**5.1 Analysis and identification of individual components**

For the chemical analysis of plants and herbs, one must choose the part of the plant containing the active compounds, and depending on the plant it may be the root (ginseng, for instance), the flowers (lavender, for example), the leaves (like bay leaf and thyme), the bark (cinnamon), the seeds, the fruit or the stem. It is also possible to observe variation of content of antimicrobial molecules in each plant due to different factors as: influence of seasonal harvest, geographic location and altitude and extraction procedures.

Fresh products can be used in order to obtain aqueous mixtures, but before extraction using organic solvents, samples are usually dried and grinded. The samples may be prepared as crude extracts or aqueous and organic solvent extracts. Further operations may include filtration and centrifugation (for clarification). Besides water, extraction is made by use of organic solvents, because most active components are not totally water-soluble. In fact, only a small portion is water-soluble, like polypeptides and polysaccharides (starch). The main organic solvents are ethanol, methanol, chloroform, dichloromethane, diethyl ether, petroleum ether and acetone. The techniques for identification of molecules and description of molecular structure include chromatography (from simple thin-layer chromatography to high-performance liquid chromatography), radioimmunoassay, mass spectrometry, nuclear magnetic resonance and X-ray crystallography.

#### **5.2 Evaluation of inhibition properties**

The differences with respect to the techniques employed to investigate the action of plant compounds and a wide variation in the chemical composition of some plant preparations can result in data difficult to compare between surveys. There is also no consensus on acceptable levels of inhibition for plants compounds, when compared with standard antibiotics. The methodologies used for the evaluation of antimicrobial properties of plants, herbs, spices and condiments do not differ significantly from those used in classic clinical microbiology. The inhibitory effects on bacteria and yeasts can be tested by broth dilution assay (evaluation can be performed using optical density or colony viable count), which allows determination and comparison of MIC and susceptibility tests by disk or agar-well diffusion. For classic antibiotics, CLSI (Clinical Laboratories Standards Institute) establishes the exact concentrations to be studied, whereas with spices or herbs there are no such specifications.

A problem that may be encountered when reviewing some scientific literature is the difference between concepts. Although MIC is a well-defined and internationally accepted concept, there are several uses of the term. In her review, Burt (Burt, 2004) gathers a number of possible definitions for MIC, MBC (minimum bactericidal concentration), bacteriostatic concentration and bactericidal concentration. These possible differences in the concepts must be taken into consideration when performing laboratorial assays. Moreover, it is also important when comparing results, in order to know with what values each researcher is comparing his results to. MIC is defined as the lowest concentration resulting in maintenance or reduction of inoculum viability; the lowest concentration required for complete inhibition of test organism up to 48 hours incubation; or as the lowest concentration resulting in a significant decrease in inoculum viability (>90%). MBC is established as being the concentration where 99.9% or more of the initial inoculum is killed or the lowest concentration at which no growth is observed after subculturing into fresh broth. Bacteriostatic concentration is defined as the lowest concentration at which bacteria

For the chemical analysis of plants and herbs, one must choose the part of the plant containing the active compounds, and depending on the plant it may be the root (ginseng, for instance), the flowers (lavender, for example), the leaves (like bay leaf and thyme), the bark (cinnamon), the seeds, the fruit or the stem. It is also possible to observe variation of content of antimicrobial molecules in each plant due to different factors as: influence of

Fresh products can be used in order to obtain aqueous mixtures, but before extraction using organic solvents, samples are usually dried and grinded. The samples may be prepared as crude extracts or aqueous and organic solvent extracts. Further operations may include filtration and centrifugation (for clarification). Besides water, extraction is made by use of organic solvents, because most active components are not totally water-soluble. In fact, only a small portion is water-soluble, like polypeptides and polysaccharides (starch). The main organic solvents are ethanol, methanol, chloroform, dichloromethane, diethyl ether, petroleum ether and acetone. The techniques for identification of molecules and description of molecular structure include chromatography (from simple thin-layer chromatography to high-performance liquid chromatography), radioimmunoassay, mass spectrometry, nuclear

