Antimicrobials Treatment

101 Toxic Food Ingredients

101 Toxic Food Ingredients

Get Instant Access

Eleftherios H. Drosinos, Panagiotis N. Skandamis, and Marios Mataragas

Introduction

The use of antimicrobials is a common practice for preservation of foods. Incorporation, in a food recipe, of chemical antimicrobials towards inhibition of spoilage and pathogenic micro-organisms results in the compositional modification of food. This treatment is nowadays undesirable for the consumer, who likes natural products. Scientific community reflecting consumers demand for natural antimicrobials has made efforts to investigate the possibility to use natural antimicrobials such us bacteriocins and essential oils of plant origin to inhibit microbial growth.

In addition, to the compositional modification of a food, antimicrobials are also used for a food surface treatment or for incorporation in the packaging material. This is especially important for cooked meat products, to decontaminate them from post-thermal processing cross-contamination. Antimicrobial substances are also used in certain stages of food process corresponding to critical control points; their presence contributes to the safety design of a food with other existing hurdles of microbial growth.

In this chapter natural (bacteriocins and essential oils) and chemical antimicrobials used in meat and meat products processing are reviewed providing in parallel basic information on antimicrobials and factors affecting their use in foods.

Laboratory of Food Quality Control and Hygiene, Department of Food Science and Technology, Agricultural University of Athens, 75, Iera Odos Street, Votanikos, Athens, GR (EL) - 118 55, Greece e-mail: [email protected]

F. Toldra (ed.), Safety of Meat and Processed Meat,

Food Microbiology and Food Safety, DOI 10.1007/978-0-387-89026-5_10,

© Springer Science+Business Media, LLC 2009

Bacteriocins

Lactic acid bacteria are widely used in fermented foods such as dairy, meat, vegetables and bakery products. The frequent use of lactic acid bacteria in foods, usually as starter cultures, is owned to the desired changes that induce flavour, odour and texture of the products as well as that positively contribute to the products' safety because they inhibit the growth of pathogens. Antimicrobial activity of lactic acid bacteria is due to pH decrease, microbial competition for nutrients and production of antimicrobial compounds such as hydrogen peroxide, lactic acid and other metabolites (e.g. bacteriocins) (Ray & Daeschel, 1992).

Bacteriocins are proteinaceous compounds, consisting of peptides and amino acids, with antimicrobial activity and are synthesized by the ribosomes of the microbial cells. Over the last decades, numerous bacteriocins produced by lactic acid bacteria (Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus and Carnobacterium) have been isolated and characterized (nisin, lactococcins, sakacins, curvaticins, carnobacteriocins, pediocins, etc.) (Nettles & Barefoot, 1993; Xiraphi et al., 2006). Bacteriocins gained increased attention because of their potential application as natural antimicrobials in foods to substitute or decrease the addition of other chemical preservatives which are considered dangerous (e.g. nitrites). On the other hand, bacteriocins are generally considered as GRAS (generally regarded as safe) substances and thus, may be added to foods or produced in situ aiming to act as natural preservatives. Nowadays, only nisin has been approved as additive and produced in industrial scale in a semi-purified form for use in dairy products (EC, 1983; Parente & Ricciardi, 1999; FDA, 2008).

Classification of Bacteriocins

Bacteriocins are extracellular compounds displaying a relative narrow antimicrobial spectrum. Tagg, Dajani, and Wannamaker (1976) termed as bacteriocins the substances that are produced by the Gram-positive bacteria and have the following properties: their molecule consist of peptides which have antimicrobial activity, they have low molecular weight which vary between the different bacteriocins as their antimicrobial spectrum and mode of action, and in general are active against close-related species to the producing strain. Bacteriocins are classified into four categories (Klaenhammer, 1993; Nes et al., 1996):

- Class I. Lantibiotics belong to this group. These are of low molecular weight peptides (<3.5 kDa) resistant to thermal treatment and are characterized by the presence of uncommon amino acids such as lanthionine and 3-methyl-lanthionine. The major representative bacteriocin from this class is nisin. Lantibiotics are divided into the following sub-categories: spiral positively charged molecules of low molecular weight (2151-4635 Da) and circular neutral or negatively charged molecules with even lower molecular weight (1825-2042 Da).

- Class II. Low molecular weight bacteriocins (<10 kDa) with 30-100 amino acids resistant to heat without lanthionine. This class is organized into three sub-groups: The Ila sub-group is the most common group including peptides active against Listeria strains. Although the bacteriocins of this group are not so active against spores are more efficient than nisin in some food categories such as meat. The most significant representative of this subgroup is pediocin PA-1/AcH. The lib sub-group includes bacteriocins that consist of two peptides (lactococcin G) and the Ilc sub-group requiring reduced cystein molecules for activity expression (lactococcin B).

- Class III. Macromolecular bacteriocins (molecular weight >30 kDa) not so stable to heat and are inactivated at high temperatures (helveticin J).

- Class IV. Bacteriocins of this category contain in their molecule a carbohydrate or fatty part needed for their activity expression (lactocin 27).

More recently, Cotter, Hill, and Ross (2005) proposed a revised classification scheme including the Classes I (lanthionine-containing lantibiotics) and II (non-lanthionine-containing bacteriocins as well as circular bacteriocins) and bacteriolysins (formerly class III). Class IV was not included in the new designation scheme because, according to the authors, bacteriocins that require non-proteinaceous moieties for activity have not yet been convincingly demonstrated.

