A. Meyer, K. Suhr and P. Nielsen, Technical University of Denmark,
6.4 Plant-derived antimicrobial agents
Plants protect themselves against microorganisms and other predators by synthesising a wide range of compounds. These compounds are also termed secondary metabolites, as they generally are not essential for the basic metabolic processes – the primary metabolism. Secondary metabolites represent a diverse array of chemical compounds mostly derived from the isoprenoid, phenylpropanoid, alkaloid or fatty acid/polyketide pathways (Dixon, 2001).
Antimicrobial compounds from plants can be divided into three groups:
1. Preinfectional agents at the plant surface (constitutive).
2. Agents bound in vacuoles and associated with hydrolytic enzyme activation systems (constitutive).
3. Phytoalexins, which are compounds produced in response to invasion (inducible).
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Compounds from the first group are normally always present in the plant as a first line of defence of the plant, in the leaf glands, for example (Harborne, 1988). The second group of compounds are highly reactive but require tissue rupture or cell collapse in order to be activated. They are stored as inactive precursors and their corresponding enzymes appear in other cell compartments.
The third group of compounds, phytoalexins, are normally not present in healthy unaffected plants. They are only produced as a response to stress (invasion by microorganism or environmental factors like drought).
It is the compounds from the first two groups that are contained in the essential oil fractions or extracts obtained from healthy plants, and they are the components generally explored for food preservation purposes.
6.4.1 Essential oils
Since ancient times antimicrobial properties of herbs and spices have been acknowledged and exploited for preservation of food and other organic matter as well as for medical treatments (Conner, 1993; Hirasa and Takemasa, 1998). In the Western world emergence and success of synthetic preservatives have practically made ‘natural’ food preservation extinct, and it is only in the last few decades that scientific interest in this area has re-emerged. The antimicrobial compounds in plant materials are commonly found in the essential oil fractions obtained by steam or supercritical distillation, pressing, or extraction by liquid or volatile solvents. Generally the oils consist of a mixture of esters, aldehydes, ketones, terpenes and phenolic compounds, and harbour the characteristic flavour and aroma of the particular spice or herb. The traditionally most well-known antimicrobial spices and herbs are clove, cinnamon, chilli, garlic, thyme, oregano and rosemary. But also bay, basil, sage, anise, coriander, allspice, marjoram, nutmeg, cardamom, mint, parsley, lemongrass, celery, cumin, fennel and many others have been reported to have an inhibitory effect toward microorganisms (Deans and Ritchie, 1987; Hili et al., 1997; Hammer et al., 1999; Elgayyar et al., 2001; and Table 6.3 below.
Many of the identified active compounds have phenolic structures, and the same compounds can be found ubiquitously in different oils, such as eugenol in cloves and cinnamon leaf, thymol and carvacrol in thyme and oregano, 1,8-cineole in sage, thyme and bay. Structures of some active compounds are presented in Figure 6.1.
6.4.2 Organic acids
The commercially most important preservatives are still the organic acids (Table 6.2). They are all naturally occurring, although the bulk amount of these substances used in foods are synthetically produced. Based on their main antimicrobial effect they can be divided into two groups. One group shows an antimicrobial activity owing primarily to their pH-reducing effect. This group includes acetic acid, citric acid, formic acid, lactic acid, malic acid, oxalic acid
and tartaric acid. They work either directly by lowering the pH of the food and thus adding stress to the microorganism, or in the undissociated form by migrating through the cell membrane into the cytoplasma of the microorganism where they dissociate and lower the internal pH of the cell. However, several reports have shown that the undissociated form of acetic acid also has
Fig. 6.1 Structures of some active compounds from essential oils.
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antimicrobial action (e.g. Rusul et al., 1987). Other reports have shown that the supplementation of up to 2.5% of citric, malic, tartaric or lactic acid to substrates adjusted to pH 2.5 or 3.5 significantly enhances fungal growth and mycotoxin production (Nielsen et al., 1989; El-Gazzar et al., 1987).
The other group of organic acid preservatives (sorbic acid, benzoic acid and propionic acid) shows antimicrobial activity only when they are present as undissociated acids. The efficiency of these acids therefore depends on the dissociation constant, pKa. As the pKaof most acids is between 3 and 5 (Table 6.2), these preservatives are only active at lower pH-values. However, some reports have shown that the dissociated acid may also have some antimicrobial activity (up to 2.5% of the undissociated compound) (Skirdal and Eklund, 1993).
Organic acids are, in practice, usually added as the corresponding sodium, potassium or calcium salts because they are more soluble in water.
Sorbic acid is an unsaturated fatty acid (CH3-CH=CH-CH=CH-COOH) with a high pKa value (4.76), which makes it active in low acid food and it is therefore applied in a great variety of food products. The compound is active against yeasts, moulds and many bacteria. Microorganisms resistant to sorbate exist and an increasing number of moulds are able to degrade sorbate, producing strong off-flavours by the decarboxylation of sorbic acid into trans-1,3-pentadiene (Liewen and Marth, 1985; Kinderlerer and Hatton, 1990).
Benzoic acid (C6H5COOH) was first described as a preservative in 1875. The compound occurs naturally in high amounts in cranberries and some other fruits and spices. Sodium benzoate has a pKavalue of 4.19, which gives optimal activity below pH 4.0. Some fungi isolates of P. roqueforti have been found to be resistant.