The differences with respect to the techniques employed to investigate the action of plant compounds and a wide variation in the chemical composition of some plant preparations can result in data difficult to compare between surveys. There is also no consensus on acceptable levels of inhibition for plants compounds, when compared with standard antibiotics. The methodologies used for the evaluation of antimicrobial properties of plants, herbs, spices and condiments do not differ significantly from those used in classic clinical microbiology. The inhibitory effects on bacteria and yeasts can be tested by broth dilution assay (evaluation can be performed using optical density or colony viable count), which allows determination and comparison of MIC and susceptibility tests by disk or agar-well diffusion. For classic antibiotics, CLSI (Clinical Laboratories Standards Institute) establishes the exact concentrations

A problem that may be encountered when reviewing some scientific literature is the difference between concepts. Although MIC is a well-defined and internationally accepted concept, there are several uses of the term. In her review, Burt (Burt, 2004) gathers a number of possible definitions for MIC, MBC (minimum bactericidal concentration), bacteriostatic concentration and bactericidal concentration. These possible differences in the concepts must be taken into consideration when performing laboratorial assays. Moreover, it is also important when comparing results, in order to know with what values each researcher is comparing his results to. MIC is defined as the lowest concentration resulting in maintenance or reduction of inoculum viability; the lowest concentration required for complete inhibition of test organism up to 48 hours incubation; or as the lowest concentration resulting in a significant decrease in inoculum viability (>90%). MBC is established as being the concentration where 99.9% or more of the initial inoculum is killed or the lowest concentration at which no growth is observed after subculturing into fresh broth. Bacteriostatic concentration is defined as the lowest concentration at which bacteria

to be studied, whereas with spices or herbs there are no such specifications.

seasonal harvest, geographic location and altitude and extraction procedures.

**5.1 Analysis and identification of individual components** 

magnetic resonance and X-ray crystallography.

**5.2 Evaluation of inhibition properties** 

fail to grow in broth but are cultured when broth is plated onto agar and the bactericidal concentration is considered to be the lowest concentration at which bacteria fail to grow in broth and are not cultured when broth is plated onto agar (Burt, 2004).

For susceptibility tests controls must be performed simultaneously, usually using the antibiotics most suited for the conventional treatment. Tetracycline was used for comparison with thirteen Thai condiments against *Vibrio parahaemolyticus* (Vuddhakul et al., 2007) and fifteen antiobiotics were used when testing the antimicrobial activity of condiments against multidrug resistant *Escherichia coli* isolated from water in Bangladesh (Rahman *et al*., 2011). Comparison with conventional antibiotics is necessary to categorize the sensibility of a specific microorganism to a plant, herb or condiment as sensible or resistant.

A rigorous evaluation of the individual MIC is sometimes difficult because of the ambiguity of results. Eugenol inhibited 9 out of 14 Gram negative and 12 out of 20 Gram positive bacteria, at a concentration of 493 ppm, by incorporating in Plate Count Agar. But the same paper reported a MIC of 32 ppm for *Candida glabrata* and *Aspergillus niger*, and a MIC of 63 ppm for *Staphylococcus aureus* and *Escherichia coli* (Cowan, 1999).

The variation in concentration of antimicrobial substances is usual and expected. This, coupled with testing different preparations contributes for the difficulty of comparing results from study to study. For instance, allicin in garlic ranges from 0.3 to 0.5%, whereas eugenol in clove ranges from 16 to 18% (Shelef, 1984).

The problem is worsened by the fact each researcher may use different methods for preparing the samples: crude, aqueous extracts, ethanolic or methanolic extracts, chloroform or other solvents, as refered above. The results from testing the inhibitory effect of essential oils is not easy to ascertain, as the hydrophobic nature of such preparations may alter the inhibition areas because of the irregular diffusion, when compared with the more hydrophilic antibiotics. Some researchers add emulsifiers, as Tween 20 or Tween 80, to the oils, but the quantity and nature of the latter must not interfere with the results, like producing false-positive ones. Thus, standardisation of methods becomes difficult to achieve (Nascimento *et al*., 2006).