Mode of Action, Molecular and Biochemical Characterization of Bacteriocins

Bacteriocin production is regulated by genes responsible for the production (Bac+) of the substance and immunity of the producer strain (Bacr) to its own antimicrobial compound. Recently, research has been driven to the isolation and transfer of genes related to bacteriocin production of wide antimicrobial spectrum from one strain to another, incapable of producing bacteriocin or producing bacteriocin with narrow antimicrobial activity (Ennahar, Sashihara, Sonomoto, & Ishizaki, 2000). Moreover, synthesis of these antimicrobial agents is regulated by four different genes (Nes et al., 1996):

- the gene responsible for the production of a precursor substance of the bacteriocin

- the gene which provides immunity to the producer strain

- the gene encoding the transport of the precursor bacteriocin outside the cell activating an ad hoc mechanism for this purpose (ABC-transporter) and finally

- the gene which activates the mechanism of production of a supplementary auxiliary protein (accessing protein) important for the extracellular transport and activation of the bacteriocin, yet its exact role is still unknown.

Besides these genes, more genes have been found for some bacteriocins of Class II responsible for the regulation of the bacteriocin production (Diep, Havarstein, & Nes, 1996).

Bacteriocins are firstly synthesized inside the ribosomes as precursor substances (pro-peptides) which are biologically inactive containing an amino acids sequence (number of amino acids vary between 14 and 30) characterized by the presence of two molecules of glycine (double-glycine leader). This sequence serves a dual purpose: to prevent the expression of the bacteriocin activity while it is still present inside the producer cell and to signal for the activation of the ABC-transporter and accessing protein mechanisms (Klaenhammer, 1993). The mechanism of bacteriocin synthesis regulation may be explained by the quorum sensing phenomenon (Cotter et al., 2005) and consists of three elements: the induction factor (IF) and two proteins, the histidine protein kinase (HPK) and the response regulator (RR). The IF, a bacteriocin-like peptide without antimicrobial activity, is required for the stimulation/activation of the genes encoding for bacteriocin synthesis. This is observed for bacteriocins belonging to Class II, whereas for lantibiotics and non-lantibiotics the bacter-iocin itself serves as external signal to regulate bacteriocin synthesis (Chen & Hoover, 2003). The N-terminal of the kinase serves as receiver of the IF and the signal is transmitted to its C-terminal leading to the activation of the enzyme and the initiation of the histidine phosphorylization. For lantibiotics and non-lantibiotics, the HPK phosphorylates the histidine residue when it senses a certain level of bacteriocin concentration in the environment (Chen & Hoover, 2003). The latter (kinase is also involved) facilitates the phosphorylization of the aspartic acid of the RR protein. The last phosphorylization causes changes in the carbon part of the RR protein activating the transcription of the corresponding genes (pre-peptide synthesis, transportation, immunity and in some cases bacteriocin production regulation) (Nes et al., 1996). The proteins involved in bacteriocin transportation (ABC-transporter system) have two regions, one hydrophobe and one carbon region for ATP binding (ATP-binding region) which have essential role during transportation. It has been proved that these proteins also carry one N-terminal extension consists of 150 amino acids with potential proteolytic activity to selectively breakdown the double-glycine leader sequence of the pre-peptides. As previously mentioned, accessory protein (470 amino acids) is also required which is believed to facilitate bacteriocin transportation and/or cleavage of the double-glycine leader sequence. However, the precise role of this protein has not yet been fully elucidated (Havarstein, Diep, & Nes, 1995).

Immunity of the bacteriocin-producing strains to their own bacteriocin is regulated by the corresponding gene encoding the extraction of immunity-related proteins. Such proteins are the LciA (encoded by the IciA gene) and

NisI (encoded by the nisi gene) offering protection against lactococcin A and nisin, respectively. These proteins deactivate the produced bacteriocin reacting (binding of the proteinic receivers of the bacteriocins) with the bacteriocin molecules (Ennahar et al., 2000).

Mode of action of the bacteriocins is characterized by two distinct phases: (1) adsorption of the bacteriocin on receivers located on the cell wall of the sensitive strains and (2) denaturation of the cytoplasmic membrane. The first step is reversible and removal of the bacteriocin (e.g. presence of proteases) maintains membrane structure intact without any damage of the bacterial cells. The second step is irreversible and the damages caused are characteristic for each bacteriocin (pore formation, lytic action or synthesis inhibition of important cell components). Cytoplasmic membrane is the main target of the bacteriocin causing extensive leakage of ions and other important cell components such as ATP or amino acid and also blockage of amino acids transportation inside the bacterial cell (Abee, Krockel, & Hill, 1995). In general, bacteriocin activity is the result of the hydrophobic and electrostatic interactions between the positively charged bacteriocins and negatively charged cytoplasmic membrane (due to the presence of many negatively charged lipids) of the sensitive strains (Ganzle, Weber, & Hammes, 1999; Moll, Konings, & Driessen, 1999).

Bacteriocins are mainly active against Gram-positive close-related species (e.g. Listeria monocytogenes). Gram-negative strains (e.g. Salmonella spp.) are more resistant to bacteriocins of the lactic acid bacteria because their membrane composition differs from that of the Gram-positive micro-organisms. Cytoplas-mic membrane of the Gram-negative micro-organisms is characterized by the presence of an additional external layer, containing phospholipids, proteins and polysaccharides lipids (LPS), impermeable to most substances. The layer facilitates the diffusion of molecules with molecular weight below 600 Da, whereas the lowest in size bacteriocin found has molecular weight approximately 3 kDa. However, the presence of various agents such as EDTA or citric acid makes the membrane sensitive to bacteriocins because of Mg2 + -binding in the LPS layer of the external membrane (Stevens, Sheldon, Klapes, & Klaenhammer, 1991).

