Beneficial Effects of Spices in Food Preservation and Safety

Spices have been used since ancient times. Although they have been employed mainly as flavoring and coloring agents, their role in food safety and preservation have also been studied in vitro and in vivo. Spices have exhibited numerous health benefits in preventing and treating a wide variety of diseases such as cancer, aging, metabolic, neurological, cardiovascular, and inflammatory diseases. The present review aims to provide a comprehensive summary of the most relevant and recent findings on spices and their active compounds in terms of targets and mode of action; in particular, their potential use in food preservation and enhancement of shelf life as a natural bioingredient.


INTRODUCTION
Plant, animal, and microbes represent an unlimited source of compounds with medicinal properties (Tajkarimi et al., 2010). Since ancient time, humans are using spices as nutritional agents (Kaefer and Milner, 2008). According to the U.S. Food and Drug Administration (FDA), spice is an "aromatic vegetable substance in the whole, broken, or ground form, the significant function of which in food is seasoning rather than nutrition" and from which "no portion of any volatile oil or other flavoring principle has been removed" (Sung et al., 2012).
More than 100 varieties of spices are produced throughout the world. Asia is the main leader for the production of spices, particularly of cinnamon, pepper, nutmeg, cloves, and ginger, while Europe grows mainly basil, bay leaves, celery leaves, chives, coriander, dill tips, thyme, and watercress. In America, instead, pepper, nutmeg, ginger, allspice, and sesame seed are mainly produced (Prasad et al., 2011).
Although spices have been used (mostly dried seed, fruit, root, bark, or vegetative material) for rituals, cosmetics and perfumery, their flavoring, coloring and, especially, preservative properties have founded wide applications both in the traditional food preparations and in the food industry. In fact, many compounds isolated from spices ( Table 1) have shown antimicrobial activity against some of the most common microorganisms that affect the food quality and shelf life (Tajkarimi et al., 2010). The introduction of spices through the meals has various beneficial effects as well. For instance, they can stimulate the secretion of saliva, promote the digestion, prevent from cold and influenza, and reduce nausea and vomiting (Ravindran, 2002;Sultana et al., 2010). In this manuscript we provide an overview on spices and their constituent as a natural food preservatives in vitro and in vivo.

IMPORTANCE OF SPICES
Spices have been important to mankind since the beginning of history. Several mythological evidence including "Epic of Gilgamaesh, " and the "Bagavad Gita, " suggest their use for several      purposes. Because of their strong preservative quality, spices were also used for embalming. According to Ayurveda, they help to maintain the balance of the body humors (Gupta et al., 2013a). Besides these, spices have been used to change the physical appearance of food. For instance, pepper and turmeric changed the color, appearance and the taste of food with many health benefits. Ginger, nutmeg and cinnamon improve digestion, considered good for spleen and sore throats (Prasad et al., 2011). Unfortunately, this beneficial effect of spices is not clinically proven. However, traditional practices emphasize the health benefits of spices. Eventually, recent studies highlighted other biological functions of spices, including antimicrobial, antioxidant, and anti-inflammatory (Tajkarimi et al., 2010).

