed in inhibition of L. monocytogenes at two different
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concentrations of monolaurine. With respect to tarragon, level of 0.4% alongside with 200 ppm monolaurine had a bactericidal effect. Moreover, at the highest level of tarragon (0.6%) and
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monolaurine (400 ppm), no L. monocytogenes was detected after 12 h till the end of storage.
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Sensory evaluation of cheese samples indicated that addition of 0.2% essential oil to cheese improved the sensory properties and a cheese with 0.4% was also acceptable by the panelists, but
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a level of 0.6% essential oil reduced the acceptability of the samples (Hamedi, et al., 2014). Chitosan coating containing rosemary and oregano essential oils was applied in semi-hard goat cheese and it was announced that in cheese samples coated twice with oregano essential oil, the greatest antimicrobial activity against Mucor and Penicllium was observed. Furthermore, the aforementioned samples were the most acceptable ones in respect of flavor and aroma from consumers’ point of view (Embuena, et al., 2016).
ACCEPTED MANUSCRIPT Dannenberg et al. (2016) evaluated the antimicrobial activity of essential oil derived from pink pepper tree fruit (Schinus terebinthifolius Raddi) against L. monocytogenes in Minas- type fresh cheese during 30 days of storage at 4°C. It was demonstrated that EO from ripe and mature fruits was more efficient toward L. monocytogenes compared to green fruits and the bacterial
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growth was reduced by 1.3 log cfu/g in 30 days.
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A nanoemulsion-based edible coating containing oregano essential oil (1.5, 2 or 2.5%
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(w/w) and mandarin fiber was prepared for coating of low-fat cheese and its antimicrobial activity against S. aureus inoculated (106 cfu/g) onto the cheese pieces was verified during 15
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days. Coating with 1.5% EO had no effect on reduction of S. aureus counts whereas the bacterial
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population decreased 1.4 and 1.5 log cfu/g at the levels of 2 and 2.5% EO, respectively. The antimicrobial activity was attributed to carvacrol, the major constituents of EO (Artiga-Artigas,
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Acevedo-Fani, & Martín-Belloso, 2017).
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5. Limitations and perspectives of EOs application in cheese Despite of the suitable efficacy of essential oils in restriction of growth and survival of
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microorganisms in cheese, some limitations have been recognized in their application due to the
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interaction of essential oil constituents with food components such as fat, carbohydrate and proteins that can alleviate the antimicrobial effects of mentioned essential oils (Burt, 2004; Gutierrez, et al., 2008; Juven, et al., 1994; Skandamis, Tsigarida, & Nychas, 2000) as well as increasing the required concentration to obtain sufficient antimicrobial activity (Hyldgaard, et al., 2012). Proteins interact with phenolic compounds present in EOs whereas fats surround the hydrophobic constituents of EOs, restricting their availability to target sites of microorganisms (Burt, 2004). In addition, the physical structure of the cheese would affect the distribution of EO
ACCEPTED MANUSCRIPT in the system and limiting its availability to microbial cells, resulting in reduction of antimicrobial effect (Gutierrez, et al., 2008; Gutierrez, Barry-Ryan, & Bourke, 2009). Moreover, it was inferred that reduction of pH, oxygen level and temperature would increase the antimicrobial effect of EOs (Skandamis, et al., 2000; Tsigarida, Skandamis, & Nychas, 2000).
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Lower pH would enhance the hydrophobicity of EOs, enabling them to easily dissolve cell
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membrane and pervade the target sites of the bacteria (Holley & Patel, 2005; Juven, et al., 1994).
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Since EOs have intense aroma, utilization at high concentrations in order to compensate their interaction with food components could result in sensory defects (Hyldgaard, et al., 2012).
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In order to rectify this shortcoming, various approaches have been proposed. One way is to
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incorporate the EOs in edible films and coatings. In this sense, transmission of antimicrobial components from film or coating to food is slow and a long-term antimicrobial activity can be
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achieved (Coma, et al., 2002; Sánchez-González, Vargas, González-Martínez, Chiralt, & Cháfer,
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2011). Micro- or nanoencapsulation of essential oils is another approach which improves the stability of EOs as well as extending the time of their antimicrobial activity. Fernandes et al.
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(2016) used inulin and whey protein isolate for microencapsulation of rosemary essential oil and
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added microcapsules to Minas Frescal cheese. It was illustrated that microencapsulated rosemary essential oil postponed the growth of mesophilic bacteria and prolonged the shelf-life of the
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cheese. It is also possible to use lower levels of EOs via combination of EO with other antimicrobial substances or technologies which act through synergistic effect (Nguefack, et al., 2012). Parsaeimehr et al. (2010) reported a remarkable synergistic effect of Z. multiflora Boiss essential oil and nisin on inhibition of enterotoxin and α-toxin generation by S. aureus during manufacturing process of a traditional Iranian white brine cheese and in BHI broth, respectively.
ACCEPTED MANUSCRIPT Essential oils are usually expensive because their production can be very costly depending on the type of plant used, method of EO extraction and facilities as well as the labor and energy costs. In some cases, it needs high amounts of plants to yield a little amount of essential oil. Moreover, some companies can present an essential oil with higher purity
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that undoubtedly affects its price. Nevertheless, many essential oils and their active
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constituents are active against bacteria and fungi and they can be produced from commonly
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available raw materials; perhaps in many cases right at the site of use so as to be rather lowcost treatments (Pandey, Kumar, Singh, Tripathi, & Bajpai, 2017). Essential oils can be used
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in agro industries instead of synthetic pesticides to control plant diseases causing severe
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destruction to crops. Using pesticides causes environmental pollution, increase the risk of pesticide residue and are threats to human health. However, application of EOs as an
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alternative antifungal agents would mitigate these disadvantages (Kotan, Kordali, Cakir,
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Kesdek, Kaya, & Kilic, 2008). In spite of high cost of EOs, due to being natural, safe, biodegradable and having little impact on non-target organisms, they can be proposed as
6. Conclusion
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potential antimicrobial agents for food commodity preservation in the near future.
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Several essential oils can be applied as natural antimicrobial agents in order to inhibit microbial deterioration of cheeses and extending the shelf-life. Major compounds including thymol, carvacrol, eugenol, carvone and cinnamaldehyde are mainly responsible for exerting antimicrobial activity through various mechanisms such as increasing the cell permeability, change of membrane fatty acids and effect on membrane proteins. However, the concentration of these substances applied in cheeses should be considered carefully because of their possible negative impacts on organoleptic properties. On the other hand, other approaches comprising
ACCEPTED MANUSCRIPT microencapsulation of essential oil, incorporation into edible film and coating or application of these oils along with other antimicrobial agents and preservatives can be more efficient in prevention of microbial growth and enhancing cheese safety and quality which should be
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investigated in future studies.
ACCEPTED MANUSCRIPT Acknowledgements This study is related to the project NO. 1395/74038 from Student Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran. We also appreciate the “Student Research Committee” and “Research & Technology Chancellor” in Shahid Beheshti University
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The authors declare that there is no conflict of interest.
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Conflict of interest
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of Medical Sciences for their financial support of this study.
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50.
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Amatiste, S., Sagrafoli, D., Giacinti, G., Rosa, G., Carfora, V., Marri, N., Tammaro, A., Bovi, E., &
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Rosati, R. (2014). Antimicrobial activity of essential oils against staphylococcus aureus in fresh sheep cheese. Italian Journal of Food Safety, 3(3), 148-150.
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Angioni, A., Barra, A., Coroneo, V., Dessi, S., & Cabras, P. (2006). Chemical composition, seasonal variability, and antifungal activity of Lavandula stoechas L. ssp. stoechas essential oils from
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stem/leaves and Flowers. Journal of Agricultural and Food Chemistry, 54(12), 4364-4370.
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Artiga-Artigas, M., Acevedo-Fani, A., & Martín-Belloso, O. (2017). Improving the shelf-life of low-fat cut cheese using nanoemulsion-based edible coatings containing oregano essential oil and
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mandarin fiber. Food Control, 76, 1-12.
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Basim, H., Yegen, O., & Zeller, W. (2000). Antibacterial effect of essential oil of Thymbra spicata L. var. spicata on some plant pathogenic bacteria/Die antibakterielle Wirkung des ätherischen Öls von spicata
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Cell wall destruction
Membrane protein damage
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Mechanisms of essential oils activity on microorganisms
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Increasing permeability
Hydrolysis of ATP and decreasing in Synthesis of ATP
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Leakage of the cell contents
Coagulation of cytoplasm
Reduction of proton motive force
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Damaging the cytoplasmic membrane
Figure 1. Mechanisms of antimicrobial activity of EO.
Reduction of the intracellular ATP pool
ACCEPTED MANUSCRIPT Table 1. Selected publications on the antimicrobial activity of essential oils in cheeses Type of essential oil
EO concentration
Microorganism type
Remarks
Reference
Wagashi
Ocimum gratissimum
200, 400, 600, 800, 1000 mg/L
Aspergillus (flavus and tamarii), Fusarium (poae and verticillioides) and Penicillium (citrinum and griseofulvum
Mycelia growth was reduced by increasing the essential oil level. Fungistatic activity against species was observed MIC values ranged from 800-1000 mg/L.
(Philippe, Souaïbou, Guy, et al., 2012)
Wagashi
Cinnamomum zeylanicum
200, 400, 600, 800, 1000 mg/L
Aspergillus terreus, Aspergillus ustus, Aspergillus niger, Aspergillus aculeatus, Penicillium brevicompactum and Scopulariopsis brevicaulis
The most susceptible strains were Scopulariopsis brevicaulis and Aspergillus terreus showing MFC of 600 to 1000 mg/L; whereas Aspergillus aculeatus was the most resistant mold to cinnamon essential oil.
(Philippe, Souaïbou, Paulin, et al., 2012)
L. monocytogenes
In the low-fat cheese, all four oils at1%reduced L. monocytogenes to <1 log10 cfu/mL. In the full-fat cheese, oil of clove was the only oil to achieve this reduction.
(SmithPalmer, et al., 2001)
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Type of cheese
'Continued'
Pimenta racemosa, Syzygium aromaticum, Cinnamomum verum and Thymus vulgaris
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Full-fat and low-fat cheeses
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O. gratissimum
0.1, 0.5 and 1%
S. enteritidis
ACCEPTED MANUSCRIPT Table 1. Continued Type of essential oil
EO concentration
Microorganism type
Remarks
Reference
Feta
Oregano Thyme combined with MAP (50% CO2 and 50% N2)
0.1 or 0.2 mL/100 g 0.1 mL/100 g
L. monocytogenes E. coli O157:H7
The growth of bacteria was restricted by the essential oils with a greater inhibitory effect toward L. monocytogenes
(Govaris, et al., 2011)
Coalho
T. vulgaris
1.25 and 2.5 µL/mL
S.aureus and L. monocytogenes Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris
Goat cheese
T.vulgari and Origanum compactum
0.05 and 0.1%
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Type of cheese
(de Carvalho, et al., 2015)
Eight spoilage and pathogenic bacteria
The highest antibacterial activity was reported for T.vulgari against S.aureus. The lowest activity was obtained by O. compactum for Yersinia enterocolitica. The maximum shelf life was obtained by 0.1% O. compactum.