Applications of Bacteriocins in Foods

Lactic acid bacteria are widely used for food preservation because they inhibit the growth of various pathogenic bacteria lowering the pH during fermentation and producing antimicrobial substances. Bacteriocins constitute a group of such antimicrobials which may find application in dairy, meat, fishery, bakery and vegetable products as well as in alcoholic beverages production for controlling pathogenic and in some cases spoilage micro-organisms (non-starter lactic acid bacteria in cheese and wine) (Daeschel, Jung, & Watson, 1991; O'Sullivan, Ross, & Hill, 2003). Bacteriocins may be applied as food preservatives directly or indirectly. Direct method is referred to the addition of bacteriocins to foods in purified/semi-purified form or incorporated into packaging film surfaces

(Appendini & Hotchkiss, 2002), whereas the addition of micro-organisms producing bacteriocins in situ is known as indirect method (Luchansky, 1999). Bacter-iocins have gained an interest as food preservatives due to the preference of consumers for safe foods with an extended shelf life but minimally processed without excess of chemical preservatives. Nisin (nisaplin, Danisco) and pediocin PA1/AcH (ALTA 2431, Quest) have been widely used in foods. Hurdle technology could be employed to extend the relatively narrow antimicrobial spectrum of the bacteriocins (Chen & Hoover, 2003). Bacteriocins may be combined with other preservative techniques (e.g. high pressure or temperature shock) or chemicals (e.g. EDTA) to include Gram-negative micro-organisms (wider antimicrobial spectrum of activity) (Stevens et al., 1991; Kalchayanand, Sikes, Dunne, & Ray, 1998; Masschalck, Deckers, & Michiels, 2003).

Dairy Products

Nitrates are commonly used to inhibit the growth of Clostridia causing problems during cheese making such as Clostridium tyrobutyricum. L. monocytogenes is another micro-organism of concern, especially for cheese such as Taleggio or Mozzarella, due to pH increase during ripening. Bacteriocins and more specifically nisin have been extensively used as alternatives for nitrate salts (Hugenholtz & de Veer, 1991; Giraffa, Picchioni, Neviani, & Carminati, 1995; Stecchini, Aquili, & Sarais, 1995; Ross et al., 1999).

Meat Products

Due to the successive application of the nisin in dairy products, an interest was developed for substitution of nitrate/nitrite salts by nisin during meat products manufacturing (Rayman, Aris, & Hurst, 1981; Taylor, Somers, & Krueger, 1985). However, the results were not as encouraging as dairy products due to low solubility of the nisin in the increased pH of the meat products (Rayman, Malik, & Hurst, 1983; Stiles, 1996; Schillinger, Geisen, & Holzaphel, 1996). Better results were obtained by the application ofbacteriocin-producing microorganisms isolated from meat products such as Pediococcus, Leuconostoc, Carnobacterium and Lactobacillus spp. Also, various bacteriocins such as saka-cin, pediocin, curvaticin or mesenterocin have been added to meat products (bologna, frankfurters and ham-type meat products) in purified/semi-purified form with promising results. These bacteriocins displayed anti-listerial activity and their addition inhibited or even reduced the growth of L. monocytogenes (Berry, Hutkins, & Mandigo, 1991; Hugas, Pages, Garriga, & Monfort, 1998; Ross et al., 1999; Laukova, Czikkova, Laczkova, & Turek, 1999; Mataragas, Drosinos, & Metaxopoulos, 2003; Drosinos, Mataragas, Kampani, Kritikos, & Metaxopoulos, 2006).

Fishery Products

An interesting bacteriocin application is the preservation of shrimps in brine (3-6% NaCl). Usually these products are preserved by the addition of sorbic or benzoic acids. Bacteriocins produced by Lactococcus lactis SIK-83 (nisin Z), Carnobacterium piscicola U149 (carnocin U149) and Lactobacillus bavaricus MI401(bavaricin A) prolonged the shelf life by 21 days (nisin), 6 days (bavar-icin) or the shelf life was similar (carnocin) with the control (10 days) without the addition of benzoate or sorbate solutions. However, best results were obtained with the use of antimicrobials (59 days) (Einarsson & Lauzon, 1995). Moreover, other bacteriocins (e.g. nisin and sakacin) alone or combined with other hurdles (e.g. low temperature, modified atmosphere and antimicrobials such as lactate or carbon dioxide) have been studied to investigate the growth of pathogenic bacteria (e.g. L. monocytogenes) in cold-smoked salmon and rainbow trout (Nilsson, Huss, & Gram, 1997; Nykanen, Weckman, & Lapvetelainen, 2000; Katla et al., 2001).

Fermented Vegetables

Bacteriocin-producing lactic acid bacteria may be applied in products of plant origin such as fermented vegetables (sauerkraut). Salt, acetate and sugar are frequently used in this kind of products to inhibit the growth of undesired micro-organisms. Anti-listerial bacteriocins (sakacin A and pediocin), produced by L. sakei Lb706 and Pediococcus acidilactici M, respectively, are used during Kimchi manufacturing and the results showed that the former bacter-iocin did not inhibit L. monocytogenes whereas pediocin readily reduced the population during fermentation at 14oC (Choi & Beuchat, 1994).

Factors Limiting Bacteriocins Efficiency in Foods

Studies have shown that bacteriocins are not so effective in foods compared to laboratory substrates. This is attributed to the fact that these studies, performed in laboratory media, have been carried out under controlled conditions without any interference as frequently happens in foods. Foods are complex systems consisting of various microenvironments which interact with each other. Interactions between bacteriocin molecules and food ingredients may negatively contribute to bacteriocin efficiency. Bacteriocins (e.g. nisin or pediocin) may initially reduce bacterial counts; however, initiation of growth occurs during storage, after an extended lag phase. Factors that are likely to interact with bacteriocins resulting in decreased activity are summarized below (Schillinger et al., 1996):

- Acidity (pH) may influence activity, solubility and stability of the bacteriocins

- Low solubility of the bacteriocins resulting in inadequate and non-homo-genate diffusion of the substances inside the food mass

- Bacteriocin binding from various food ingredients such as fat molecules

- Inactivation of the bacteriocin molecules by other food additives

- Presence of various enzymes that breakdown bacteriocins such as proteases

- Mechanisms such as lipid oxidation destabilizing bacteriocin molecules.