Table 6.2 Natural antimicrobial organic acids
Organic acids E number1 Produced by pKa MIC/allowed
dose (ppm)2
Acetic acid E260, 261–3 Bacteria, fungi, animals 4.74 q.s.
Carbonic acid E290 Bacteria, fungi, plants, 6.37; 10.25 q.s.
animals
Citric acid E330, 331–3 Bacteria, fungi, plants 3.13; 4.77; 6.39 q.s.
Formic acid E236, 237–8 Plants, animals 3.75 q.s.
Lactic acid E270, 325–7 Bacteria, fungi, animals 3.08 q.s.
Malic acid E296 Bacteria, fungi, plants 3.40; 5.11 q.s.
Oxalic acid Bacteria, fungi, plants 1.23; 4.19
Tartaric acid E334, 335–7 Plants 2.98; 4.34 q.s.
Benzoic acid E210, 211–13 Cranberries, cinnamon, 4.19 200–6,000 clove a.o.
Propionic acid E280, 281–3 Bacteria, plants 4.87 1,000–3,000
Sorbic acid E200, 201–3 Plants 4.76 200–6,000
Medium chain Plants, animals
fatty acids + microbial fermentation *3
products
Notes: 1. E number as listed in Directive 95/2/EU. 2. The maximum level of use depends on the foodstuff. *3. Kabara, 1993.
Benzoates have an advantage of low cost compared to other preservatives. An adverse flavour effect is often encountered at higher use levels.
Propionic acid is produced by several lactic acid bacteria and is also found in some fruits and spices. It is effective against moulds and some bacteria but has very limited activity against yeasts. Propionates are able to inhibit Bacillus subtilis, which causes ‘rope’ in bread. Propionates have been used particularly in preservation of grains, bread and other baked products. Levels used are generally higher than with benzoate and sorbate.
6.4.3 Enzyme-released antimicrobial agents
In common food plants two major types of antimicrobial compounds activated upon hydrolytic cleavage exist. In the Allium family (garlic, onion, leek) sulfoxides are converted into pungent and lachrymatory sulfides such as diallyl disulfide upon tissue rupture (Walker, 1994). Most potent is garlic. It contains alliin (propenyl-cysteine sulfoxide), which is hydrolysed by the enzyme alliinase to allicin (2-propenyl-2-propenethiol sulfinate). A recent study concluded that 0.5% (5,000 ppm) of a garlic extract was needed to inhibit important pathogens (Unal et al., 2001). Other studies have shown that these substances inhibit most microorganisms, but only at relatively high concentrations (Beuchat, 1994;
Conner, 1993), and the main hindrance of use is sensory effects.
In plants of Cruciferae (cabbage, mustard, horseradish, brussels sprout) glucosinolates are the substrates for hydrolytic enzymes. A number of different isothiocyanates are identified in the Cruciferous family, and some of the compounds are also believed to exert anti-carcinogenic effects (Verhoeven et al., 1997; Mithen et al., 2000). Sinigrin stored in mustard seeds are cleaved by myrosinase to yield allyl isothiocyanate (AITC), which constitutes the main compound of mustard essential oil (95%). Penicillium expansum, Aspergillus flavus and Botrytis cinerea were unable to grow in the presence of 100g AITC /L gas-phase, and Salmonella typhimurium, Listeria monocytogenes and Escherichia coli O157:H7 were inhibited by 1000g /L (Delaquis and Sholberg, 1997). These authors found E. coli O157:H7 to be the most resistant of the bacteria tested, whereas Lin et al. (2000) found L. monocytogenes to be more resistant than E. coli O157 : H7 and Salmonella montevideo in broth solution.
Applied through the gas phase, the volatile AITC has proved effective in very low doses against food spoilage fungi (Delaquis and Mazza, 1995; Nielsen and Rios, 2000). The gaseous form seems to have higher antimicrobial potential than the liquid (Lin et al., 2000).
6.4.4 Phytoalexins and other plant-derived compounds
An active defence system of plants against invading microorganisms is the de novo synthesising of phytoalexins. At least 200 phytoalexins have been characterised from over 20 plant families (Harborne, 1988), and certain chemical types seem to be associated with some plant families. Examples of Natural food preservatives 133
these are isoflavonoids from Leguminosa as pisatin from garden pea (Pisum sativum) and phaseollin from beans (Phaseolus vulgaris). Others are rishitin from potatoes and tomatoes (Solanaceae), falcarindiol and 6-methoxy-mellein from carrots (Walker, 1994; Smid and Gorris, 1999). Use of phytoalexins for food preservation has been suggested by many, but practical attempts are lacking or scarce (Smid and Gorris, 1999).
Investigation of plant antimicrobial compounds has been focused on food plants or herbs and spices traditionally known to contain activity. Some authors argue that a number of potentially useful plant types are ignored by this approach (Wilkins and Board, 1989). Also the innate antimicrobial systems of some food products could be brought to play important roles in the preservation of the product itself if minimal processing schemes conserving or enhancing the responsible compounds are used. Examples of these systems are the content of protective polycyclic compounds, the proanthocyanidins, in strawberries. They protect against grey mould, Botrytis cinerea, until the berries are ripe and the concentration decreases to non-inhibitory levels (Wilkins and Board, 1989).
Similar effects are seen in ripening fruit (peaches and plums) where volatile aldehydes and esters have been shown to inhibit B. cinerea and other phytopathogens (Wilson et al., 1987).