(Zantar, et al., 2014)
Microorganism type
Remarks
Reference
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No inhibitory effect was observed at 1.25 µL/mL. At 2.5 µL/mL S.aureus, L. monocytogenes and Lactococcus ssp. were inhibited.
'Continued'
Table 1. Continued Type of cheese
Type of essential oil
EO concentration
ACCEPTED MANUSCRIPT
Z. multiflora Boiss
0.05, 0.1, 0.2 and 0.4%
Mesophilic lactobacilli enterobacteriaceae
Lower microbial counts were observed compared to the control at the end of storage.
(Es'haghi Gorji, et al., 2014)
Iranian ultra-filtered white cheese
Z. multiflora Bioss
50, 100, 200, 400, 600 and 1000 ppm
Aspergillus flavus
No fungal growth was observed at 400 ppm and higher and a dose of 1000 ppm had a fungcidal effect. Z. multiflora Boiss essential oil had an inhibitory effect on radial fungal growth and aflatoxin production at all levels.
(Gandomi, et al., 2009)
Coalho
Rosemary
20% v/v
E. coli
A decline of 2.3 log cycle in the bacterial population within the first 24 hour of storage.
(Ribeiro, et al., 2013)
Appenzeller
Cinnamon
2, 5, 10 and 20% v/v
Penicillium spp
EO inhibited the growth of all fungi at levels more than 10% v/v.
(Jeong, et al., 2014)
2, 2, 5% v/w
L. monocytogenes
Higher level of EO was more efficient in bacterial count reduction.
(Moosavy, et al., 2013)
EO concentration
Microorganism type
Remarks
Reference
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Lighvan
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Gouda
Mentha spicata
'Continued' Table 1. Continued Type of cheese
Type of essential oil
ACCEPTED MANUSCRIPT
Bunium persicum
1 and 2% w/v
E. coli O157:H7 L. monocytogenes
The bacterial number decreased during storage period and higher level was more effective.
(Ehsani, et al., 2016)
Iranian white-brined cheese
Mentha pulegium
7.5, 15 and 30 µL/mL
L. monocytogenes
The highest antibacterial effect was observed at the level of 0.03% but, 0.015% was more acceptable by panelists. The maximum growth lasted for 14 days following a log reduction in bacterium count.
(Sadeghi, et al., 2016)
Iranian white-brined cheese
Pimpinella anisum
750, 1500 and 3000 ppm 500, 1000 and 2000 ppm
E. coli
The highest reduction was obtained at the highest level.
(Ehsani, et al., 2012)
S. aureus
The greatest inhibitory activity was achieved in samples containing 30 µL/100 mL essential oil plus 0.5% probiotic.
(Sadeghi, et al., 2013)
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7.5, 15 and 30 µL/100 mL
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Cuminum cyminum L. plus Lactobacillus acidophilus
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Allium ascalonicum Iranian white-brined cheese
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Iranian white-brined cheese
ACCEPTED MANUSCRIPT Table 2. Major compounds of essential oils used in different cheeses Essential oil used
Main components
Reference
Wagashi
Ocimum gratissimum
thymol (28.1%), γ-terpinene (21.30%) and p-cymene (16.5%)
(Philippe, Souaïbou, Guy, et al., 2012)
Feta
Origanum vulgare
Carvacrol (80.05%), p-cymene (5.25%), thymol (4.81%) and γterpinene (2.90%)
(Govaris, et al., 2011)
Thymus vulgaris
Thymol (44.3%), p-cymene (19.8%), carvacrol (14.2%), thymoquinone (6%)
Coalho
Thymus vulgaris
Thymol (43.19%), p-cymene (28.55%), γ-terpinene (6.36%), linalool (5.57%), carvacrol (3.14%)
(de Carvalho, et al., 2015)
Gouda
Zataria multiflora
Carvacrol (71.12%), γ-terpinene (7.34%), α-pinene (4.26%)
(Es'haghi Gorji, et al., 2014),
Coalho
Eugenia caryophyllata Thumb
Eugenol (74%), α–humullene (9.62%), d-cadinene (4.64%)
(Trajano, et al., 2010)
Lighvan
Mentha spicata
Carvone (78.76%), Limonene (11.5%), cis-dihydro Carveol (1.43%)
(Moosavy, et al., 2013)
Cheese yogurt
Carum carvi
Carvone (74.4%), Limonene (15.5%), α-pinene (8.29%)
(Mohamed, et al., 2013)
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Cheese type
Carvone (64.59%), Limonen (20.64%), Dillapiol (12.04%)
Origanum compactum
Carvacrol (75.6%), p-cymene (8.3%), γ-terpinene (6.6%)
Thymus vulgaris
Carvacrol (81.2%), p-cymene (3.8%), γ-terpinene (2.7%), α-Humulene(2%)
Iranian white brined
Mint essential oil
neo Menthol (38.7%), IsoMenthone (31.32%) and 1.8 Cineole (7. 87%)
(Tehrani, et al., 2015)
Iranian white
Mentha pulegium
pulegone (36.68%), piperitenone (16.88%), and 1,8 cineole (14.58%)
(Sadeghi, et al., 2016)
Goat
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Anethum graveolens
'Continued' 48
(Zantar, et al., 2014)
ACCEPTED MANUSCRIPT Table 2. Continued Essential oil used
Main components
Reference
White brined
Cuminum cyminum
Cuminaldehyde (29.02%), αterpinene-7-al (20.7%), γ- terpinene (12.94%)
(Sadeghi, et al., 2013)
Traditional Argentinean
Oreganum vulgare L. var hirtum
γ-terpinene (15.1%), terpinen-4-ol (15.5%) and thymol (13.0%)
(Marcial, et al., 2016)
Kashar
Zingiber officinale Roscoe
Zingiberene (39.12%), geranial (13.44%), camphene (11.15%), β-sesquiphellandrene (10.64%)
(N. Kavas, et al., 2016)
Lor
Mentha spicata
Carvone (40.5%), 1, 8-cineole (23.3%), cis-carveol (19.1%), β-caryophyllene (3.9%)
(Gökhan Kavas, et al., 2014)
Feta
Zataria multiflora Boiss
Caryophyllene (25.1%), carvacrol (22.8%), thymol (22.5%), α-Terpineol (19.1%)
(Ghasemi, et al., 2015)
Coalho
Origanum vulgare L.
Carvacrol (69.0%), thymol (14.12%), γ-terpinene (3.71%), and p-cymene (3.67%)
(De Souza, et al., 2016)
Iranian white cheese
Bunium Persicum
Frescal
Rosmarinus officinalis
White brined
Pimpinella anisum
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Cheese type
Cuminaldehyde (11.4%), γ- terpinene (11.37%), α-Pinene (11.27%) and α– terpinene (11.13%)
(Ehsani, et al., 2016)
1, 8-Cineole (40.8%), Camphor (Fernandes, et al., (28.8%), Α-Pinene (10.9%), Β-Pinene 2016) (8.1%) Benzene (37.8%), Longifolene-(V4) (28.8%), Phenol, 2-methoxy-4-(1propenyl) (11.2%)
Allium ascalonicum
Diallyl disulphide (20%), Trisulfide, methyl 2-propenyl (18.1%), Trisulfide, di-2-propenyl (15.3%)
Zataria multiflora Boiss
Carvacrol (71.12%), γ-terpinene (7.34%), α-pinene (4.26%), eucaliptol (3.37%)
49
(Ehsani, et al., 2012)
(Noori, et al., 2012)
ACCEPTED MANUSCRIPT Highlights:
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This paper gives an overview of cheese spoilage and pathogen bacteria. Antimicrobial properties of essential oils are reviewed. Application of essential oils as natural antimicrobial agents in different cheeses are investigated The main components of essential oils that have antimicrobial properties are overviewed
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essential
oils
as
antimicrobial
Nasim Khorshidian, Mojtaba Yousefi, Elham Khanniri, Amir Mohammad Mortazavian PII: DOI: Reference:
S1466-8564(17)30321-1 doi:10.1016/j.ifset.2017.09.020 INNFOO 1858
To appear in:
Innovative Food Science and Emerging Technologies
Received date: Revised date: Accepted date:
16 March 2017 15 August 2017 28 September 2017
Please cite this article as: Nasim Khorshidian, Mojtaba Yousefi, Elham Khanniri, Amir Mohammad Mortazavian , Potential application of essential oils as antimicrobial preservatives in cheese. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Innfoo(2017), doi:10.1016/ j.ifset.2017.09.020
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ACCEPTED MANUSCRIPT
Potential application of essential oils as antimicrobial preservatives in cheese
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Nasim Khorshidian1, 2, Mojtaba Yousefi1, 2, Elham Khanniri1, Amir Mohammad Mortazavian3*
Student Research Committee, Department of Food Technology, Faculty of Nutrition Sciences and Food Technology/National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
2
Food Safety Research Center (Salt), School of Nutrition and Food Sciences, Semnan University of Medical Sciences, Semnan, Iran
3
Department of Food Technology, Faculty of Nutrition Sciences and Food Technology/National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Corresponding authors:
Amir M. Mortazavian (ph.D)
Ph: + 98-912-7114977
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Fax: +98 21 22360657 E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract Increasing demand of consumers to use food products without preservatives or natural preservatives as possible has compelled the food industries for utilization of preservatives with herbal and microbial origins instead of artificial preservative in their production. Essential oils
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are volatile, natural plant-derived substances that are used in medicine, food flavoring and food preservation. These diverse compounds represent considerable potential antioxidant, antibacterial
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and antifungal activities via various mechanisms. This review represents an overview on the
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impact of essential oils and their constituents as natural antimicrobials versus common pathogenic and spoilage microorganisms in cheese along with the related mechanisms of actions.
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Industrial Relevance: Natural preservatives have proven popularity such that interest continues
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in substituting artificial additives with natural. Therefore, production of safe food without or with low amounts of synthetic preservatives is one of the most important challenges in the food
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industry. This review, introduces the potential application of essential oils as natural
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antimicrobial agents for reduction of common spoilage and pathogenic bacteria as well as molds and yeasts in cheese-making industry.