Future Considerations on Bacteriocins Applications in Foods

When bacteriocin application is combined with conventional preservation techniques and good hygiene practices (GHP), pathogenic bacteria or spoilage microorganisms may be effectively controlled. However, the addition ofbacteriocins in purified form is not used by food industries in extensive scale because of the high cost of this application. Bacteriocin addition as additives comprises an attractive alternative solution for minimally processed foods to ensure their safety. Also, bacteriocins may be used as substitutes of chemical preservatives such as nitrite/ nitrate salts and sorbate/benzoate. Bacteriocin efficiency or spectrum activity may be increased by combining bacteriocins with other substances (e.g. chelators) or new preservation techniques (e.g. Ultra Hydrostatic Pressure and Pulsed Electric Field) which may lead to substitution of some chemicals or to the application of milder methods of processing (e.g. thermal treatment). Various molecular techniques (e.g. transfer of genes responsible for bacteriocin production to other non-bacteriocin-producing strains, mutation of genes responsible for bacteriocin production, technology of protein engineering, etc.) may be employed to develop proteinic molecules with improved solubility and stability, broader antimicrobial spectrum and higher antimicrobial activity. Furthermore, these techniques may serve as means to develop bacterial strains capable of producing such improved proteinic molecules (Abee et al., 1995).

Recently, mathematical models have been developed to describe growth and bacteriocin production of bacteriocin-producing strains added (Messens, Neysens, Vansieleghem, Vanderhoeven, & De Vuyst, 2002; Messens, Verluyten, Leroy, & De Vuyst, 2003). Modelling contributes to the determination of how environmental factors affect the growth and bacteriocin production and also to predict the bacteriocin efficiency (Leroy, Verluyten, Messens, & De Vuyst, 2002; Leroy & De Vuyst, 2003). Commercial use of bacteriocins requires optimization of their production in order to make cost-effectively their application. One method that is usually followed for the optimization of bacteriocin production is by varying one factor in turn while the other factors are kept constant. This method is laborious and requires a lot of time in case of that several factors are under study. Hence, statistical experimental designs have been developed to evaluate the influence of substrate composition and environmental conditions (e.g. temperature and/or pH) on growth and bacter-iocin production (Rollini & Manzoni, 2005; Dominguez, Bimani, Caldera-Olivera, & Brandelli, 2007).

Naturally Occurring Compounds from Plants

Nowadays, there is an increasing demand worldwide for environmental friendly and more natural antimicrobials to be used for mild preservation. This is due to the negative attitude of consumers towards preservatives of chemical origin. Nature, and especially plants, has been recognized as a remarkable source of antimicrobial compounds, which are primarily intended to increase natural preinfectional and postinfectional defence of plants against micro-organisms and insects (Smid & Gorris, 1999). Such systems include prohibitins, inhibitins, postinhibitins, phytoalexins, phenolics and essential oils. Over 1300 plants are considered as potential sources of antimicrobials (Nychas, 1995). Phenolics and essential oils (EOs) are the major compounds which have also been proven promising for food preservation, since the 1920s (Shelef, 1983). They are commonly obtained by steam- or hydro-distillation as well as by solvent extraction (e.g. with ethanol) from spices and herbs (Davidson & Naidu, 2000; Coma, 2008).

Since ancient times (with the earliest report in 1550 BC), spices and herbs have been used for their perfume and flavour as seasoning additives and as preservatives due to their strong antimicrobial and antioxidant properties (Tassou, Lambropoulou, & Nychas, 2004; Coma, 2008). Herbs are distinguished from spices in that herbs commonly constitute portions of aromatic, soft stemmed plants and aromatic shrubs and trees, whereas spices are rhizomes, roots, barks, flower buds, fruits and seeds (Davidson & Naidu, 2000). The antimicrobial activity of spices and herbs is primarily attributed to the phenolic component of their essential oil fraction (phyto-phenols; Davidson & Naidu, 2000). In particular, essential oils mainly consist of terpenes (e.g. mono-terpenes, sesquerpitenes), terpenoides and other aromatic compounds (e.g. simple phenols, such as eugenol and thymol, aldehydes, esters and alcohols) (Davidson, 1997; Smid & Gorris, 1999; Bakkali, Averbeck, Averbeck, & Idaomar, 2008). Other plant extracts include isothiocyanate derivatives (e.g. found in cabbage, horseradish, mustard, broccoli) and phenolic compounds, such as di- or tri-phenols, phenolic acids, such as hydroxucinnamic acid, and flavonoids (Davidson, 1997). Based on toxicological studies, the majority of active components of herbs and spices are considered as food-grade or generally recognized as safe (GRAS) (Smid & Gorris, 1999). Various mechanisms of inhibition have been suggested for essential oils, damaging structural and functional properties of bacterial membranes being the most dominant. In particular, EOs penetrate cell envelope, dissolve in the lipid layer of cellular membranes, bind to the hydrophobic sites of membrane proteins, and by increasing the permeability of the cell membrane, they cause loss of vital intracellular material or inhibit nutrients intake via dissipation of pH gradient and the electrical potential (compounds of proton motive force; Tassou et al., 2004; Burt, 2004; Nychas & Skandamis, 2005; Oussalah, Caillet, Salmieri, Saucier, & Lacroix, 2006). Additional modes of action include inhibition of oxygen uptake, inhibition of nucleic acid synthesis and inactivation of membrane proteins (e.g. ATPase) or other intracellular enzymes (Lemay et al., 2002; Burt, 2004). Gram-positive bacteria are considered more susceptible to EOs than Gram-negative bacteria (Smith-Palmer, Stewart, & Fyfe, 1998; Fisher & Phillips, 2006). Of the Gram-positive bacteria, lactic acid bacteria have been reported as the most resistance to EOs (Ouattara, Simard, Piette, Begin, & Holley, 2000; Lemay et al., 2002).