SPICES FOR FOOD PRESERVATION AND SAFETY
Food spoilage refers to an irreversible modification in which food becomes not edible or its quality is compromised. Such changes can be driven by different factors, either physical (oxygen, temperature, light) and/or biological (enzymatic activity and microbial growth). Despite the current technologies available in the production chain (for instance freezing, pasteurization, drying, preservatives), it seems impossible to eliminate completely the risk of food spoilage (Gutierrez et al., 2009). Lipid oxidation is one of the main issues of food spoilage. Hence, food industries have applied antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) to prevent spoilage (Stoilova et al., 2007). However, their safety is doubtful and consumers are progressively demanding natural compounds. For this reason spices represent a potent tool for the food industry, thanks to their natural properties (Hyldgaard et al., 2012). Indeed spices possess antioxidant capacity, mainly due to the presence of phenolic compounds (Figures 1A,B). They exhibit antioxidant property by scavenging free radicals, chelating transition metals, quenching of singlet oxygen, and enhancing the activities of antioxidant enzymes (Rubió et al., 2013). Stoilova et al. (2007) reported that the CO 2 extract of ginger had in vitro activity comparable with that of BHT in inhibiting the lipid peroxidation both at 37 and 80 • C. Moreover, pimento and black pepper extracts reduced the formation of acrylamide up to 75 and 50%, respectively, in a model mixture simulating heated potato matrix (180 • C for 20 min). Eugenol, the main component of pimento essential oil, limited the formation of acrylamide by 50% (Ciesarová et al., 2008). Some other studied antioxidants are: quercetine (dill), capsaicin (red chilli), curcumin (turmeric), carvacrol (oregano, thyme, marjoram), thymol (oregano, thyme), piperine (black pepper), gingerol, etc (ginger, marjoram; Figures 1A,B; Rubió et al., 2013;Przygodzka et al., 2014;Srinivasan, 2014). The relationship between antioxidant properties of spices and food spoilage has been well-documented. Another issue in food spoilage is the microbial growth. Spices can also exert antimicrobial activity in two ways: by preventing the growth of spoilage microorganisms (food preservation), and by inhibiting/regulating the growth of those pathogenic (food safety; Tajkarimi et al., 2010). Studies regarding in vitro and in vivo antimicrobial activities of spices have been reported in the following sections.

Antimicrobial Activity In vitro
Numerous articles published in the last few decades have described the antimicrobial activities of spices in vitro. Extracts of entire plants, or part of them, obtained with diverse solvents (such as ethanol, methanol, ethyl acetate, and water) have been tested against microbes (Tajkarimi et al., 2010). Their essential oils or active compounds, alone or in combination, were also used to test the activity against different microbes (Singh et al., 2007;Weerakkody et al., 2010;Bassolé and Juliani, 2012). Disc-diffusion, drop-agar-diffusion, broth microdilution, and direct-contact technique in agar represent the most common methods utilized for screening Malik, 2010a, 2011).
According to these reports, spices possess a very wide spectrum of activity against Gram-positive and Gram-negative bacteria, yeasts and molds (Tajkarimi et al., 2010; Table 1). Alves-Silva et al. (2013) reported that the bush-basil essential oils have antimicrobial activity against Listeria innocua, Serratia marcenscens, Pseudomonas fragi, P. fluorescens, Aeromonas hydrophila, Shewanella putrefaciens, Achromobacter denitrificans, Enterobacter amnigenus, En. gergoviae, and Alcaligenes faecalis, and against the yeasts Yarrowia lipolytica, Saccharomyces cerevisiae, Candida zeylanoides, Debaryomyces hansenii, and Pichia carsonii. Moreover, they were able to inhibit molds such as Mucor racemosus and Penicillium chrysogenum. In the same study, celery and coriander essential oils also showed a very similar antimicrobial activity against the tested strains.
Although the antimicrobial activity of spices may vary according to the types of spice (origin and bioactive compounds), different bacteria can react in different ways (Hyldgaard et al., 2012). Oregano essential oil showed higher antimicrobial activity against Listeria monocytogenes compared to Escherichia coli (Siroli et al., 2014b). Huacatay and basil essential oils were   active against Staphylococcus aureus and Bacillus subtilis (Shirazi et al., 2014). Essential oil of angelica roots were effective against Clostridium difficile, Cl. perfringens, Enterococcus faecalis, Eubacterium limosum, Peptostreptococcus anaerobius, and in a lower extent against E. coli and Bacteroides fragilis (Fraternale et al., 2014). Nigella sativa extracts were more effective on St. aureus (5th day inhibition zone 34 mm) as compared to E. coli (5th day inhibition zone, 13 mm) and P. aeruginosa (5th day inhibition zone, 30 mm; Islam et al., 2012). Rosmarinus officinalis essential oil showed a strong antimicrobial effect against Ls. monocytogenes and S. aureus compared with E. coli (Jordán et al., 2013). A list of spices and their effects on most relevant bacteria is reported in Table 1.
Spices, essential oils and extracts have also been known for their anti-fungal activity ( Table 1; Tajkarimi et al., 2010). Huacatay and basil essential oils were active against Candida albicans (Shirazi et al., 2014). Radwan et al. (2014) reported that among 22 common spice extracts, turmeric, and nutmeg extracts were the most active against different plant pathogens belonging to the genus Colletotrichum. In another study, where 23 spice extracts were studied, Olax subscorpioidea extract showed the highest antifungal activity, particularly against C. albicans and C. tropicalis (Dzoyem et al., 2014). A reduction of mycelial growth and inhibition of conidial germination and aflatoxin production by A. flavus were described by Nerilo et al. (2016) when 150, 10 and 15 µg/mL of ginger EO were applied, respectively. Ferreira et al. (2013) also reported a decrease (99.9 and 99.6%) of aflatoxin B1 and B2 when 0.5% of turmeric EO was employed while the same EO completely inhibited the biomass of Fusarium graminearum and its zearalenone production, at 3.5 and 3 mg/mL, respectively (Kumar et al., 2016).