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Keywords: Cheese; Essential oil; Spoilage; Antimicrobial; Foodborne pathogen
ACCEPTED MANUSCRIPT 1. Introduction Food safety is one of the main issues in the food industry and apart from spoilage of foodstuff, there is always concern about the outbreak of foodborne illnesses among food manufacturers, regulatory agencies, researchers and consumers. Therefore, producing safer food is one of the
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most important priorities in the food industry (Friedman, Henika, & Mandrell, 2002; Mohamed,
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Zaky, Kassem, Abbas, Salem, & Said-Al Ahl, 2013; Smith-Palmer, Stewart, & Fyfe, 2001).
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Different methods, including thermal processing, lowering of water activity, various methods of
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packaging, irradiation, high pressure, high-intensity pulsed electric field processing and the addition of chemical preservatives have been utilized to produce safer foodstuffs (Čvek, Markov,
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Frece, Dragičević, Majica, & Delaš, 2010; Delves-Broughton, 2005; Jeong, et al., 2014; Lee,
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Yeo, Park, Kang, & Hahm, 2010; López, Sánchez, Batlle, & Nerín, 2005). Food-grade and GRAS food additives have been normally used by the food industry for some time; however,
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synthetic preservatives are now being replaced by natural preservatives as consumers seek foods
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for a healthier lifestyle (Zantar, Yedri, Mrabet, Laglaoui, Bakkali, & Zerrouk, 2014). Although a specific category for natural additives has not been defined, there are various natural
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antioxidants, antimicrobials, sweeteners and coloring agents that are derived from animals, plants
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and microorganisms (Carocho, Morales, & Ferreira, 2015). Bacteriocins, natamycin, reuterin from microbial sources; lysozyme, lactoperoxidase, lactoferrin from animal sources; polyphenols and essential oils from plant sources can be named as examples of natural preservatives (Carocho, et al., 2015). Essential oils (EO) that are lipophilic liquids, extracted from divers plants containing different natural, biologically active components have antimicrobial and antioxidant properties (Amatiste, et al., 2014; Hamedi, Razavi-Rohani, & Gandomi, 2014; Yousefi Asli, Khorshidian, Mortazavian, & Hosseini, 2017). Antimicrobial properties of
ACCEPTED MANUSCRIPT essential oils against a wide range of microorganisms have been reported in various studies (Čvek, et al., 2010; Delves-Broughton, 2005; Jeong, et al., 2014; Kotzekidou, Giannakidis, & Boulamatsis, 2008; Lee, et al., 2010; Smith-Palmer, et al., 2001; Yahyazadeh, Omidbaigi, Zare, & Taheri, 2008). Due to the hydrophobicity of essential oils’ components, they easily pass
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through the bacterial cell membrane interfering with molecular transport mechanisms leading to
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cell inactivation (Burt, 2004; Goñi, López, Sánchez, Gómez-Lus, Becerril, & Nerín, 2009).
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Essential oils commonly exhibit greater inhibitory properties against Gram-positive than Gramnegative bacteria due to the lipopolysaccharide barrier in the outer membrane of Gram-negative
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bacteria (Techathuvanan, Reyes, David, & Davidson, 2014; Tehrani & Sadeghi, 2015). As
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mentioned before, essential oils have antibacterial and antifungal activities against different microorganisms and as no review has been published in this context, the aim of this paper was to
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review the antimicrobial properties of essential oils derived from different plant sources used in
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different types of cheeses.
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2. Spoilage and pathogenic microorganisms in cheese
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Cheese is one of the most popular dairy products which is a rich source of essential nutrients such as proteins, vitamins, minerals, short-chain fatty acids and certain trans-fatty acids that can
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be regarded as a part of healthy diet (López-Expósito, Amigo, & Recio, 2012). Cheese spoilage can be caused by both bacteria and fungi. However, the type of the spoilage varies depending on the type of cheese. Common spoilage molds in cheeses belong to the genera Penicillium, Aspergillus, Cladosporium, Mucor, Fusarium, Monilia and Alternaria (Johnson, 2002). Yeasts frequently found in spoiled cheese include Candida spp., Yarrowia lipolytica, Pichia spp., Kluyveromyces marxianus, Geothricum candidum and Debaryomyces hansenii (Filtenborg, Frisvad, & Thrane, 1996; Fleet, 1990; Johnson, 2002; Kosse, Seiler, Amann, Ludwig, & Scherer,
ACCEPTED MANUSCRIPT 1997). The growth of mold is associated with undesirable taints and odors, liquefaction of the curd and in some cases, production of mycotoxins. In fresh cheeses such as cottage cheese with a higher pH compared to other types of cheeses, Gram-negative psychrotrophic species, such as Pseudomonas and some coliforms can cause spoilage. The most important psychrotrophic
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species are Pseudomonas spp., Alcaligenes spp., Achromobacter spp. and Flavobacterium spp.
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Other bacteria involved in cheese spoilage are Enterobacteriaceae, Bacillus spp., Clostridium
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butyricum, Clostridium tyrobutyricum and Clostridium sporogenes which cause blowing (Robinson, Tamime, & Wszolek, 2002).
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Although cheese is a safe food, occasionally recalls and outbreaks do occur (Kousta,
Listeria
monocytogenes,
Salmonella
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Mataragas, Skandamis, & Drosinos, 2010). The most serious outbreaks have been caused by and enteropathogenic
Escherichia coli
(EPEC).
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Enterohaemorrhagic strains of E. coli (EHEC), such as E. coli O157:H7 may cause serious
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infections and fatalities in the very young and the elderly. E. coli O157:H7 is considered to be a potentially high-risk pathogen in cheese due to its high tolerability to low pH for a long period of
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time and its association with unpasteurized milk. In this regard, soft cheeses made from raw milk
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are high-risk products (Reitsma & Henning, 1996). L. monocytogenes is a psychrotrophic and fairly heat-tolerant pathogen that can be present
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in raw milk. It may enter the cheese process if hygienic conditions are not maintained in the processing. In surface-ripened cheeses, the pH of the cheese is increased from around 5 to 6-7 because of desired surface mold growth in these types of cheeses. High pH along with high moisture content and temperature of the ripening rooms (8-12°C) provide suitable conditions for rapid growth of L. monocytogenes (Carpentier & Cerf, 2011; Gould, Mungai, & Barton Behravesh, 2014; Melo, Andrew, & Faleiro, 2015).
ACCEPTED MANUSCRIPT Salmonella can be found in cheeses made from raw milk and do not survive pasteurization (Villarruel-L, et al., 2016). However, they may also enter cheese as post-pasteurization contaminants if the process is not properly controlled. If acid production during manufacture is slow, Salmonella may grow during cheese making and have been shown to survive for more than
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60 days in some cheeses (Fernandes, 2009). Staphylococcus aureus is able to tolerate salt and
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moderate acidity and can multiply during cheese manufacture and ripening in soft cheeses. It
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may survive for long periods even in hard cheeses and if high enough populations are developed, enterotoxin may be produced which persists for many months even after the cells have lost their
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viability (Fernandes, 2009).
3. Essential oils and mechanisms of their antimicrobial activity
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Essential oils are natural aromatic and volatile liquids which might be derived from different
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parts of the plants, including flowers, roots, barks, leaves, seeds, peel, fruits, wood and the whole plants (Bozin, Mimica-Dukic, Simin, & Anackov, 2006; Hammer, Carson, & Riley, 1999;
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Hyldgaard, Mygind, & Meyer, 2012). The International Organization for Standardization (ISO)
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has defined essential oils as ‘product obtained from a natural raw material of plant origin, by steam distillation, by mechanical processes from the epicarp of citrus fruits, or by dry distillation,
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after separation of the aqueous phase if any by physical processes’, going on to specify that ‘the essential oil can undergo physical treatments, which do not result in any significant change in its composition’ (ISO/DIS9235, 2013). Essential oils are a mixture of various compounds characterized by aromatic smell, generally liquid colorless to slightly yellowish color and insoluble in water, but soluble in organic solvents (Nazzaro, Fratianni, De Martino, Coppola, & De Feo, 2013). There are almost 3000 different essential oils with approximately 300 used commercially in the flavoring and fragrances market (Burt, 2004). Their composition varies
ACCEPTED MANUSCRIPT depending on the type of the plant species, geographic origin of the plant, climate conditions, soil composition, vegetative cycle stage and the part of the plant that was used for EO extraction (Angioni, Barra, Coroneo, Dessi, & Cabras, 2006; Masotti, Juteau, Bessière, & Viano, 2003). They are usually secreted as secondary metabolites having roles in pollination or defense
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mechanisms against bacteria and fungi (Tajkarimi, Ibrahim, & Cliver, 2010). Essential oils are
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extracted by means of water or steam distillation, maceration and supercritical fluid extraction
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(Burt, 2004; Edris, 2007; Shannon, Milillo, Johnson, & Ricke, 2011). Although EOs are used primarily as flavoring agents in the food industry, due to possessing antimicrobial activity, they
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can be incorporated into food products in order to extend the shelf-life. However, the
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prerequisites of this application are knowledge about EOs properties, minimum inhibitory concentrations (MIC), the target microorganisms, the mode of action and the probable
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interactions with food matrix as well as sensory quality of the food. The antimicrobial activity of
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EOs can be completely associated with their active constituents (Hyldgaard, et al., 2012). About 90-95% of the whole EO comprises volatile components consisting monoterpenes and
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sesquiterpene hydrocarbons and their oxygenated derivatives besides aliphatic aldehydes,
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alcohols and esters. The non-volatile portion constitutes about 5-10% of the whole EO that includes mainly hydrocarbons, fatty acids, sterols, carotenoids, waxes, cumarines and flavonoids
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(Luque De Castro, Jiménez-Carmona, & Fernández-Pérez, 1999). The most active antimicrobial compounds present in EOs can be divided into four groups based on the chemical structure: terpens (e.g., p-cymene, limonene), terpenoids (e.g., thymol, carvacrol), phenylpropenes (e.g., eugenol, vanillin) and other compounds such as allicin or isothiocyanates (Hyldgaard, et al., 2012).
ACCEPTED MANUSCRIPT The mechanism of action of EOs against microorganisms has not yet been elucidated entirely and cannot be attributed to a single mechanism. There are several locations in the microorganisms which are supposed to be the sites of the action for EOs (Nazzaro, et al., 2013). The fundamental mechanisms of EOs antimicrobial activity are shown in Figure 1. The
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antimicrobial activity is pertained to the hydrophilic or lipophilic character of the EOs’
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components, the microorganism type and its cell wall structure. It is assumed that EOs are more
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effective against Gram-positive bacteria compared to Gram-negative ones (Cimanga, et al., 2002; Hyldgaard, et al., 2012; Smith-Palmer, Stewart, & Fyfe, 1998). The cell wall of Gram-positive
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bacteria consists of peptidoglycan (90-95%) along with teichoic acid and proteins linked to it.