Factors Affecting the Effectiveness of Essential Oils in Foods

Even though the results of most in vitro assays suggest that essential oils have a substantial antimicrobial effectiveness, when used in food systems, the amounts required are considerably higher (10- to 100-fold) or the concentration of the targeted micro-organisms quite lower (Shelef, 1983; Burt, 2004). Given that effective levels in foods may often have negative sensory impact, the commercial application of EOs in foods is currently limited. For instance, when oregano or nutmeg EOs were added at the maximum acceptable organoleptic level on cooked chicken breast they showed limited activity even at refrigeration temperatures (Firouzi, Shekarforoush, Nazer, Borumand, & Jooyandeh, 2007). The performance of EO in foods is the additive (potentially synergistic) or antagonistic outcome of several factors, and specifically: (i) certain intrinsic properties of foods, such as fat, pH, salt, water and proteins, which determine the solubility of EOs in the water phase (Kabara, 1991; Juven, Kanner, Schved, & Weisslowicz, 1994; Nychas, 1995; Smith-Palmer, Stewart, & Fyfe, 2001); (ii) the structure and viscosity of the foods (solid vs. liquid foods) (Skandamis, Tsigarida, & Nychas, 2000); (iii) the decomposition of some EOs constituents (e.g. allyl isothiocyanate) in aqueous face and/or their interaction with certain hydrophilic substances, such as thiols, and sulphydryl or terminal amino groups of proteins (Ward, Delaquis, Holley, & Mazza,, 1998); and (iv) factors affecting the physiology of the micro-organisms, such as composition of bacterial membranes, availability of nutrients, oxygen tension and incubation temperature (Kabara, 1991; Juven et al., 1994; Smid & Gorris, 1999; Gill, Delaquis, Russo, & Holley, 2002; Nychas & Skandamis, 2005).

The major limiting factor for the activity of EOs in foods, even when they are applied at concentrations highly above those required for inhibition based on in vitro studies, is their reduced solubility, due to the presence of apolar constituents in their composition. For instance, highly hydrophobic constituents of EOs (e.g. thyme, mint and bay oil) may show limited effectiveness in foods of high fat content, such as liver pate (30-40%), full-fat cheese or adipose meat tissue, because EOs will partition in the lipid fraction of food phase, thereby reducing the residual EO concentration in the hydrophilic portion where micro-organisms are partitioned (Tassou, Drosinos, & Nychas, 1995; Cutter, 2000; Smith-Palmer et al., 2001; Holley & Patel, 2005). However, the opposite may also occur with less hydrophobic EOs, such as clove oil, which was more effective against L. monocytogenes and S. enteritidis in full-fat than in low-fat cheese (Smith-Palmer et al., 2001). In this respect, the octanol/water partitioning coefficient of an EO (Smid & Gorris, 1999) may be a reliable indicator of the expected antimicrobial effectiveness. Furthermore, the activity of EOs may be quenched by other macromolecules which form hydrophobic cavities and hydrogen bonds, as exemplified by Tween 80 (Juven et al., 1994). Low pH increases the hydrophobicity of essential oils, enhancing their potential to bind onto hydro-phobic sites of membrane proteins, and EOs also become more soluble in the lipid-rich membranes of the target micro-organisms (Juven et al., 1994). Nevertheless, low pH may also increase the solubility of the EOs in the lipid phase and hence counteract the antimicrobial effectiveness. Furthermore, given that the phenolic group may be active both as un-ionized (e.g. at pH <5.0) and ionized, it has been suggested that phenolic preservatives may maintain effectiveness over a wide range of pH values, such as pH 3.5-8.0 (Kabara, 1991).

Apart from pH and fat, the high amounts of protein and the reduced water content of foods may also decrease the effectiveness of EOs (Burt, 2004). Complex formation between EOs constituents and proteins (e.g. bovine serum albumin or casein up to 6%) may reduce the probability of EO to attack the target micro-organisms, as exemplified in cheese and broth (Rico-Munoz & Davidson, 1983; Juven et al., 1994; Smith-Palmer et al., 2001). Furthermore, low water content may hamper the transfer of EO to the active sites in the microbial cells (Smith-Palmer et al., 2001). In this respect, a comparative evaluation of 0.03% oregano essential oil against Salmonella typhimurium in broth and within solid medium containing 20% gelatin showed that the counts and metabolism of the bacterium were considerably suppressed in liquid culture compared to gelatin medium (Skandamis et al., 2000). Thus, it may be postulated that broth facilitated contact of EO with Salmonella. Recent evidence suggested that the strong attachment of pathogens on the rough surface of chicken skins accounted for the limited effectiveness of citral, linalool and bergamot oil added at the minimum inhibitory concentrations according to in vitro data (Fisher & Phillips, 2006). It has also been speculated that the greater nutrient availability of foods compared to laboratory media may increase bacterial resistance to antimicrobial agents, including EOs (Gill et al., 2002).

Lowering oxygen tension in packages increases the effectiveness of EOs. Juven et al. (1994) showed that anaerobic conditions significantly enhanced the antimicrobial activity of 350 mg/ml thyme essential oil, 140 mg/ml thymol or 200 mg/ml carvacrol against S. Typhimurium on nutrient agar. This was associated either with the oxidation of phenolic constituents of EOs or the lower energy yields of bacterial metabolism under aerobic conditions (Juven et al., 1994). The fact that 0.8% oregano essential oil in ground meat caused more pronounced inhibition to L. monocytogenes, S. Typhimurium and spoilage flora under 40% C02/30% 02/30% N2 and 100% CO2 compared to that observed in aerobic packages supports the above explanations (Skandamis & Nychas, 2001a; Skandamis, Tsigarida, & Nychas, 2002; Tsigarida, Skandamis, & Nychas, 2000). In addition to oregano essential oil, the activity of coriander oil against Aeromonas hydrophila in cooked pork was significantly enhanced under vacuum as compared to aerobic storage at 10°C (Stecchini, Sarais, & Milani, 1993).