Enhancement of the Antimicrobial Activity In vitro
To enhance the antimicrobial potential of spices or their constituents, the use of mixed extracts or natural compounds having different origins have been reported (Bassolé and Juliani, 2012). In most of the cases spices showed synergistic activities/effects. For instance, the antimicrobial activity of basil, oregano, bergamot, and perilla essential oils alone or in combinations, were tested. Basil and oregano essential oils alone had MICs of 1.25 and 0.625 µL/mL against E. coli, respectively, while their values were 0.313 µL/mL when used in combination.
The MIC values against St. aureus for basil and bergamot EOs alone were for both 1.25 µL/mL, whereas the MICs of the two essential oils decreased to 0.313-0.156 µL/mL when combined, indicating higher antimicrobial activity. MICs of oregano and bergamot essential oils were 0.625 and 1.25 µL/mL against B. subtilis, respectively, whereas 0.313 µL/mL was determined for combined effect. Finally, the MIC values of oregano and perilla were 0.625 µL/mL for both against S. cerevisiae, while the mixture needed MICs of 0.313-0.156 µL/mL (Lv et al., 2011). In another study, Tabanelli et al. (2014) demonstrated the additive effect of citral and linalool against S. cerevisiae. In fact, linalool (250 mg/L) reduced markedly the amount of citral needed for the same effect (from around 150 to 50 mg/L). However, Tejeswini et al. (2014) reported antagonistic effects when cinnamaldehyde was combined with clove essential oils for molds inhibition.
The use of spice oils together with other preservation techniques has been also assessed. For example, low pressure atmosphere enhanced the susceptibility of E. coli and S. enteritidis to oregano, lemongrass or cinnamon essential oils in vitro. In particular, the MIC of cinnamon vapors for S. enteritidis decreased from 0.512 to 0.128 µL/mL (Frankova et al., 2014). Tabanelli et al. (2014) reported that the decrease of a w potentiated the antimicrobial effect of citral (but not linalool) while lower pH favored the antimicrobial power of linalool (but not citral) against S. cerevisae. Some other hurdle technologies were also used for the enhancement of antimicrobial potential of essential oils. Tyagi and Malik (2010a, 2011 described the enhancement in antimicrobial potential of essential oils in combination of negative air ions (NAI) against food spoilage microorganisms.