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Since the major parts of EOs are hydrophobic, they interact with the cell membrane and easily pass through it to the cytoplasm. The cell wall structure in Gram-negative bacteria is more
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complex comprised of a monolayer of peptidoglycan surrounded by an outer membrane
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consisting of proteins and lipopolysaccharide (LPS). This outer cell boundary is charged, so it has a hydrophilic nature; however, hydrophobic compounds can pass through this barrier
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(Nazzaro, et al., 2013; Nikaido, 1994; Vaara, 1992).
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The lipophilic nature of the hydrocarbon skeleton and hydrophilic nature of functional groups of EOs play substantial roles in the antimicrobial effects of these compounds. The
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greatest antimicrobial activity in EOs is found with phenolic compounds, following in order by aldehydes, ketones, alcohols, ethers and hydrocarbons (Kalemba & Kunicka, 2003). The activity of phenols is attributed to the acidic characteristic of the hydroxyl group. These compounds change the cell permeability, interfere with the enzymes involved in energy production and interrupt the protein motive force that eventually leads to the cell death (Basim, Yegen, & Zeller,
ACCEPTED MANUSCRIPT 2000). The shape of the bacteria also can be determinative of the activity of EOs and it has been stated that rod-shaped cells are more susceptible than coccoid-shape (Nazzaro, et al., 2013).
4. Application of EOs in various types of cheese
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Table 1 summarizes selected publications on the antimicrobial activity of different
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essential oils applied in cheeses. The major compounds of each essential oil used in cheeses are
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shown in Table 2.
Effect of essential oil from Ocimum gratissimum (200, 400, 600, 800 or 1000 mg/L) was
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assessed against six fungi including Aspergillus flavus, Aspergillus tamarii, Fusarium poae,
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Fusarium verticillioides, Penicillium citrinum and Penicillium griseofulvum isolated from a traditional cheese named wagashi. Wagashi is a soft, brine-pickled cheese made from whole
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cow’s milk by adding the juice of crushed stems of Bryophylum and no rennet or lactic starter is
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used. It is almost fried before consumption. It was noted that mycelia growth was reduced by increasing the essential oil level. A significant fungistatic activity against all the examined
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species was observed with MIC values ranged from 800-1000 mg/L. Penicillium species and F.
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poae were the most susceptible fungi to the essential oil. O. gratissimum essential oil efficacy was attributed to its major compounds, including thymol, γ-terpinene and p-cymene (Philippe,
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Souaïbou, Guy, et al., 2012). Similarly, antifungal activity of O. gratissimum essential oil was attributed to thymol. It was demonstrated that thymol interacts with cell envelope to impair vesicles and cell membranes as well as ergosterol synthesis which has a role in membrane integrity and fluidity (Ahmad, et al., 2011; Hyldgaard, et al., 2012). In vitro antifungal activity of Cinnamomum zeylanicum and O. gratissimum (200, 400, 600, 800 or 1000 mg/L) were explored against six molds including Aspergillus terreus, Aspergillus ustus, Aspergillus niger, Aspergillus aculeatus, Penicillium brevicompactum and
ACCEPTED MANUSCRIPT Scopulariopsis brevicaulis isolated from wagashi cheese. Results showed that O. gratissimum had fungistatic activity against all species with MIC values in the range of 400-1000 mg/mL and fungicidal activity against A. terreus (MFC of 1000 mg/mL) and S. brevicaulis (MFC of 600 mg/mL) which were the most susceptible strains. C. zeylanicum showed only inhibitory effect on
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A. terreus, S. brevicaulis and P. brevicompactum. A. aculeatus was the most resistant strain to
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this essential oil. It was announced that O. gratissimum was more active against all the tested
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strains due to its prominent constituent, thymol (Philippe, Souaïbou, Paulin, Issaka, & Dominique, 2012).
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Essential oils (Pimenta racemosa, Syzygium aromaticum, Cinnamomum verum and Thymus
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vulgaris) were added to full-fat and low-fat cheeses at the levels of 0.1, 0.5 and 1% to evaluate their influence on L. monocytogenes and Salmonella Enteritidis growth at 4 and 10°C,
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respectively during 14 days of storage. It was concluded that at 1%, clove and cinnamon
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essential oils were the most effective oils regarding reduction of L. monocytogenes count in lowfat cheese within three days compared to the bay and thyme essential oils which reduced L.
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monocytogenes to less than 1 log10 cfu/mL during 10 days. In the case of full-fat cheese, only
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clove oil (at 1%) was effective in reducing L. monocytogenes to less than 1 log10 cfu/mL and at 0.1%, inhibiting the growth in low-fat and full-fat cheese. Clove oil at 0.5% in low-fat cheese,
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reduced S. Enteritidis to ≤ 1 log10 cfu/g, but recovered to 4.7 log10 cfu/g at day 14 (Smith-Palmer, et al., 2001). In a study by Govaris et al. (2011), antibacterial effects of oregano (0.1 or 0.2 mL/100 g) and thyme (0.1 mL/100 g) essential oils against L. monocytogenes and E. coli O157:H7 in feta cheese stored under modified atmosphere packaging (50% CO2 and 50% N2) at 4°C were determined. The results revealed that at 0.1 mL/100 g of oregano essential oil, L. monocytogenes and E. coli O157:H7 could survive up to 18 and 22 days of storage, respectively,
ACCEPTED MANUSCRIPT while at the level of 0.2 mL/100 g, the counts of survivors were up to 14 and 16 days. A similar trend as found with the oregano essential oil study was reported in the case of thyme essential oil at 0.1 mL/100 g. Moreover, both essential oils possessed a greater inhibitory effect toward L. monocytogenes compared to E. coli O157:H7. These results were consistent with other studies
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which evaluated the antimicrobial efficiency of oregano against E. coli O157:H7 and L.
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monocytogenes (Elgayyar, Draughon, Golden, & Mount, 2001; Gutierrez, Barry-Ryan, &
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Bourke, 2008; Santurio, De Jesus, Zanette, Schlemmer, Fraton, & Fries, 2014). In studies conducted by Burt and Reinders (2003), Sikkema et al. (1995) and De Sousa et al. (2012), cells
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of E. coli O157:H7 exposed to oregano essential oil were inactivated due to cell lysis.
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In a study by Amatiste et al. (2014), the antimicrobial activity of T. vulgaris L. and Origanum vulgare L. essential oils against S. aureus in fresh sheep cheese was assessed. The
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determined MIC and MBC of the essential oils were 4 and 8 µL/mL, respectively. In addition, it
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was observed the aforementioned essential oils had no effect on S. aureus count in cheese
EOs with cheese matrix.
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samples during 7 days of storage which was attributed to the interaction of active components of
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In another study, the effects of T. vulgaris essential oil toward S. aureus, L. monocytogenes and cheese starter cultures Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris
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were examined through MIC determination and measurement of viability in a Coalho cheesebased broth medium and a coalho cheese model. Coalho cheese is a semi-hard, a medium to high-moisture cheese typical of the northeast region of Brazil. It has a mild acidic flavor and fat content in the range of 35-60% in dry matter. The lower MIC for starter culture (1.25 µL/mL) compared to S. aureus and L. monocytogenes (2.5 µL/mL) was an indicative of a higher sensitivity of mesophilic starter culture. Determination of cell viability in coalho cheese-based
ACCEPTED MANUSCRIPT broth medium at various concentrations of EO (1.25, 2.5 or 5 µL/mL) showed no effect on S. aureus at 1.25 µL/mL, but at 2.5 µL/mL, both pathogenic bacteria and starter culture counts were reduced. At the level of 5 µL/mL, the counts of pathogenic bacteria decreased sharply and starter cultures were inhibited. Determination of cell viability in coalho cheese model showed
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that at 1.25 µL/mL, the essential oil had no impact on the counts of S. aureus and L.
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monocytogenes and the counts of Lacococcus ssp. were almost the same as the initial count.
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Essential oil at 2.5 µL/mL decreased S. aureus, L. monocytogenes and Lactococcus ssp. varying from 0.3 to 1 log cfu/g after 72 h of exposure. It was deduced that Lactococcus ssp. were more
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susceptible to essential oil and the selection of essential oil doses should be performed
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cautiously. Besides, a food matrix with higher fat and protein would impair the efficacy of antimicrobial components in essential oils (de Carvalho, et al., 2015).
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In a work performed by Zantar et al. (2014), essential oils of T. vulgari and Origanum
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compactum (0.05 and 0.1%) were added to goat cheese and their effects on some pathogenic and spoilage microorganisms were studied. Assessing antibacterial activity by disk diffusion method
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showed that the highest antibacterial activity for T. vulgari against S. aureus and the lowest
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activity for O. compactum toward Yersinia enterocolitica. Determination of antifungal activity by agar diffusion method at the concentrations of 1% and 3% indicated that O. compactum
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thoroughly inhibited the growth of A. flavus and Fusarium solani. EOs were added to goat cheese (0.05 and 0.1%) and it was observed that in samples fortified with 0.1% O. compactum, coliforms were not present from the first day of storage and from day 1 in the case of T. vulgari. In shelf-life estimation of cheese by monitoring the growth of molds and yeasts, it was announced that EOs extended the shelf-life of cheeses and O. compactum was more efficient in
ACCEPTED MANUSCRIPT this respect. Sensory evaluation of cheeses showed that samples containing T. vulgari were more acceptable by the panel group. De Souza et al. (2016) measured inhibitory effects of Origanum vulgare L. (0.6, 1.25, 2.5 or 5 µL/mL) on S. aureus, L. monocytogenes, L. lactis ssp. lactis and L. lactis ssp. cremoris in
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coalho cheese broth and slurry. MIC of essential oil against S. aureus and L. monocytogenes
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were 2.5 µL/mL and 0.6 µL/mL for mesophilic lactic acid bacteria. During 24 h, 0.6 µL/mL
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essential oil had no inhibitory effect toward both pathogenic bacteria in cheese broth whilst viable cell counts of starter culture decreased 1 log cfu/mL. A reduction of 1.8 and 1.5 log
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cfu/mL were observed at 1.25 µL/mL in L. monocytogenes and S. aureus, respectively after 24 h.
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The above-mentioned concentration showed the bactericidal effect on the lactic acid bacteria by decreasing 4 log cfu/mL of cell counts. O. vulgare at 2.5 µL/mL decreased S. aureus and L.