Regarding the effect of temperature, it is suggested that low temperatures reduce the activity of phenolic preservatives, either lowering the solubility of phenolics in the lipids of cell membrane or due to reduction in the rate of interaction with the membranes (Kabara, 1991). Reports by Tassou et al. (1995) on the application of mint essential oil in tzatziki (pH 4.5) at 4 and 10°C, against L. monocytogenes, or by Skandamis and Nychas (2001b) on the application of oregano essential oil in eggplant salad (pH 4.0-5.0) at 0-15°C, against E. coli 0157:H7, and by Stecchini et al. (1993) for clove and coriander against A. hydrophila on cooked pork support these hypotheses. However, at growth-permitting conditions, it is expected that low temperature would enhance activity of EOs by delaying bacterial growth, as compared with higher temperatures (Hao, Brackett, & Doyle, 1998a, 1998b; Nadarajah, Han, & Holley, 2005a; Solomakos, Govaris, Koidis, & Botsoglou, 2008). Finally, salt (e.g. 3%) may have a potentiating effect on some phenolic compounds, as has been shown for butylated hydroxyanisole, a well-known antioxidant agent (Kabara, 1991).

Applications of EOs in Meat and Meat Products Direct Application of EOs in the Product

The majority of studies evaluating the antimicrobial activity of phenolic compounds, essential oils or their constituents are performed in vitro. Evidence on their activity in perishable foods, such as meat and meat products, is essential in order to establish their use. An overview of pertinent studies is provided in Table 10.1, whereas major issues and outcomes of these studies are detailed in the next paragraphs.

A total of nine essential oils (20% in alcohol), namely angelica root, banana puree, bay leaf, caraway seed, carrot root, eugenol (from clove), marjoram, pimento leaf and thyme were evaluated for their ability to inhibit growth of A. hydrophila and L. monocytogenes on cooked beef (internal temperature of 74°C) and chicken (internal temperature 85°C) at 5 and 15°C (Hao et al., 1998a,

Table 10.1 Effectiveness of essential oils or their components applied directly in foods

Antimicrobial

agent or plant

Experimental

Growth

essential oil

Food

Concentration21

condi tionsb

Microorganism

inhibition0

Inactivationd

References

Eugenol

Cooked

0.

1 ml (of 20%

5, 15°C

L. monocytogenes

L

-

Hao et al.

Pimento leaf

chicken

solution in

10 and 105 CFU/g

(1998a)

Caraway seeds

ethanol) on 25 g of slice

A. hydrophila

M-H

Eugenol

Cooked beef

0.

1 ml (of 20%

5, 15°C

L. monocytogenes

N-L

-

Hao et al.

Pimento leaf

solution in ethanol) on 25 g of slice

A. hydrophila 10 and 105 CFU/g

L-H

-

(1998b)

Mustard

Acidified

0.

1%

22°C

Escherichia coli

-

1.5 logs

Lemay et al.

chicken

Inoculum:

Brochothrix

L

-

(2002)

meat model

103—104 CFU/g

thermosphacta

(sausage;

Lb. alimentarius.

L

-

pH 5.o",

lactic acid

aw>0.96,

bacteria

190 ppm

nitrite.

cooked at

55°C)

Table 10.1

(continued)

Antimicrobial

agent or plant

Experimental

Growth

essential oil

Food

Concentration21

condi tionsb

Microorganism

inhibition0

Inactivationd

References

Rosemary

Pork liver sausage

0.5% ground rosemary

5°C

L. monocytogenes Inoculum:

L-M

-

Pandit & Shelef

1% EO

102-103 CFU/g

L-M

-

(1994)

5% encap

H

-

sulated EO

(spray dried

on modified

starch)

0.1-0.5%

H

-

antioxidant

extract by

co2

extraction

Sage

Chicken

Up to 2.5%

35°C

B. cereits

H

-

Shelef et al.

noodles and

rubbed sage

Inoculum:

S. aureus

H in chicken

-

(1984)

strained

105-106 CFU/g

Nin beef

beef

Pseudomonas sp. S. typhimurium

M in chicken Nin beef N

Clove

Minced mutton

0.5%, 1%

7, 30°C

L. monocytogenes

N-L

Vrinda-Menon & Garg (2001)

Clove

Cooked pork

500 |ig/cnr

2°C; air or

A. hydrophila

-

2-3 logs

Stecchini et al.

(75°C for

vacuum

Inoculum:

within 8

(1993)

30 min)

10°C; air or vacuum

106 CFU/cm2

Growth in the control

days 4-6 logs within 8 days

Table 10.1

(continued)

Antimicrobial

agent or plant

Experimental

Growth

essential oil

Food

Concentration®

condi tionsb

Microorganism

inhibition0

Inactivationd

References

Coriander

Cooked pork (75°C for 30 min)

10°C; air or vacuum

A. hydrophila Inoculum: 106 CFU/cnr

Growth in the control

4 logs within 8 days

6 logs within 8 days

Mint

Pâté

0.5-2%

4, 10°C Inoculum: 107 CFU/g

L. monocytogenes S. enteritidis

N-L N-L

Tassou et al. (1995)

Thyme

Minced pork

Up to 0.25%

4, 8°C

L. monocytogenes Inoculum:

H

2-2.3 logs initial

Aureli, Costantini

104 CFU/cnr

reduction

& Zolea

(1992)

Oregano

Minced pork

Up to 0.4 |il/g

Vacuum with or without nitrites

C. botulinum spores

Inoculum: 300-3000 spores/g

N without nitrites

Ismaiel & Pierson (1990)

Garlic

Fresh sausage Beef burger

3-5% ground garlic

25°C

S. typhimurium Inoculum: 104 CFU/cnr

M-H

El-Khateib & El-Rahman (1987)

Clove

Beef

1% dry mass

4, 24° C

L. monocytogenes

-

1 log only at 24° C '

Ting & Deibel (1992)

Oregano

Minced beef

- 100% CO,

M in CO2/O2/N2 H in CO,

Skandamis et al. (2001a)

Table 10.1

(continued)

Antimicrobial

agent or plant

Experimental

Growth

essential oil

Food

Concentration

condi tionsb

Microorganism

inhibition0

Inactivationd

References

Oregano

Beef fillets

0.8%

5°C under

Spoilage flora

L in air

1 log

Skandamis

(i) With

-Air

L. monocytogenes

M in vacuum/

reduction

et al. (2002)

natural flora

- Vacuum

S. typhimurium

MAP-high

of

Tsigarida et al.