There are other methodologies available to use in order to ascertain the effects of antibacterial activity of certain phytochemicals. The rate of inhibition and cell death can be observed through time-kill analysis and survival curves. The physical aspects of antibacterial activity concerning the structural modifications achieved can be observed by the use of scanning electron microscopy (Burt, 2004).

Yeasts susceptibility can be evaluated by techniques similar to those used for bacteria, however the evaluation of antifungal ability of phytochemicals must be performed by other methods. One of these methods is the spore germination assay. Spores are put in contact with the testing compound for a period of time; afterwards they are observed microscopically in a slide (usually fixed with lactophenol cotton blue) and spore germination (or its absence) is observed.

In terms of antiviral ability of plant products, they can be determined by observation of cytopathic effects or plaque formation in cells infected and put in contact with the phytochemicals. Other option is, in the same conditions, to use molecular techniques for detection of products resulting from viral replication, as nucleic acids.

Antimicrobial Activity of Condiments 125

Marjoram oil Several species

Eugenol *Listeria monocytogenes*,

Mint oil *Listeria monocytogenes,* 

Cilantro oil *Listeria monocytogenes*  Oregano oil *Clostridium botulinum* spores Rosemary *Listeria monocytogenes* 

Thyme oil, cinnamaldehyde *Pseudomonas putida*  Carvacrol, citral, geraniol *Salmonella typhimurium*  Oregano oil *Photobacterium phophoreum*  Mint oil *Salmonella enteritidis*  Eugenol, linalool, oregano Increased shelf-life

Clove oil, carvacrol *Listeria monocytogenes* 

peppermint oil *Streptococcus thermophilus*  Mint oil *Salmonella enteritidis* 

Thyme oil *Escherichia coli* O157:H7

Oregano oil *Escherichia coli* O157:H7 Cinnamaldehyde *Escherichia coli* O157:H7

Sage oil *Bacillus cereus, Staphylococcus* 

Basil methyl chaviol (BMC) Natural flora

Carvacrol Natural flora

Fruits Carvacrol, cinnamic acid Natural flora

**7. Commercial availability and legislation** 

Table 2. List of herbs/spices/condiments or natural antimicrobial molecules used in

Basil Rice storage pests

different groups of foodstuff and their potential targets (adapted from Tajkarimi *et al*., 2010).

Some essential oils have been registered by the European Commission as flavouring agents to be used in foodstuff. In terms of legislation related to foodstuff, both the European Union (through European Food Safety Agency and its national branches) and the United States

Chinese cinnamon Pathogenic microorganisms Oregano, pimento, oregano+pimento *Escherichia coli* O157:H7,

Sage oil *Bacillus cereus, Staphylococcus* 

*Listeria monocytogenes*, *Aeromonas hydrophila*, *Escherichia coli* O157:H7

*aeruginosa*, *Listeria monocytogenes*

*Pseudomonas* spp.

*Aeromonas hydrophila*

*Salmonella enteritidis* 

*aureus, Salmonella typhimurium* 

*aureus, Salmonella typhimurium* 

*Bacillus cereus*, *Pseudomonas* 

**Food Group Plant/spice/herb or active compound Microbial target** 

Clove oil, eugenol+coriander, oregano, thyme oils, rosemary oil, clove, tea tree

Oregano+thyme, oregano+marjoram,

Clove, cinnamon, cardamom,

thyme+sage

Meat Products

Fish

Dairy

Rice

Vegetables

Researchers must have in mind, as mentioned earlier, that different plants may have the same antimicrobial phytochemicals, although they may be in different concentrations, resulting in different MIC for the same molecule. Moreover, the same plant may have more than an antimicrobial molecule, resulting in effects that can not be easily evaluated. An inhibition or decrease in bacterial population may be due to different mechanisms.