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monocytogenes by 2 and 2.5 log cfu/mL after 24 h and a sharp decrease (4 log cfu/mL) in the
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counts of starter culture after 1 h, occurred. The essential oil at 5 µL/mL showed a bactericidal effect against both pathogenic bacteria after 24 h. In cheese slurries, 0.6 µL/g essential oil had no
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impact on pathogenic and lactic acid bacteria counts. At 1.25 µL/g, the viable cell counts of both
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pathogenic bacteria and Lactococcus spp. decreased 1.5 and 2 log cfu/g, respectively after 72 h. Exposure to 2.5 µL/g essential oil caused a reduction of 2.5 and 2.8 log cfu/g in S. aureus and L.
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monocytogenes after 72 h and a sharp decrease in Lactococcus spp. counts after 24 h. It was also noted that in order to decrease the bacteria count the same as the broth, higher concentrations were needed (De Souza, et al., 2016). Higher protein and fat in food compared to simulated condition could interact with essential oil constituents being unavailable to act on cell membrane (Burt, 2004). Furthermore, higher nutrients in cheese would provide a suitable condition for repairing the damaged cells of bacteria (Burt, 2004; Gill, Delaquis, Russo, & Holley, 2002).
ACCEPTED MANUSCRIPT In an experiment, zein-based edible film was prepared by incorporation of Zataria multiflora Bioss essential oil (1, 2, 3 or 4% w/v) in order to control the growth of S. Enteritidis, L. monocytogenes, E. coli and S. aureus in Iranian Feta cheese during 14 days of storage. The results showed that a sharp decrease in the bacterial count occurred in samples coated with
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fortified films (Ghasemi, Javadi, Moradi, & Khosravi-Darani, 2015). In another attempt, Z.
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multiflora Boiss essential oil (50, 100, 200, 400, 600 or 1000 ppm) antifungal activity toward A.
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flavus ATCC 15546 was reported in the culture media and Iranian ultra-filtered white cheese. Radial growth and spore production analysis in agar medium demonstrated that fungal growth
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and spore production were reduced 21.3%, 42.7% by using 50 ppm, 79.4%5 and 21.7% in the
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case of 100 ppm and 75.8% and 92.5% by using 200 ppm essential oil. Z. multiflora Boiss essential oil showed a greater impact on spore production compared to mycelia growth. At the
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concentrations higher than 400 ppm, no fungal growth was observed and a dose of 1000 ppm had
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a fungicidal effect. Also, reduction of 22.6%, 82% and 90% in mycelial dry mass at the concentrations of 50, 100 and 150 ppm were reported while aflatoxin secretion was reduced
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31%, 97.6% and 99.4% at the same concentrations. Z. multiflora Boiss essential oil had an
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inhibitory effect on the radial fungal growth and aflatoxin production at all levels used in cheese samples, but fungal growth and aflatoxin production were not completely inhibited at any
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concentration (Gandomi, et al., 2009). Accordingly, Z. multiflora Boiss effect on growth and citrinin production by P. citrinum in culture media and mozzarella cheese was explored. MIC and MFC were reported as 200 and 400 ppm, respectively. In YES broth containing 100 and 200 ppm essential oil, citritin level was reduced 69 and 92%, respectively. Evaluation of the efficiency of EO activity (50, 100, 200, 400, 600, 800 or 1000 ppm) on fungal radial growth and citrinin production in mozzarella cheese depicted that radial growth and citrinin production were
ACCEPTED MANUSCRIPT inhibited at all levels of EO. However, P. citrinum was not completely inhibited by EO at 1000 ppm and an inhibition of 89.94% for fungi growth and 87% for citrinin production were reported. (Noori, et al., 2012) Eshaghi et al. (2014) added Z. multiflora Boiss essential oil at various amounts (0.05, 0.1,
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0.2 or 0.4%) to the milk for preparation of Gouda cheese and its influence on microbiological
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characteristics and formation of biogenic amines was studied during 90 days of ripening.
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According to the obtained results, increasing the essential oil level led to a decrease in tyramine and histamine amounts. At 0.05%, no significant reduction was observed in tyramine and
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histamine levels compared to the control samples, while 5, 22 and 44% reduction in tyramine
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and 14, 29 and 46% reduction in histamine were observed at the levels of 0.1, 0.2 and 0.4% at the end of storage. The counts of mesophilic lactobacilli decreased 0.5, 1.2 and 1.5 log cfu/g in
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cheese samples at 0.1, 0.2 and 0.4% on the day 15 of ripening. At 0.2 and 0.4% essential oil,
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Enterobacteriaceae reduced 0.68 and 0.75 log cfu/g at the end of ripening (90 days). In cheese samples containing 0.1, 0.2 and 0.4% essential oil, lactococci counts reduced 0.27, 0.58 and 1.3
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log cfu/g compared to the control group at the end of storage. At the same concentrations, the
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aerobic mesophilic counts were reduced 0.57, 0.77 and 1.57 log cfu/g and a reduction of 0.58, 0.84 and 2.2 log cfu/g in yeast counts were noticed. Regarding sensory evaluation, samples
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treated by 0.2% essential oils were the most preferable ones (Eshaghi Gorji, Noori, Nabizadeh Nodehi, Jahed Khaniki, Rastkari, & Alimohammadi, 2014). The impact of thyme and clove essential oil (1.5% v/v) incorporated into the whey protein isolate-based edible film applied for Kashar cheese on the growth of E. coli, L. monocytogenes and S. aureus during 60 days of storage was investigated. It was pointed out that thyme essential oil had a higher antimicrobial activity in comparison with clove essential oil. In cheese samples
ACCEPTED MANUSCRIPT coated with edible films containing thyme and clove essential oil, the number of E. coli were 5.86 and 6.12 log10 cfu/g on day 0 of storage which were decreased 2.25-4.49 and 1.57-4.91 log10 cfu/g on day 60. In the case of L. monocytogenes, the bacterial counts in thyme-coated cheese samples reduced from 5.66 on day 0 of storage to 4.40 log10 cfu/g on day 60. This suppression
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was from 5.93 to 4.81 log10 cfu/g from day 0 to 60 of storage in samples coated with clove-
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fortified edible films. S. aureus counts in samples coated with films containing thyme essential
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oil were 5.49 and 4.16 log10 cfu/g on day 0 and 60 of storage, respectively. In cheeses coated
respectively (Kavas, Kavas, & Saygili, 2015).
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with clove essential oil, S. aureus on day 0 and 60 of storage were 5.64 and 4.63 log10 cfu/g,
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The main components of essential oils in abovementioned studies include thymol and carvacrol. It is presumed that thymol would disrupt outer and inner cell membranes and interact
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with membrane proteins and intracellular targets (Hyldgaard, et al., 2012; Nazzaro, et al., 2013).
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The interaction of thymol with membrane results in the release of K+ and ATP due to the change in the permeability of membrane (Walsh, Maillard, Russell, Catrenich, Charbonneau, & Bartolo,
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2003; Xu, Zhou, Ji, Pei, & Xu, 2008). Moreover, thymol integrates with polar head-groups
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located in the lipid bilayer that would cause alterations in the cell membrane (Di Pasqua, Betts, Hoskins, Edwards, Ercolini, & Mauriello, 2007; Turina, Nolan, Zygadlo, & Perillo, 2006). It was
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assumed that thymol could form a complex membrane or periplasmic proteins through hydrogen bonds and hydrophobic interactions (Juven, Kanner, Schved, & Weisslowicz, 1994). Di Pasqua et al. (2010) noted that thymol would damage the citrate metabolic pathway in addition to influencing the enzymes involved in ATP synthesis. Carvacrol is supposed to change the cell membrane structure as well as its fatty acids profile and therefore, increase the permeability and fluidity of the membrane. It has the capability of influencing the outer
ACCEPTED MANUSCRIPT membrane of Gram-negative bacteria and releasing LPS. It was also suggested that its hydroxyl group acts as a carrier for transportation of monovalent cations by carrying H+ into the cytoplasm and transporting K+ back out. Contrarily, Veldhuizen et al. (2006), announced that hydroxyl group in carvacrol was not fundamental in the revealing of antimicrobial activity, but instead, it
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was related to non-hydroxyl groups. Furthermore, it was observed that carvacrol could affect
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protein motive force and synthesis of flagellin thus, decreasing motility of bacteria. It was
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announced that components such as eugenol, carvacrol and cinnamaldehyde would prevent the membrane-bound ATPase activity in E. coli and L. monocytogenes (Sikkema, et al., 1995).
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Ribeiro et al. (2013) studied rosemary essential oil (20% v/v) as a way to control
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multidrug-resistant E. coli in coalho cheese. MIC of essential oil was 200 µL/mL which was added to cheese samples inoculated with 105 cfu/mL of test strain and stored at 8°C for 7 days. It
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was revealed that incorporation of essential oil caused a reduction of 2.3 log cycles in the
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bacterial population within the first 24 h of storage that was stable during the whole period of storage. In a similar study, the inhibitory effect of Eugenia caryophyllata Thumb leaves essential
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oil on contaminating microorganisms of coalho cheese was investigated. MIC and Minimum
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Cidal Concentration (MCC) of essential oil were in the range of 2.5-5 and 5-20 µg/mL, respectively. The lowest MIC levels were associated with Y. enterocolitica, Candida albicans,
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Candida parapsilosis and Candida krusei. Cidal concentration of E. caryophyllata essential oil against yeasts was 2-4 fold higher than MIC, while it was 4-8 fold higher for bacteria. This point showed the higher sensitivity of yeasts to essential oil in comparison with bacteria. No cidal effect was reported for Pseudomonas aeruginosa because of being Gram-negative and having low sensitivity toward essential oil. Clove essential oil was also added at 2.5, 5, 10 or 20 µg/g after whey drainage and salting of the cheese and its impact on mesophilic bacteria and fungi in
ACCEPTED MANUSCRIPT vacuum-packed cheese during 15 days of refrigerated storage was determined. Essential oil at 5, 10 or 20 µg/g significantly lowered the count of mesophilic bacteria compared to the control cheese samples. The essential oil at 2.5-20 µg/g showed a static effect on fungi count in comparison to the control samples. It was also implied that due to the oil loss during cheese
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pressing in addition to the presence of fat and protein in the cheese matrix and interaction with
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the essential oil components, antimicrobial effect of the essential oil was alleviated. Furthermore,
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because of a higher availability of nutrients in food matrix compared to laboratory media, repairing the bacterial damaged cells occurred faster (Trajano, Lima, de Souza, & Travassos,
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2010). The main constituent of E. caryophyllata essential oil is eugenol which is believed to
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change the membrane and fatty acid profile, influence the transport of ions and ATP as well as the inhibition of the enzymes such as ATPase, histidine decarboxylase, amylase and protease
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(Nazzaro, et al., 2013; Thoroski, Blank, & Biliaderis, 1989; Wendakoon & Morihiko, 1995).