Antimicrobial Potential in Real Food Model System (In vivo)
Numerous natural compounds of spices with defined antimicrobial properties have been isolated. However, in vitro studies represent only one part of the use of active compounds as preservatives in food. Moreover, their physical and biochemical properties have been changed in real food systems due to the complexity of the food matrices (Tajkarimi et al., 2010). Therefore, whether spices or their components have the potential to inhibit the food spoilage and act as a food preservative has been determined in different studies.
As summarized in Table 2, the use of spices as preservatives has been assessed in multiple foods: meat, fish, dairy products, vegetables, rice, fruit, and animal food (Tajkarimi et al., 2010;Jayasena and Jo, 2013). Hernández-Ochoa et al. (2014) reported that cumin and clove essential oils inhibited the growth of total bacteria by 3.78 log CFU/g when used on meat samples for 15 days at 2 • C. The antimicrobial activity of different spice extracts in raw chicken meat during storage for 15 days at 4 • C was also studied. It has been found that the treatment of raw chicken meat with extracts of clove, oregano, cinnamon, and black mustard was effective against microbial growth (Radha et al., 2014). Essential oils of marjoram and coriander showed above 50% protection of chickpea seed from Aspergillus flavus infestation (Prakash et al., 2012). In an in vivo assay with cherry tomatoes (Lycopersicon esculentum), bay oil was effective against Alternaria alternata infection (Xu et al., 2014). In another experiment, Da Silveira et al. (2014) treated fresh Tuscan sausages with bay leaf essential oil. Comparing to the non-treated control, the essential oil was able to reduce the population of total coliforms (reduction of 2.8 log CFU/g) and extended the shelf life for 2 days. Rattanachaikunsopon and Phumkhachorn (2008) applied basil oil in nham, a fermented pork sausage, inoculated with S. enteritidis SE3 at 4 • C. Basil oil reduced the number of bacteria from 5 to 2 log CFU/g after 3 days and the sensory evaluation suggested that these concentrations of oil were acceptable for the consumers. The isothiocyanates derived from oriental mustard reduced aflatoxins biosynthesis in A. parasiticus by 60.5-89.3% during Italian piadina storage (Saladino et al., 2016). Finally, Patrignani et al. (2015) reviewed the use of spices and their constituents in minimally processed fruits and vegetables.
Although several studies proved possible applications for spices and their derivatives as food preservatives, only few of them are currently applied on the market. For instance, rosemary is already employed for its preservative properties in meat products. Essential oil of rosemary has been used not only for its flavoring compounds but also for its antimicrobial and antioxidant activity. In fact, carnosic acid, one of its main component, is not only antimicrobial but it possesses an antioxidant activity higher than the common food additives, butylated hydroxytoluene (BHT), and butylated hydroxyanisole (BHA; De La Torre Torres et al., 2015).
Allyl isothiocyanate (AITC), a bioactive organosulfur compound found in cruciferous, plants, such as mustard, is known for its anticarcenogenic properties. It has been tested for effectiveness in preservation of fresh beef, sliced raw tuna and cheese. It possesses a strong antimicrobial activity against E. coli O157:H7, Salmonella enterica serovar Montevideo, S. enterica ser. Typhimurium, P. corrugata, Campylobacter jejuni, St. aureus, and Ls. monocytogenes. Moreover it has the generally recognized as safe (GRAS) status provided by the regulatory agencies of U.S. However, its application is sometimes limited because of its poor aqueous solubility, instability at high temperature, and susceptibility to degradation by nucleophilic molecules (Kim et al., 2002;Li et al., 2015).