(ii) Flame

- 40%C02/

Inoculum:

permeability

pathogens

(2000)

sterilized

30%02/~ 30%N2 Films of low and high permeability

103 CFU/g

H in vacuum/ MAP-low permeability

in flame sterilized fillets

Protecta II

Chicken

2%

Spray chilling

Natural flora

-

2-3 logs

Dickens (2000)

(herbal mix)

broilers

(1°C 30 min)

Bell pepper

Minced beef

0.02-2.5 ml/

7°C with 0-4%

S. typhimurium

H at 1.5 ml/

3 logs at

Careaga et al.

(Capsicum

100 g minced

NaCl

Inoculum:

100 g

>1.5 ml/

(2003)

annuum

meat

103 CFU/g

100 g

extract)

0.02-5 ml/100 g minced meat

P. aeruginosa Inoculum: 103 CFU/g

>1.5 ml/ 100 g

Thyme

Minced beef

0.6%

4, 10°C alone or

L. monocytogenes

H

3 logs initial

Solomakos

(Flame

with nisin

(2-strains

reduction

et al. (2008)

sterilized)

cocktail) Inoculum: 104 CFU/g

Savory

Pork fillets

50 |il/100 g meat

4°C alone or

L. monocytogenes

-

3 logs

Ghalfi et al.

Oregano

curvatus

Inoculum: 104 CFU/g

(2007)

Table 10.1 (continued)

Antimicrobial agent or plant Experimental Growth essential oil Food Concentration21 conditions'3 Microorganism inhibition0 Inactivationd References

Table 10.1 (continued)

Antimicrobial agent or plant Experimental Growth essential oil Food Concentration21 conditions'3 Microorganism inhibition0 Inactivationd References

Oregano

Beef patties

200-500 ppm

2°C with ascorbic acid and lycopene, 70%02/ 20% CO 2/ 10%N2

Natural flora

M

Sánchez-Escalante et al. (2003)

Rosemary

- 60%OV 40%C02

Natural flora

H

Djenane et al. (2003a, b)

Oregano

Chicken

- 70%C02/ 30%N2

Natural flora

H

Chouliara et al. (2007)

Oregano Nutmeg

Barbecued chicken breast

1-3 pl/g

3, 8, 20°C Inoculum: 106 CFU/g

Yersinia enterocolitica L. monocytogenes

N

Firouzi et al. (2007)

Cilantro oil

Ham

0.1-6% in canola oil

10°C vacuum Inoculum: 104 CFU/cm2

L. monocytogenes (5-strains cocktail)

N

Gill et al. (2002)

Table 10.1

(continued)

Antimicrobial

agent or plant

Experimental

Growth

essential oil

Food

Concentration21

condi tionsb

Microorganism

inhibition0

Inactivationd

References

Protecta I, II

Lean beef

Spray (15s) with

4°C

E. coli 0157:H7

M

1 log initial

Cutter (2000)

(herbal

tissues

2.5% Protecta

Inoculum:

L. monocytogenes

reduction

mixtures)

I and II

105 CFU/cnr

S. Typhimurium

Ground beef

Mixed with

Aerobic plate

N-L

-

(lean and

2.5% Protecta

counts

adipose)

I and II liquid, powder, spray

Clove

Buffalo meat steaks

Dipped in 0.1%

4°C with 2% lactic acid and Vitamin C

Natural flora

L-M

Naveena et al. (2006)

Bergamot

Chicken skin

Dipped in oils at

Room

E. coli 0157:H7

-

1-3 logs

Fisher &

Citral

MICC for

temperature

L. monocytogenes

Phillips

Linalool

15-60 s

for 60 s Inoculum: 108 CFU/g

S. aureus B. cereus

(2006)

Mustard flour

Ground beef

5%, 10%, 20%

4°C, 100% N2

Natural flora E. coli 0157:H7 (5-strains cocktail) 10-100, 103,

106 CFU/g

Elimination No at 5%, 10% Elimination at 20%

Nadarajah et al. (2005a)

Table 10.1

(continued)

Antimicrobial

agent or plant essential oil

Food

Concentration®

Experimental condi tionsb

Microorganism

Growth inhibition0

Inactivationd References

Carvacrol

Steak tartare

5 mmol/g

Inocula

107 CFU/g

N

Veldhuizen et al. (2007)

Carvacrol

Cooked

0.1-2%

Abuse chilling

CI. perfringens

Inhibition of

Juneja &

Thymol Oregano Cinnamal-

ground turkey

(54.4 to 7.2°C for 12-21 h)

germination

Friedman (2007)

dehyde

a When no exact concentration could be calculated by the study, description of the experimental procedures is provided. b When no modified atmosphere packaging is reported, the study was performed under aerobic conditions.

c The following classification has been used: N, no inhibition (similar increase in logs to the control): L, low inhibition (<1.5 log lower than the control): M, medium inhibition (1.5-2.5 log lower than the control): H, high inhibition (>2.5 log lower than the control).

d The following classification has been used: "-", no such response was observed. Log numbers refer to the total reduction compared to the control by the end of storage.

c Minimum inhibitory concentration.