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Jeong et al. (2014) examined antimicrobial activity of 8 essential oils against Penicillium spp. during cheese ripening. According to the antifungal activity assays, cinnamon (bark and
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leaf) had the highest activity and was selected for incorporation at 2, 5, 10 or 20% (v/v) into the
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cheese. It inhibited the growth of all fungi at levels more than 10% v/v. The cinnamon essential oils effect on cheese starters (FD-DVS ABT-5, KAZU 1 and FD-DVS FLORA-DANICA) was
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also determined. Lactobacilli in FD-DVS ABT-5 starter were not influenced by cinnamon essential oils at any concentration whereas cinnamon leaf essential oil (≥ 20%) and cinnamon bark oil (≥ 10%) inhibited the growth of lactobacilli in KAZU 1 and at ≥ 5%, both essential oils had an inhibitory effect against lactobacilli in FD-DVS FLORA-DANICA starter. The antifungal activity was ascribed to the major components including eugenol, cinnamaldehyde and linalool (Jeong, et al., 2014). Cinnamaldehyde represents three possible mechanisms of action at different
ACCEPTED MANUSCRIPT concentrations. At low levels, it prevents the enzymes involved in cytokine interactions, at sublethal levels, prohibits ATPase and at lethal concentrations disturbs cell membrane. The mode of action in fungi is impairing of cell division (Hyldgaard, et al., 2012; Nazzaro, et al., 2013). Moosavy et al. (2013) described the antibacterial effect of Mentha spicata essential oil (2
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or 2.5% v/w) on L. monocytogenes in traditional Lighvan cheese at different ripening
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temperature (4 or 14°C) and salt water concentrations (12 or 15%) during 60 days. Essential oil
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reduced the growth of L. monocytogenes significantly, but there was no significant difference between the two concentrations used. The number of L. monocytogenes was reduced with
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increasing the essential oil level, salt water percentage, ripening temperature and storage period.
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It was inferred that some environmental factors such as temperature and salt percentage would act as synergists and enhance the efficiency of the essential oil.
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Antimicrobial activity of some plants’ essential oils, including dill, caraway, coriander,
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basil and lemon balm against L. monocytogenes, S. aureus, Bacillus cereus, E. coli O157:H7 and Salmonella typhimurium was surveyed in cheese yogurt (Mohamed, et al., 2013). It was
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illustrated that caraway essential oil had the highest inhibitory effect followed by dill, coriander,
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basil and lemon balm. Therefore, caraway and dill were selected for further studies. The MIC of caraway and dill essential oils were 0.003 and 0.005 mL/mL, respectively. The aforementioned
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essential oils showed the most inhibitory activity against L. monocytogenes and S. typhimurium after 7 and 14 days in cheese yogurt samples. A reduction of 1.3 and 4 log cycles after 7 and 14 days in S. aureus counts were reported by caraway. In addition, it reduced E. coli O157:H7 counts 1.7 log cycles after 7 days and completely inhibited the growth of bacterium after 14 days of storage. Dill essential oil decreased S. aureus number by 0.9 and 1.5 log cycles after 7 and 14 days and inhibited E. coli O157:H7 by 1 log cycle after 7 days and no bacterium was detected
ACCEPTED MANUSCRIPT after 14 days of storage. B. cereus was inhibited thoroughly by caraway and dill essential oils after 7 and 14 days of storage, respectively. These two essential oils were not effective on the growth of lactic acid bacteria (L. delbrueckii ssp.bulgaricus and S. thermophilus) that was in agreement with the results obtained by Ehsani and Mahmoudi (2012) and Zantar et al. (2014),
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implying no influence of Pimpinella anisum, Allium ascalonicum, T. vulgari and Origanum
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essential oils on the growth and metabolic activity of lactic acid bacteria. In contrast, the
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antibacterial effect of Origanum vulgare toward mesophilic lactic acid bacteria was reported by De Souza et al. (2016). In addition, the inhibitory effect of Origanum vulgare var hirtum
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essential oil on lactic acid bacteria was assessed in vitro and in two types of cheeses. The
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essential oil (50, 100, 150 or 200 µg/g) was added to the milk containing S. thermophilus CRL 728 and CRL 813, L. delbruekii ssp. bulgaricus CRL 656 and CRL 468 and Lactococcus lactis
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ssp. lactis CRL 597. Evaluation of cell viability and biological activity showed no change in the
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growth, acidifying activity and fermentative activity of lactic acid bacteria even at the highest concentration of essential oil (Marcial, Gerez, De Kairuz, Araoz, Schuff, & De Valdez, 2016).
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Jay et al. (2005) also pointed out that in comparison with other Gram-positive bacteria, lactic
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acid bacteria were very resistant to EOs. The main component in M. spicata, caraway and dill essential oils is carvone. It would disrupt pH gradient and membrane potential of cells.
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Oosterhaven et al. (1995) announced that carvone reduced the growth of E. coli, S. thermophilus and L. lactis through interrupting the metabolic energy status of the cells. Helander et al. (1998) observed no effect of carvone on E. coli and S. typhimurium as well as the intracellular ATP pool. Fernandes et al. (2016) prepared microencapsulated form of rosemary essential oil and examined its antimicrobial effect in Minas frescal cheese during 15 days at 6°C. Addition of
ACCEPTED MANUSCRIPT 0.5% microencapsulated essential oil reduced the mesophilic bacterial count 1.36 log cycles after three days and 0.73 log cycles after 15 days of storage. In a study carried out by Sadeghi et al. (2016), essential oil of Mentha pulegium at various concentrations (7.5, 15 or 30 µL/mL) on the growth of L. monocytogenes inoculated at 103 cfu/mL in Iranian white-brined cheese during 60
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days was examined. The results showed that in control samples without essential oil up to day 7,
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the L. monocytogenes growth was enhanced, but it was decreased gradually during 60 days. In
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treated samples, the maximum growth lasted for 14 days following a log reduction in bacterium count in other days. The highest antibacterial effect was observed at the level of 0.03%, although
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0.015% essential oil was more acceptable from consumers’ point of view. The compounds
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responsible for the antibacterial effect were pulegone, piperitenone and 1, 8 cineole. Pimpinella anisum (750, 1500 or 3000 ppm) and Allium ascalonicum (500, 1000 or 2000
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ppm) were added to cheese milk and their influence on the growth of E. coli was studied in
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Iranian white-brined cheese during 60 days of ripening. The highest bacterial count reduction was observed at the highest essential oils concentration and A. ascalonicum was more effective
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in this respect. On the other hand, A. ascalonicum essential oil at 750 ppm was the most
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preferred sample by the panelists. The antimicrobial activity of P. anisum essential oil and A. ascalonicum were attributed to benzene derivatives and organo sulfide compounds, respectively;
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that constituted the major parts of the oils (Ehsani et al. 2012). Bunium persicum (black cumin) essential oil was studied in terms of in vitro antibacterial activity against S. typhimurium, E. coli O157:H7, S. aureus and L. monocytogenes as well as its effect at 1 and 2% (w/v) on E. coli O157:H7 and L. monocytogenes in Iranian white cheese. MIC and MBC for E. coli O157:H7, L. monocytogenes, S. typhimurium and S. aureus were 10, 5, 10, 1.25 µL/mL and 20, 10, 20 and 2.5 µL/mL, respectively. In cheese samples containing essential
ACCEPTED MANUSCRIPT oil, the number of E. coli O157:H7 and L. monocytogenes colonies decreased during storage period (45 days) and in this regard, a higher level of essential oil was more effective. It was noted that L. monocytogenes was more sensitive compared to E. coli O157:H7. The authors emphasized that although the higher dose of essential oil reduced the bacterial number in cheese
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samples significantly, but it was not enough to consider the cheese as safe and it was suggested
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that these natural preservatives should be accompanied with other preservation methods. Sensory
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evaluation of un-inoculated cheese samples containing black cumin EO revealed that fortified cheese with essential oil received higher scores than control samples in all sensory characteristics
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(flavor, texture, odor, color and general acceptability) and lower level (1%) of essential oil was
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more acceptable during 45 days of storage (Ehsani, Hashemi, Naghibi, Mohammadi, & Khalili Sadaghiani, 2016). In agreement with the study, Hassanein et al. (2013) reported that
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incorporation of black cumin essential oil (0.1 or 0.2%) in Domiati soft cheeses increased the
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acceptability of products compared to control samples during 30 days of storage. Cuminum cyminum L. essential oil (7.5, 15 or 30 µL/100 mL) was applied alone and in
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combination with a probiotic bacteria Lactobacillus acidophilus (0.5% w/v) in Iranian white-
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brined cheese in order to determine their antibacterial activity against S. aureus during 75 days of storage. It was deduced that essential oil and the probiotic bacteria had synergistic effects on
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decreasing the growth of S. aureus in cheese. The greatest inhibitory activity during 75 days of storage was found in samples containing 30 µL/100 mL essential oil plus 0.5% probiotic. However, regarding cheese acceptability, the cheese manufactured by the milk containing 15 µL/100 mL essential oil received the highest score by the panelists (Sadeghi, Akhondzadeh Basti, Noori, Khanjari, & Partovi, 2013).
ACCEPTED MANUSCRIPT An edible coating based on whey protein isolate and alginate fortified with 1.5 (v/v) ginger essential oil was applied in coating of Kashar cheese and its antimicrobial impact on S. aureus and E. coli O157:H7 was analyzed during 30 days of storage at 4°C. Bacteriostatic effect of the edible coating containing ginger essential oil against S. aureus was observed on the 7th
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day that the bacterial count was reduced to 2.48 log10 cfu/g while in the case of E. coli, this effect
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was detected from the 1st day of storage and the highest level was reported on the 30th day. The
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bactericidal effect was only considered in S. aureus on the 30th day without any effect on E. coli in this time (N. Kavas, Kavas, & Saygili, 2016). In another study by the same authors, an edible
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film based on egg white protein was developed and various levels of sage and lemon balm
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essential oils (0.5, 1 or 2% v/v) were incorporated in order to investigate their antimicrobial efficacy against E. coli O157:H7, L. monocytogenes, S. aureus and yeast and molds in Lor
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cheese during 30 days. It was indicated that sage essential oil had a higher antibacterial effect
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compared to balm essential oil at all concentrations whereas the latter possessed a greater antifungal activity. Edible films with essential oils (0.5%) exhibited a bacteriostatic effect against
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all microorganisms from the 1st day of storage. Sage essential oil had a bactericidal effects
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against L. monocytogenes, S. aureus and yeast-molds on day 15 and 30 of storage, respectively. At 1% (v/v), sage and lemon balm essential oil demonstrated a bacteriostatic effect against S.