Enhancement of the Antimicrobial Activity In vivo
Although some in vivo studies ended up with products acceptable for the consumers, the sensory aspect represents a critical point Frontiers in Microbiology | www.frontiersin.org  Sessou et al., 2012 in the use of spices and their active compounds in food. In fact, sometimes MIC values were three or four times higher than those estimated in vitro, have been applied to have a measurable or stable antimicrobial effect in vivo. This aspect can dramatically affect the physical characteristics and organoleptic properties of the food products. To overcome these issues, several strategies have been exploited for the enhancement of antimicrobial potential of spices in vivo.
The synergistic effect of spices together with their constituents or other natural products has been tested. Water extracts of clove, cinnamon, and oregano were applied, alone (10 mg/L) or in combination (3.3 g/L each), in raw chicken meat and several characteristics were followed during storage for 15 days at 4 • C. The mixture of the three extracts had the strongest impact on the bacterial load due to the synergistic actions of antimicrobial compounds present in the mixed spices (Radha et al., 2014). Siroli et al. (2014a) examined citral, carvacrol, citron essential oil, hexanal and 2-(E)-hexenal, alone (250 mg/L) or in combination (125 + 125 mg/L, except for the combination of citron essential oil/carvacrol, 200 + 50 mg/L, respectively), to sanitize minimally processed apples. The treatment with citral/2-(E)-hexenal and hexanal/2-(E)-hexenal maintained a good retention of color parameter within the 35 days and there were no yeast spoilage in any treated sample. Gabriel and Pineda (2014) studied the effect of different concentrations of vanillin and licorice root extract (LRE) on the mild heat decimal reduction times (D55-values) of a cocktail of E. coli O157:H7 in young coconut liquid endosperm. They found that the combined effect was most significant only at concentrations above 250 and 210 mg/L, respectively for vanillin and LRE. The efficacy of thymol (0.1% w/w) in combination with sodium lactate (1 and 2% v/w) was evaluated in fish patty samples stored at 4 • C for 5 days. The presence of thymol plus 2% of sodium lactate had a synergetic effect against S. enterica ser. Typhimurium (Ilhak and Guran, 2014). Tejeswini et al. (2014) evaluated the antifungal activity of cinnamaldehyde, eugenol, peppermint, and clove essential oils and their combinations in tomato fruit system. While different concentrations of eugenol in combination with peppermint showed either additive or non-significant effect on mold inhibition, combination of cinnamaldehyde with clove essential oil produced nonsignificant or antagonist effects. Barbosa et al. (2014) also assessed the impact of basil essential oil alone or in combination with sodium hexametaphosphate (SHMP), on the shelf life of chicken sausage. Concentrations of 0.3 or 0.03% of essential oil inhibited the coliforms for 15 days at 4 • C (P < 0.05). On the contrary, this effect was inhibited when SHMP was combined.
The synergistic effect of spices on other food preservation systems, such as mild thermal processing, has been also explored. Ngang et al. (2014) studied how to reduce the thermal impact during juice production. They demonstrated that pasteurizing pineapple juice at 60 • C in presence of long coriander essential oil, lowered the time required for a 97% reduction of Ls. monocytogenes compared with treatment without essential oil. Similarly, mint, lemon grass, or eucalyptus essential oils worked synergistically with mild thermal treatment to inhibit the microbial growth in real food systems. Therefore, subsequent lower doses of oils were required for the food preservation (Tyagi et al., 2013(Tyagi et al., , 2014a. The use of spices together with additional high tech/cuttingedge technologies has also been studied. Pina-Pérez et al. (2012) demonstrated the applicability of Pulsed Electric Fields (PEF) in combination with cinnamon against S. enterica ser. Typhimurium to enhance the safety of dairy beverages. The maximum synergistic effect was achieved by 10 kV/cm-3000 µs PEF treatment with 5% (w/v) cinnamon. The maximum inactivation level (1.97 log 10 cycles) was achieved at 30 kV/cm-700 µs plus 5% cinnamon. Patrignani et al. (2013) enhanced the effect of high-pressure homogenization (HPH) treatment (100 MPa for 1-8 successive passes) with citral into inoculated apricot juices, extending their shelf life in turn. Abriouel et al. (2014), instead, potentiated the effect of high hydrostatic pressure (HHP) on brined olives using thyme and rosemary essential oils. In other cases, novel technologies have been used to preserve the functional compounds. For instance, the use of AITC can be limited by its poor aqueous solubility, degradation by nucleophilic molecules, high volatility, and strong odor. Koa et al. (2012) masked the odor and volatility of AITC through its microencapsulation with Arabic gum and chitosan. In addition, Li et al. (2015) developed nanoemulsions that allowed a better aqueous solubility and chemical stability. Eventually, new packaging systems (active packaging) have been studied where essential oils or their main compounds were incorporated into the films. However, until now the research did not provide consistent results (Maisanaba et al., 2016). All these studies showed that the antimicrobial and food preservative potential of natural compounds can be enhanced or maintained by applying physical technologies.