1998b). Of the essential oils tested, eugenol and pimento leaf, followed by caraway seed, were the most effective in suppressing the maximum population and/or delaying growth of A. hydrophila and to a lesser extent L. monocytogenes (Table 10.1), even though the latter showed negligible growth within 14 days at 5°C. The inhibitory effect was more evident at 5°C than at 15°C and at lower initial inoculation levels (10 CFU/g) compared to 105 CFU/g. Moreover, more essential oils seemed to be effective in chicken than in beef, especially against A. hydrophila inoculated at low cell density. Nadarajah et al. (2005a) reported that 5-20% mustard flour was capable of eliminating 3 log CFU/g of E. coli O157:H7 in ground beef packaged under 100% N2 and stored at 4°C, whereas elimination of 6 logs required at least 20% flour. Bell pepper is another spice with very active EO. In particular, essential oil of bell pepper at concentrations 1.5 ml/100 g and 0.3 ml/100 g of minced meat was capable of completely inhibiting growth of S. typhimurium and Pseudomonas aeruginosa, respectively, whereas higher concentrations could exert bactericidal effect (Careaga et al., 2003). Moreover, addition of 1% NaCl reduced the required levels of capsicum extract (bell pepper) for inhibition of P. aeruginosa. The antimicrobial activity of mustard and horseradish is highly attributed to allyl isothiocyanate (AIT). AIT is considered more effective against Gram-negative bacteria, such as E. coli O157:H7 and Vibrio parahaemolyticus than Gram-positive bacteria, such as lactic acid bacteria (Ward et al., 1998; Muthukumarasamy, Han, & Holley, 2003; Holley & Patel, 2005).

In order to reduce the binding of EO by food ingredients, and moderate its sensory impact, a promising alternative application is the encapsulation within edible matrices or surfactant micelles. For instance, rosemary oil up to 5% encapsulated in modified starch was more capable of inhibiting L. monocytogenes in pork liver sausage than 1% pure EO (Pandit & Shelef, 1994). Encapsulation within surfactant micelles, likely increases the water solubility of hydrophobic EOs, e.g. carvacrol and eugenol, and hence, facilitates their dispersion in the aqueous phase (Gaysinsky, Davidson, Bruce, & Weiss, 2005; Gaysinsky, Taylor, Davidson, Bruce, & Weiss, 2007). Recent studies have also demonstrated that the activities of thyme (Solomakos et al., 2008) or savory and oregano (Ghalfi, Benkerroum, Doguiet, Bensaid, & Thonart, 2007) essential oils in pork and beef may be significantly enhanced, when combined with nisin or bacteriocin-producing lactic acid bacteria (e.g. L. curvatus). Moreover, modified atmosphere and lactic acid may also positively contribute to the activity of EOs, e.g. rosemary and clove, against both Gram-negative and Gram-positive members of meat microbial association (Djenane, Sanchez-Escalante, Beltran, & Roncales, 2003a, 2003b; Naveena, Muthukumar, Sen, Babji, & Murthy, 2006). In this manner, EOs may also exhibit a protective effect on lipids and/or myoglobin oxidation, and at levels that limit the negative impact on taste and flavour (Chouliara, Karatapanis, Savvaidis, & Kontominas, 2007; Naveena et al., 2006).

The activity of essential oils from sage, clove, rosemary, oregano and thyme is extensively documented in meat and chicken under aerobic, modified atmosphere packaging (MAP) or vacuum storage (Table 10.1). For instance, rubbed sage at levels up to 2.5% in chicken noodles and strained beef showed considerable inhibition on germination of B. cereus spores, no activity was obtained with sage oil up to 5000 ppm (Shelef, Jyothi, & Bulgarelli, 1984). In addition, clove and oregano essential oils possess a wide antimicrobial spectrum, including pathogens and spoilage flora of meat (Table 10.1). Oregano oil not only delayed growth of meat microbial association of ground meat at 5°C, especially Gram-positive flora (e.g. Brochothrix thermosphacta and lactic acid bacteria), but also reduced the rate of glucose consumption, the release of a-amino acids and the production of organic acids, compared to the control (Skandamis & Nychas, 2001a). Such effects were more pronounced under 100% CO2, followed by 40% C02/30% 02/30% N2 and then aerobic storage, suggesting enhanced activity due to multiple hurdles, namely low temperature, oregano oil and MAP. Moreover, the effectiveness of essential oil increased with concentration from 0.05 to 1% (v/w). Similar effects have been observed with chicken (Chouliara et al., 2007). Levels of oregano oil up to 0.8% also exerted a profound antimicrobial effect against S. typhimurium and L. monocytogenes and spoilage flora of beef slices under vacuum or MAP, when packaging film of low permeability was used. Limited activity was observed in high permeable pouches, in which MAP and vacuum collapsed during storage (Tsigarida et al., 2000; Skandamis et al., 2000). Furthermore, a beneficial attribute of oregano and thyme oils is their compatibility with the sensory properties of many meat products, such as beef-burgers and smoked sausages. This characteristic renders them promising additives for use in meat preservation. The same may also be the case with garlic, mustard or pepper extracts (El-Khateib & El-Rahman, 1987; Careaga et al., 2003).

Application of Essential Oils in Active Packaging

It is well known that packaging protects foods from microbial or chemical contamination, it ensures mechanical resistance and in some cases, such as vacuum or modified atmosphere packaging, it may delay biological and chemical reactions, t

Was this article helpful?

0 0
Going Green Foods

Going Green Foods

What Is The First Essential Step For Going Green With Food? Get Everything You Need To Know To Get Started With Helping The Earth And Going Green With Food. This Book Is One Of The Most Valuable Resources In The World When It Comes To Everything You Need To Know About Green Agriculture.

Get My Free Ebook


Post a comment