Herbs and spices and antimicrobials

Herbs and spices are used widely in the food industry as flavours and fragrances. However, they also exhibit useful antimicrobial and antioxidant properties. Many plant-derived antimicrobial compounds have a wide spectrum of activity against bacteria, fungi and mycobacteria and this has led to suggestions that they could be used as natural preservatives in foods. Although more than 1300 plants have been reported as potential sources of antimicrobial agents, such alternative compounds have not been sufficiently exploited in foods to date.

In this article, the antimicrobial compounds from herbs and spices are reviewed and the barriers to the adoption of these substances as food preservatives are discussed. The mode of action of essential oils and the potential for development of resistance are also discussed. The focus is primarily on bacteria and fungi in prepared foods.

Barriers to the use of herb and spice essential oils as antimicrobials in foods:

Since ancient times, spices and herbs have not been consciously added to foods as preservatives but mainly as seasoning additives due to their aromatic properties. Although the majority of essential oils from herbs and spices are classified as Generally Recognized As Safe, their use in foods as preservatives is limited because of
flavour considerations, since effective antimicrobial doses may exceed organoleptically acceptable levels. This problem could possibly be overcome if answers could be given to the following questions:

• Can the inhibitory effect of an essential oil (a mixture of many compounds) be attributed to one or several key constituents?
• Does the essential oil provide a synergy of activity, which simple mixtures of components cannot deliver?
• What is the minimum inhibitory concentration (MIC) of the active compound(s) of the essential oil?
• How is the behaviour of the antimicrobial substance(s) affected by the homogeneous (liquid, semisolid) or heterogeneous (emulsions, mixtures of solids and semisolids)
  structure of foodstuffs?
• Could efficacy be enhanced by combinations with traditional (salting, heating, acidification) and modern (vacuum packing, VP, modified atmosphere packing, MAP) methods of food preservation?

An in-depth understanding of the antimicrobial properties of these compounds is needed to answer these questions but such understanding has been lacking, despite the burgeoning literature on the subject. Methodological limitations (discussed in more detail below) in the evaluation of antimicrobial activity in vitro have led to many contradictory results. Moreover, there have been too few studies in real foods (these are considered la borious and often lead to negative outcomes). There is also a need to investigate the appropriate mode of application of an essential oil in a foodstuff. For instance, immersion, mixing, encapsulation, surface-spraying, and evaporating onto active packaging are some promising methods of adding these compounds to foods that have not been extensively investigated.

Measuring antimicrobial activity:

The antimicrobial activity of plant-derived compounds against many different microorganisms, tested individually and in vitro, is well documented in the literature. However, the results reported in different studies are difficult to compare directly. Indeed, contradictory data have been reported by different authors for the same antimicrobial compound. Also, it is not always apparent whether the methods cited measure bacteriostatic or bactericidal activities, or a combination of both. Antimicrobial assays described in the literature include measurement of:

• the radius or diameter of the zone of inhibition of bacterial growth around paper discs impregnated with (or wells containing) an antimicrobial compound on agar media;
• the inhibition of bacterial growth on an agar medium with the antimicrobial compound diffused in the agar;
• the minimum inhibitory concentration (MIC) of the antimicrobial compound in liquid media;
• the changes in optical density or impedance in a liquid growth medium containing the antimicrobial compound.

Three main factors may influence the outcome of the above methods when used with essential oils of plants:

1. the composition of the sample tested (type of plant, geographical location and time of the year)
2. the microorganism (strain, conditions of growth, inoculum size, etc.), and
3. the method used for growing and enumerating the surviving bacteria.

Many studies have been based on subjective assessment of growth inhibition, as in the disc diffusion method, or on rapid techniques such as optical density (turbidimetry) without accounting for the limitations inherent in such methods. In the disc method, the inhibition area depends on the ability of the essential oil to diffuse uniformly through the agar as well as on the released oil vapours. Other factors that may influence results involve the presence of multiple active components. These active  compounds at low concentrations may interact antagonistically, additively or synergistically with each other. Some of the differences in the antimicrobial activity of oils observed in complex foods compared with their activity when used alone in laboratory media could be due to the partitioning of active components between lipid and aqueous phases in foods.

Turbidimetry is a rapid, non-destructive and inexpensive method that is easily automated but has low sensitivity. Turbidimetry detects only the upper part of growth curves, and requires calibration in order to correlate the results with viable counts obtained on agar media The changes in absorbance are only evident when population levels reach 106 –107 CFU/ml, and are influenced by the size of the bacterial cells at different growth stages. The physiological state of the cells (injured or healthy), the state of oxidation of the essential oil as well as inadequate dissolution of the compound tested may also affect absorbance measurements in growth media.

Unlike the plate counting technique, impedance-based methods can be used to monitor microbial metabolism in real time mode. The impedimetric method is recognized as an alternative rapid method not only for screening the biocide activity of novel antimicrobial agents but also for estimation of growth kinetics in mathematical modelling. The technique depends on using a medium that offers a sharp detectable impedimetric change as the bacterial population grows and converts the low
conductivity nutrients into highly charged products. As with turbidometry, calibration of impedimetric data with plate counts is necessary.