MODE OF ANTIMICROBIAL ACTION OF SPICES
Although the antimicrobial effects of spices and their derivates have been tested against a wide range of microorganisms over the years, their mode of action is still not completely understood. In fact, spices and their essential oils can contain many different bioactive compounds present in variable amounts. Basically, the bioactive constituents of spices can be divided into volatile and non-volatile compounds (Figures 1A,B). The first ones are mainly responsible for the antimicrobial activity of spices. They can be divided in four groups: terpens, terpenoids, phenylpropenes, and "others" (such as products of degradation; Hyldgaard et al., 2012). Terpens are evaluated as lesser active antimicrobial compounds amongst the other compounds. For instance, the weak activity of ρ-cymene, one of the main component of thyme, is mainly related to its action as a substitutional membrane impurity. It can affect the melting temperature and the membrane potential, which in turn causes a decrease in cell motility (Hyldgaard et al., 2012). On the other hand, terpenoids, such as the well-studied thymol and carvacrol, exert their antimicrobial activity due to their functional groups (hydroxyl groups and delocalized electrons). For instance, thymol can interact with the membrane both with the polar head-group region of the lipid layer, affecting the permeability, or with the proteins, determining an accumulation of misfolded structures (Hyldgaard et al., 2012;Marchese et al., 2016). These changes can lead to cell leakages that in turn can bring the cell to death (O'Bryan et al., 2015). Once it is inside the cells, thymol can also disrupt important energy-generating processes such as the citrate metabolic pathway and the synthesis of ATP (Hyldgaard et al., 2012;O'Bryan et al., 2015). Carvacrol acts mainly at the level of the membrane as a transmembrane carrier of monovalent cations, exchanging K+ with H+ in the cytoplasm (O'Bryan et al., 2015). Other organic compounds present in spices are phenylpropenes, such as eugenol and cinnamaldhehyde. The antimicrobial activity of eugenol is performed mainly at the level of the membranes and proteins, inducing permeabilization and enzyme inactivation. On the contrary cinnamaldheyde, although less powerful than eugenol, can react and cross-link with DNA and proteins other than interact with cell membranes. Eventually, spices possess other degradation compounds originating from unsaturated fatty acids, lactones, terpenes, glycosides, and sulfur-and nitrogen-containing molecues. For instance, the mode of action of AITC, a nitrogen-containing compound, is generally considered as a non-specific inhibition of periplasmic or intracellular targets. In fact, due to its highly electrophile central carbon atom, it can inhibit enzymes and affect proteins by oxidative cleavage of disulfide bonds (Hyldgaard et al., 2012). AITC is the main constituent of mustard essential oil. Clemente et al. (2016) reported that mustard EO induced cell cycle arrest, resulting in bacterial filamentation.
Other than affecting membrane and intracellular stability, Szabo et al. (2010) reported that clove, oregano, lavender, and rosemary essential oils possess quorum sensing inhibitory activity. For instance, molecules such as furanones can be internalized by bacteria, bind to LuxR-type proteins, and destabilize them (Camilli and Bassler, 2006). In this way spices could impact the motility, swarming, and biofilm production of bacteria. Overall, antimicrobial activity of spices cannot be confirmed based only on the action of one compound. The final activity is a synergistic effect of more components.

CONCLUSION
Starting from the food preparation, spices can affect both food spoilage microorganisms (food preservation) and human pathogens (food safety) due to the antimicrobial and antifugal activity of their natural constituents. Spices are provided from natural herbs and plants and generally recognized as safe (GRAS) by the American Food and Drug Administration (FDA). However, the need of high amount of natural compounds represent the main limitation for effective performance against microorganisms. Mostly, their organoleptic characteristics may impact the results of in vitro and in vivo trials. For this reason, combinations of spices or their pure natural compounds, applied with or without additional technologies, represent a promising alternative to avoid this problem. Synergistic effects can lead to a reduction of both natural compounds used and treatment applied. In several cases, additive activities have been also reported. The study of spices, natural compounds, and novel combination technologies can be source of inspiration for developing novel or enhanced molecules acting against spoilage microorganisms.

AUTHOR CONTRIBUTIONS
DG: Data compilation, manuscript writing, DB: Data compilation, table formation, SP: Data compilation, manuscript writing, and formating, AT: Data compilation, manuscript writing, editing and formatting, and final approval.

ACKNOWLEDGMENTS
We thank Prof. V. Craig Jordan (Father of Tamoxifen) for providing the facility to revise the manuscript. This research was supported by a grant from the Ministry of Education, Science and Technological Development of Serbia (Project No. 173029).