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aureus, L. monocytogenes and E. coli from the 1st day of storage. On day 15, a bactericidal effects of sage essential oil against bacteria and antifungal activity were detected. With respect to lemon balm essential oil, no yeasts were observed in cheese samples and a bactericidal effect against L. monocytogenes was reported on day 30. Similarly, at 2% (v/v), sage and lemon balm essential oil demonstrated a bacteriostatic effect against S. aureus, L. monocytogenes and E. coli from the first day of storage. Sage essential oil in the edible film inhibited the growth of S.
ACCEPTED MANUSCRIPT aureus, L. monocytogenes and yeast and molds on day 7 and E. coli was inhibited on day 15. Lemon balm essential oil depicted bactericidal effect against S. aureus and L. monocytogenes on day 15 while in the case of E. coli, this effect was reported on day 30. The most resistant bacterium to essential oils was E. coli O157:H7, followed by S. aureus and L. monocytogenes
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(Kavas & Kavas, 2016). In edible films, greater levels of antimicrobial agents of essential oils
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would remain in the film and at the surface of the food which prevents the microorganisms for a
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longer period (Cagri, Ustunol, & Ryser, 2002; Coma, Martial-Gros, Garreau, Copinet, Salin, & Deschamps, 2002).
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In another similar study by Kavas and Kavas (2014), whey protein isolate-based edible
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films containing mint essential oil (1, 2, 3 or 4% v/v) were applied in Lor cheese and microbiological quality was assessed during 15 days of storage. Antimicrobial effect was
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augmented by increasing the mint essential oil concentration and storage duration. Mint oil at the
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level of 4% had bactericidal and antifungal activity and all of the microorganisms were prohibited. The highest antimicrobial activities at the level of 3% were for S. aureus (0.83 log10
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cfu/g with 5.9 log10 cfu/g decrease), yeast-molds (1.41 log10 cfu/g with 4.8 log10 cfu/g decrease),
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L. monocytogenes (2.25 log10 cfu/g with 4.53 log10 cfu/g reduction) and E. coli (2.89 log10 cfu/g with 4.01 decrease). The most resistant microorganism to essential oil was E. coli and the most
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sensitive was S. aureus. The bactericidal effect against S. aureus started from the day 15 at a dose of 2% and on day 7 at a dose of 3% essential oil. This effect at the level of 3% essential oil were recorded on day 10 and 15 of storage for yeast-molds and L. monocytogenes, respectively. Antibacterial effectiveness of tarragon, spearmint and pennyroyal essential oils individually and in combination with monolaurine was evaluated in culture medium and white cheese against L. monocytogenes. Determination of MIC in Mueller Hinton broth showed the
ACCEPTED MANUSCRIPT values of 50 ppm, 0.4, 2 and 0.2% for monolaurine, spearmint, tarragon and pennyroyal essential oils, respectively. Fractional inhibitory concentration indices (FIC) determined for combination of monolaurine along with spearmint, tarragon and pennyroyal essential oils were 0.75, 0.66 and 1, respectively. The results were indicative of an additive effect of these combinations. The
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results of antibacterial activity of essential oils (0.2, 0.4 or 0.6% v/v) individually or in
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combination with monolaurine (200 or 400 ppm) in soft cheese during 168 h revealed that by
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increasing the essential oil level, antilisterial activity was enhanced whilst in the case of monolaurine, no significant further reduction at the level of 400 ppm was reported compared to
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200 ppm. The highest inhibition was observed in a combination of 0.6% pennyroyal and 400
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ppm monolaurine. Addition of spearmint essential oil to milk caused a significant reduction in L. monocytogenes count and this reduction was amplified in the presence of monolaurine.
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Nevertheless, no difference was observed in inhibition of L. monocytogenes at two different
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concentrations of monolaurine. With respect to tarragon, level of 0.4% alongside with 200 ppm monolaurine had a bactericidal effect. Moreover, at the highest level of tarragon (0.6%) and
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monolaurine (400 ppm), no L. monocytogenes was detected after 12 h till the end of storage.
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Sensory evaluation of cheese samples indicated that addition of 0.2% essential oil to cheese improved the sensory properties and a cheese with 0.4% was also acceptable by the panelists, but
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a level of 0.6% essential oil reduced the acceptability of the samples (Hamedi, et al., 2014). Chitosan coating containing rosemary and oregano essential oils was applied in semi-hard goat cheese and it was announced that in cheese samples coated twice with oregano essential oil, the greatest antimicrobial activity against Mucor and Penicllium was observed. Furthermore, the aforementioned samples were the most acceptable ones in respect of flavor and aroma from consumers’ point of view (Embuena, et al., 2016).
ACCEPTED MANUSCRIPT Dannenberg et al. (2016) evaluated the antimicrobial activity of essential oil derived from pink pepper tree fruit (Schinus terebinthifolius Raddi) against L. monocytogenes in Minas- type fresh cheese during 30 days of storage at 4°C. It was demonstrated that EO from ripe and mature fruits was more efficient toward L. monocytogenes compared to green fruits and the bacterial
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growth was reduced by 1.3 log cfu/g in 30 days.
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A nanoemulsion-based edible coating containing oregano essential oil (1.5, 2 or 2.5%
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(w/w) and mandarin fiber was prepared for coating of low-fat cheese and its antimicrobial activity against S. aureus inoculated (106 cfu/g) onto the cheese pieces was verified during 15
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days. Coating with 1.5% EO had no effect on reduction of S. aureus counts whereas the bacterial
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population decreased 1.4 and 1.5 log cfu/g at the levels of 2 and 2.5% EO, respectively. The antimicrobial activity was attributed to carvacrol, the major constituents of EO (Artiga-Artigas,
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Acevedo-Fani, & Martín-Belloso, 2017).
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5. Limitations and perspectives of EOs application in cheese Despite of the suitable efficacy of essential oils in restriction of growth and survival of
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microorganisms in cheese, some limitations have been recognized in their application due to the
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interaction of essential oil constituents with food components such as fat, carbohydrate and proteins that can alleviate the antimicrobial effects of mentioned essential oils (Burt, 2004; Gutierrez, et al., 2008; Juven, et al., 1994; Skandamis, Tsigarida, & Nychas, 2000) as well as increasing the required concentration to obtain sufficient antimicrobial activity (Hyldgaard, et al., 2012). Proteins interact with phenolic compounds present in EOs whereas fats surround the hydrophobic constituents of EOs, restricting their availability to target sites of microorganisms (Burt, 2004). In addition, the physical structure of the cheese would affect the distribution of EO
ACCEPTED MANUSCRIPT in the system and limiting its availability to microbial cells, resulting in reduction of antimicrobial effect (Gutierrez, et al., 2008; Gutierrez, Barry-Ryan, & Bourke, 2009). Moreover, it was inferred that reduction of pH, oxygen level and temperature would increase the antimicrobial effect of EOs (Skandamis, et al., 2000; Tsigarida, Skandamis, & Nychas, 2000).
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Lower pH would enhance the hydrophobicity of EOs, enabling them to easily dissolve cell
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membrane and pervade the target sites of the bacteria (Holley & Patel, 2005; Juven, et al., 1994).
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Since EOs have intense aroma, utilization at high concentrations in order to compensate their interaction with food components could result in sensory defects (Hyldgaard, et al., 2012).
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In order to rectify this shortcoming, various approaches have been proposed. One way is to
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incorporate the EOs in edible films and coatings. In this sense, transmission of antimicrobial components from film or coating to food is slow and a long-term antimicrobial activity can be
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achieved (Coma, et al., 2002; Sánchez-González, Vargas, González-Martínez, Chiralt, & Cháfer,
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2011). Micro- or nanoencapsulation of essential oils is another approach which improves the stability of EOs as well as extending the time of their antimicrobial activity. Fernandes et al.
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(2016) used inulin and whey protein isolate for microencapsulation of rosemary essential oil and
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added microcapsules to Minas Frescal cheese. It was illustrated that microencapsulated rosemary essential oil postponed the growth of mesophilic bacteria and prolonged the shelf-life of the
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cheese. It is also possible to use lower levels of EOs via combination of EO with other antimicrobial substances or technologies which act through synergistic effect (Nguefack, et al., 2012). Parsaeimehr et al. (2010) reported a remarkable synergistic effect of Z. multiflora Boiss essential oil and nisin on inhibition of enterotoxin and α-toxin generation by S. aureus during manufacturing process of a traditional Iranian white brine cheese and in BHI broth, respectively.
ACCEPTED MANUSCRIPT Essential oils are usually expensive because their production can be very costly depending on the type of plant used, method of EO extraction and facilities as well as the labor and energy costs. In some cases, it needs high amounts of plants to yield a little amount of essential oil. Moreover, some companies can present an essential oil with higher purity
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that undoubtedly affects its price. Nevertheless, many essential oils and their active
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constituents are active against bacteria and fungi and they can be produced from commonly
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available raw materials; perhaps in many cases right at the site of use so as to be rather lowcost treatments (Pandey, Kumar, Singh, Tripathi, & Bajpai, 2017). Essential oils can be used
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in agro industries instead of synthetic pesticides to control plant diseases causing severe
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destruction to crops. Using pesticides causes environmental pollution, increase the risk of pesticide residue and are threats to human health. However, application of EOs as an
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alternative antifungal agents would mitigate these disadvantages (Kotan, Kordali, Cakir,
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Kesdek, Kaya, & Kilic, 2008). In spite of high cost of EOs, due to being natural, safe, biodegradable and having little impact on non-target organisms, they can be proposed as
6. Conclusion
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potential antimicrobial agents for food commodity preservation in the near future.
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Several essential oils can be applied as natural antimicrobial agents in order to inhibit microbial deterioration of cheeses and extending the shelf-life. Major compounds including thymol, carvacrol, eugenol, carvone and cinnamaldehyde are mainly responsible for exerting antimicrobial activity through various mechanisms such as increasing the cell permeability, change of membrane fatty acids and effect on membrane proteins. However, the concentration of these substances applied in cheeses should be considered carefully because of their possible negative impacts on organoleptic properties. On the other hand, other approaches comprising
ACCEPTED MANUSCRIPT microencapsulation of essential oil, incorporation into edible film and coating or application of these oils along with other antimicrobial agents and preservatives can be more efficient in prevention of microbial growth and enhancing cheese safety and quality which should be
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investigated in future studies.
ACCEPTED MANUSCRIPT Acknowledgements This study is related to the project NO. 1395/74038 from Student Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran. We also appreciate the “Student Research Committee” and “Research & Technology Chancellor” in Shahid Beheshti University
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The authors declare that there is no conflict of interest.