Studies in vitro:

Almost all essential oils from spices and herbs inhibit microbial growth as well as toxin production. The antimicrobial effect is concentration dependent and may become strongly bacteriocidal at high concentrations. Gram-positive bacteria, Gram-negative bacteria, yeasts and moulds are all affected by a wide range of essential oils. Well-known examples include the essential oils from allspice, almond, bay, black pepper, caraway, cinnamon, clove, coriander, cumin, garlic, grapefruit, lemon, mace, mandarin, onion, orange, oregano, rosemary, sage and thyme. The antimicrobial activity of these compounds is influenced by the culture medium, the temperature of incubation and the inoculum size. In addition, a strong synergism with some membrane chelators acting as permeabilizing agents against Gram-negative bacteria has been
reported.

Applications in food systems:

There have been relatively few studies of the antimicrobial action of essential oils in model food systems and in real foods. The efficacy of essential oils in vitro is often
much greater than in vivo or in situ, i.e. in foods. For example, the essential oil of mint (Mentha piperita) has been shown to inhibit the growth of Salmonella enteritidis and Listeria monocytogenes in culture media for 2 days at 30ºC. However, the effect of mint essential oil in the traditional Greek appetizers tzatziki (pH 4.5) and taramasalata (pH 5.0) and in paté (pH 6.8) at 4ºC and 10ºC was variable. Salmonella enteritidis died off in the appetizers under all conditions examined but not when inoculated in paté and maintained at 10ºC. Similarly, L. monocytogenes numbers declined in the appetizers but increased in paté.

Growth of Escherichia coli, Salmonella spp., L. monocytogenes and Staphylococcus aureus was inhibited by oregano essential oil (EO) in broth cultures. However, the
antimicrobial action of this EO in an emulsion or pseudoemulsion type of food such as aubergine salad, taramasalata and mayonnaise depended on environmental factors such as pH, temperature and oil (vegetable or olive) used. Homemade aubergine salad and taramasalata were inoculated with E. coli O157:H7 and Salmonella enteritidis, respectively. The pH of these products was adjusted to 4–5.3. A range of concentrations (0–2.1%) of oregano essential oil was added and the foods were incubated at temperatures from 0 to 20ºC. The survival curves for E. coli O157:H7 in aubergine salad at 0 and 15ºC, A reduction in viable counts for both pathogens in both foods tested was observed and their death rate depended on the pH, the storage temperature and the essential oil concentration.

The type of oil or fat present in a food can affect the antimicrobial efficacy of essential oils. This was evident when the efficiency of four plant essential oils was assessed in low-fat and full-fat soft cheese against L. monocytogenes and Salmonella enteritidis at 4ºC and 10ºC, respectively, over a 14-day period. In the low-fat cheese, all four oils at 1% reduced L. monocytogenes to below the detection limit of the In contrast, in the full-fat cheese, the oil of clove was the only substance to achieve such reduction. The oil of thyme was ineffective against Salmonella enteritidis in the full-fat cheese, despite the fact that this organism was completely inhibited in broth culture. Thyme oil was as effective as the other three oils in the low fat cheese, reducing Salmonella Enteritidis to less than 1 log CFU/g from day 4 onwards.

Mode of action and development of resistance:

In general, the mode of action of essential oils is concentration dependent. Low concentrations inhibit enzymes associated with energy  production while higher amounts may precipitate proteins. However, it is uncertain whether membrane damage is quantitatively related to the amount of active antimicrobial compound to which the cell is exposed, or the effect is such that, once small injuries are caused, the breakdown of the cell follows Essential oils damage the structural and functional properties of membranes and this is reflected in the dissipation of the two components of the proton motive force: the pH gradient (∆pH) and the electrical potential (∆ψ). Carvacrol, an active component of many essential oils, has been shown to destabilize the cytoplasmic and outer membranes and act as a ‘proton exhanger’, resulting in a reduction of the pH gradient across the cytoplasmic membrane. The collapse of the proton motive force and depletion of the ATP pool eventually led to cell death. Like other many preservatives, the essential oils cause leakage of ions, ATP, nucleic acids and amino acids. Nutrient uptake, nucleic acid synthesis and ATPase activity may also be affected,
leading to further damage to the cell. Several reports have demonstrated that most essential impair the respiratory activity of bacteria and yeasts.

Antibiotics and related drugs have substantially reduced the threat posed by infectious diseases in the last century. However, the emergence and spread of antibiotic-resistant bacteria has, more recently, become a major concern. This concern has widened to include all microorganisms exposed to antimicrobial agents, including the so-called ‘natural’ compounds. However, there is relatively little information on the resistance mechanisms of microorganisms against plant-derived antimicrobial  compounds.

Legislation:

Many essential oils from herbs and spices are used widely in the food, health and personal care industries and are classified as GRAS substances or are permitted food additives. A large number of these compounds have been the subject of extensive toxicological scrutiny. However, their principal function is to impart desirable flavours and aromas and not necessarily to act as antimicrobial agents. Therefore, it is possible that additional safety and toxicological data would be required before regulatory approval for their use as novel food preservatives would be granted.

Future prospects and multifactorial preservation:

Given the high flavour and aroma impact of plant essential oils, the future for using these compounds as food preservatives lies in the careful selection and evaluation of their efficacy at low concentrations but in combination with other chemical preservatives or preservation processes. Synergistic combinations have been identified between garlic extract and nisin, carvacrol and nisin, vanillin or citral and sorbate, thyme oil and/or cinnamaldehyde in an edible coating, and low-dose gamma irradiation and extracts of rosemary or thyme.

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