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Conflict of interest
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of Medical Sciences for their financial support of this study.
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Cell wall destruction
Membrane protein damage
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Mechanisms of essential oils activity on microorganisms
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Increasing permeability
Hydrolysis of ATP and decreasing in Synthesis of ATP
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Leakage of the cell contents
Coagulation of cytoplasm
Reduction of proton motive force
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Damaging the cytoplasmic membrane
Figure 1. Mechanisms of antimicrobial activity of EO.
Reduction of the intracellular ATP pool
ACCEPTED MANUSCRIPT Table 1. Selected publications on the antimicrobial activity of essential oils in cheeses Type of essential oil
EO concentration
Microorganism type
Remarks
Reference
Wagashi
Ocimum gratissimum
200, 400, 600, 800, 1000 mg/L
Aspergillus (flavus and tamarii), Fusarium (poae and verticillioides) and Penicillium (citrinum and griseofulvum
Mycelia growth was reduced by increasing the essential oil level. Fungistatic activity against species was observed MIC values ranged from 800-1000 mg/L.
(Philippe, Souaïbou, Guy, et al., 2012)
Wagashi
Cinnamomum zeylanicum
200, 400, 600, 800, 1000 mg/L
Aspergillus terreus, Aspergillus ustus, Aspergillus niger, Aspergillus aculeatus, Penicillium brevicompactum and Scopulariopsis brevicaulis
The most susceptible strains were Scopulariopsis brevicaulis and Aspergillus terreus showing MFC of 600 to 1000 mg/L; whereas Aspergillus aculeatus was the most resistant mold to cinnamon essential oil.
(Philippe, Souaïbou, Paulin, et al., 2012)
L. monocytogenes
In the low-fat cheese, all four oils at1%reduced L. monocytogenes to <1 log10 cfu/mL. In the full-fat cheese, oil of clove was the only oil to achieve this reduction.
(SmithPalmer, et al., 2001)
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Type of cheese
'Continued'
Pimenta racemosa, Syzygium aromaticum, Cinnamomum verum and Thymus vulgaris
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Full-fat and low-fat cheeses
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O. gratissimum
0.1, 0.5 and 1%
S. enteritidis
ACCEPTED MANUSCRIPT Table 1. Continued Type of essential oil
EO concentration
Microorganism type
Remarks
Reference
Feta
Oregano Thyme combined with MAP (50% CO2 and 50% N2)
0.1 or 0.2 mL/100 g 0.1 mL/100 g
L. monocytogenes E. coli O157:H7
The growth of bacteria was restricted by the essential oils with a greater inhibitory effect toward L. monocytogenes
(Govaris, et al., 2011)
Coalho
T. vulgaris
1.25 and 2.5 µL/mL
S.aureus and L. monocytogenes Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris
Goat cheese
T.vulgari and Origanum compactum
0.05 and 0.1%
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Type of cheese
(de Carvalho, et al., 2015)
Eight spoilage and pathogenic bacteria
The highest antibacterial activity was reported for T.vulgari against S.aureus. The lowest activity was obtained by O. compactum for Yersinia enterocolitica. The maximum shelf life was obtained by 0.1% O. compactum.
(Zantar, et al., 2014)
Microorganism type
Remarks
Reference
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No inhibitory effect was observed at 1.25 µL/mL. At 2.5 µL/mL S.aureus, L. monocytogenes and Lactococcus ssp. were inhibited.
'Continued'
Table 1. Continued Type of cheese
Type of essential oil
EO concentration
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Z. multiflora Boiss
0.05, 0.1, 0.2 and 0.4%
Mesophilic lactobacilli enterobacteriaceae
Lower microbial counts were observed compared to the control at the end of storage.
(Es'haghi Gorji, et al., 2014)
Iranian ultra-filtered white cheese
Z. multiflora Bioss
50, 100, 200, 400, 600 and 1000 ppm
Aspergillus flavus
No fungal growth was observed at 400 ppm and higher and a dose of 1000 ppm had a fungcidal effect. Z. multiflora Boiss essential oil had an inhibitory effect on radial fungal growth and aflatoxin production at all levels.
(Gandomi, et al., 2009)
Coalho
Rosemary
20% v/v
E. coli
A decline of 2.3 log cycle in the bacterial population within the first 24 hour of storage.
(Ribeiro, et al., 2013)
Appenzeller
Cinnamon
2, 5, 10 and 20% v/v
Penicillium spp
EO inhibited the growth of all fungi at levels more than 10% v/v.
(Jeong, et al., 2014)
2, 2, 5% v/w
L. monocytogenes
Higher level of EO was more efficient in bacterial count reduction.
(Moosavy, et al., 2013)
EO concentration
Microorganism type
Remarks
Reference
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Lighvan
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Gouda
Mentha spicata
'Continued' Table 1. Continued Type of cheese
Type of essential oil
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Bunium persicum
1 and 2% w/v
E. coli O157:H7 L. monocytogenes
The bacterial number decreased during storage period and higher level was more effective.
(Ehsani, et al., 2016)
Iranian white-brined cheese
Mentha pulegium
7.5, 15 and 30 µL/mL
L. monocytogenes
The highest antibacterial effect was observed at the level of 0.03% but, 0.015% was more acceptable by panelists. The maximum growth lasted for 14 days following a log reduction in bacterium count.
(Sadeghi, et al., 2016)
Iranian white-brined cheese
Pimpinella anisum
750, 1500 and 3000 ppm 500, 1000 and 2000 ppm
E. coli
The highest reduction was obtained at the highest level.
(Ehsani, et al., 2012)
S. aureus
The greatest inhibitory activity was achieved in samples containing 30 µL/100 mL essential oil plus 0.5% probiotic.
(Sadeghi, et al., 2013)
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7.5, 15 and 30 µL/100 mL
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Cuminum cyminum L. plus Lactobacillus acidophilus
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Allium ascalonicum Iranian white-brined cheese
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Iranian white-brined cheese
ACCEPTED MANUSCRIPT Table 2. Major compounds of essential oils used in different cheeses Essential oil used
Main components
Reference
Wagashi
Ocimum gratissimum
thymol (28.1%), γ-terpinene (21.30%) and p-cymene (16.5%)
(Philippe, Souaïbou, Guy, et al., 2012)
Feta
Origanum vulgare
Carvacrol (80.05%), p-cymene (5.25%), thymol (4.81%) and γterpinene (2.90%)
(Govaris, et al., 2011)
Thymus vulgaris
Thymol (44.3%), p-cymene (19.8%), carvacrol (14.2%), thymoquinone (6%)
Coalho
Thymus vulgaris
Thymol (43.19%), p-cymene (28.55%), γ-terpinene (6.36%), linalool (5.57%), carvacrol (3.14%)
(de Carvalho, et al., 2015)
Gouda
Zataria multiflora
Carvacrol (71.12%), γ-terpinene (7.34%), α-pinene (4.26%)
(Es'haghi Gorji, et al., 2014),
Coalho
Eugenia caryophyllata Thumb
Eugenol (74%), α–humullene (9.62%), d-cadinene (4.64%)
(Trajano, et al., 2010)
Lighvan
Mentha spicata
Carvone (78.76%), Limonene (11.5%), cis-dihydro Carveol (1.43%)
(Moosavy, et al., 2013)
Cheese yogurt
Carum carvi
Carvone (74.4%), Limonene (15.5%), α-pinene (8.29%)
(Mohamed, et al., 2013)
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Cheese type
Carvone (64.59%), Limonen (20.64%), Dillapiol (12.04%)
Origanum compactum
Carvacrol (75.6%), p-cymene (8.3%), γ-terpinene (6.6%)
Thymus vulgaris
Carvacrol (81.2%), p-cymene (3.8%), γ-terpinene (2.7%), α-Humulene(2%)
Iranian white brined
Mint essential oil
neo Menthol (38.7%), IsoMenthone (31.32%) and 1.8 Cineole (7. 87%)
(Tehrani, et al., 2015)
Iranian white
Mentha pulegium
pulegone (36.68%), piperitenone (16.88%), and 1,8 cineole (14.58%)
(Sadeghi, et al., 2016)
Goat
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(Zantar, et al., 2014)
ACCEPTED MANUSCRIPT Table 2. Continued Essential oil used
Main components
Reference
White brined
Cuminum cyminum
Cuminaldehyde (29.02%), αterpinene-7-al (20.7%), γ- terpinene (12.94%)
(Sadeghi, et al., 2013)
Traditional Argentinean
Oreganum vulgare L. var hirtum
γ-terpinene (15.1%), terpinen-4-ol (15.5%) and thymol (13.0%)
(Marcial, et al., 2016)
Kashar
Zingiber officinale Roscoe
Zingiberene (39.12%), geranial (13.44%), camphene (11.15%), β-sesquiphellandrene (10.64%)
(N. Kavas, et al., 2016)
Lor
Mentha spicata
Carvone (40.5%), 1, 8-cineole (23.3%), cis-carveol (19.1%), β-caryophyllene (3.9%)
(Gökhan Kavas, et al., 2014)
Feta
Zataria multiflora Boiss
Caryophyllene (25.1%), carvacrol (22.8%), thymol (22.5%), α-Terpineol (19.1%)
(Ghasemi, et al., 2015)
Coalho
Origanum vulgare L.
Carvacrol (69.0%), thymol (14.12%), γ-terpinene (3.71%), and p-cymene (3.67%)
(De Souza, et al., 2016)
Iranian white cheese
Bunium Persicum
Frescal
Rosmarinus officinalis
White brined
Pimpinella anisum
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Cheese type
Cuminaldehyde (11.4%), γ- terpinene (11.37%), α-Pinene (11.27%) and α– terpinene (11.13%)
(Ehsani, et al., 2016)
1, 8-Cineole (40.8%), Camphor (Fernandes, et al., (28.8%), Α-Pinene (10.9%), Β-Pinene 2016) (8.1%) Benzene (37.8%), Longifolene-(V4) (28.8%), Phenol, 2-methoxy-4-(1propenyl) (11.2%)
Allium ascalonicum
Diallyl disulphide (20%), Trisulfide, methyl 2-propenyl (18.1%), Trisulfide, di-2-propenyl (15.3%)
Zataria multiflora Boiss
Carvacrol (71.12%), γ-terpinene (7.34%), α-pinene (4.26%), eucaliptol (3.37%)
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(Ehsani, et al., 2012)
(Noori, et al., 2012)
ACCEPTED MANUSCRIPT Highlights:
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This paper gives an overview of cheese spoilage and pathogen bacteria. Antimicrobial properties of essential oils are reviewed. Application of essential oils as natural antimicrobial agents in different cheeses are investigated The main components of essential oils that have antimicrobial properties are overviewed
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