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- Materials and Methods
Aims: To determine structure–function relationships of antibacterial phenolic acids and their metabolites produced by lactic acid bacteria (LAB).
Methods and Results: Minimum inhibitory concentrations (MICs) of 6 hydroxybenzoic and 6 hydroxycinnamic acids were determined with Lactobacillus plantarum, Lactobacillus hammesii, Escherichia coli and Bacillus subtilis as indicator strains. The antibacterial activity of phenolic acids increased at lower pH. A decreasing number of hydroxyl groups enhanced the activity of hydroxybenzoic acids, but had minor effects on hydroxycinnamic acids. Substitution of hydroxyl groups with methoxy groups increased the activity of hydroxybenzoic, but not of hydroxycinnamic, acid.
Metabolism of chlorogenic, caffeic, p-coumaric, ferulic, protocatechuic or p-hydroxybenzoic acids by L. plantarum, L. hammesii, Lactobacillus fermentum and Lactobacillus reuteri was analysed by LC-DAD-MS. Furthermore, MICs of substrates and metabolites were compared. Decarboxylated and/or reduced metabolites of phenolic acids had a lower activity than the substrates. Strain-specific metabolism of phenolic acids generally corresponded to resistance.
Conclusions: The influence of lipophilicity on the antibacterial activity of hydroxybenzoic acids is stronger than that of hydroxycinnamic acids. Metabolism of phenolic acids by LAB detoxifies phenolic acids.
Significance and Impact of the Study: Results allow the targeted selection of plant extracts for food preservation, and selection of starter cultures for fermented products.
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- Materials and Methods
Phenolic compounds are secondary plant metabolites that possess aromatic rings with one or more hydroxyl or methoxy groups. Phenolic acids are a subclass of phenolic compounds, encompassing hydroxybenzoic acids (C6–C1 structures) and hydroxycinnamic acids (C6–C3 structures) (Schieber and Aranda Saldana 2009; Dai and Mumper 2010). Phenolic acids have antimicrobial activity and hold promise for application as preservatives in food and food-packing materials. Phenolic acids or plant extracts containing these compounds gave satisfactory results when added to beef and food-packing materials such as hand sheets and pea starch (Ejechi and Akpomedaye 2005; Elegir et al. 2008; Corrales et al. 2009).
The antimicrobial activity of phenolic acids is determined by their chemical structure, in particular the number and position of substitution in the benzene ring, and the saturated chain length (Cueva et al. 2010). Phenolic acids had lower antimicrobial activity compared with their butyl and methyl esters (Cueva et al. 2010). The antimicrobial effect increased with increasing length of the alkyl chain (Merkl et al. 2010). Oligomers show higher activity than the corresponding phenolic acid monomers (Elegir et al. 2008). Hydroxybenzoic and hydroxycinnamic acids occurring in plants exhibit diversity with respect to the number of hydroxyl or methoxy groups (Fig. 1); however, current knowledge on structure–function relationships of the antimicrobial activity of phenolic acids does not account for this diversity of compounds. Moreover, additional derivatives of these compounds are produced from bacterial metabolism (Fig. 1).
Figure 1. Hydroxybenzoic and hydroxycinnamic acids and their decarboxylated and reduced metabolites used in this study.
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Lactobacillus spp. are more resistant to phenolic compounds when compared to other groups of bacteria such as Clostridium spp. and Bacteroides spp. (Lee et al. 2006). The tolerance of lactobacilli to phenolic acids and their ability to metabolize phenolic acids are strain or species specific (Van Beek and Priest 2000; Cueva et al. 2010; Curiel et al. 2010; Svensson et al. 2010). However, the strain-specific tolerance of LAB to phenolic acids has not been related to the metabolism of phenolic acids. Moreover, the antibacterial activity of the products of phenolic acid metabolism by LAB remains unknown. Knowledge on structure–function relationships of phenolic acids is important for application of these compounds as food preservatives as well as the selection of starter cultures for food fermentations. Therefore, this study aimed to elucidate the structure–function relationships of phenolic acids with model organism isolated from food. Moreover, it was determined whether phenolic acid metabolism by LAB is a mechanism of detoxification of noxious compounds that LAB encounter in their natural habitat.
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Despite the substantial body of literature related to the antibacterial activity of phenolic acids (Table 4), a systematic evaluation of the effect of hydroxyl and methoxy groups on the antibacterial activity of phenolic acids has not been reported. Moreover, different studies used different methodologies for the determination of antibacterial activity, and organisms of the same species or genus exhibit substantial strain-to-strain variation in the sensitivity to phenolic acids (Table 4). This study determined structure–function relationships of hydroxycinnamic and hydroxybenzoic acids by taking into account the effects of hydroxyl and methoxy groups as well as the contribution of carboxyl groups and the double bond in hydroxycinnamic acids. Our data generally agree with MICs of phenolic acids that were previously published (Table 4). Our results particularly confirm and extend for a large selection of structurally diverse phenolic acids that lactobacilli are more resistant against their antibacterial activity compared to E. coli, B. subtilis and other bacteria relevant in food (Table 4, Lee et al. 2006).
Table 4. Antibacterial activity of phenolic acids
|Phenolic acid||Lactobacillus spp.||MIC (g l−1)||References|
|Escherichia coli||Bacillus spp.|
| || ||Hydroxybenzoic acids|| |
|Benzoic acid*||1–1·8||0·50||0·05–0·12||Chipley (2005) and Cueva et al. (2010)|
|p-Hydroxybenzoic acid||0·125–13·8||0·34–0·55||0·40–0·69||Campos et al. (2003), Tuncel and Nergiz (1993), Landete et al. (2008), Cueva et al. (2010) and Merkl et al. (2010)|
|Protocatechuic acid||7·7–14·1||0·55–2·67||0·4–2·67||Tuncel and Nergiz (1993), Taguri et al. (2006), Landete et al. (2008) and Merkl et al. (2010)|
|Gallic acid||< 0·5†||0·60||0·02–1·6||Campos et al. (2003), Taguri et al. (2006) and Wansi et al. (2010)|
|Syringic acid||4·95–4·99||0·55||0·40||Tuncel and Nergiz (1993), Landete et al. (2008)|
| || ||Hydroxycinnamic acids|| |
|Chlorogenic acid|| ||0·10|| ||Xia et al. (2010)|
|Caffeic acid||0·5†–1||0·32–2·67||0·22–1·60||Stead (1993), Wen et al. (2003), Lee et al. (2006), Taguri et al. (2006), Almajano et al. (2007) and Merkl et al. (2010)|
|p-Coumaric acid*||0·5†–1||0·45||0·40||Stead (1993) and Tuncel and Nergiz (1993)|
|t-Cinnamic acid||7·4||1·33|| ||Landete et al. (2008) and Rastogi et al. (2008)|
|Ferulic acid||0·5†–1||0·45–1·94||0·40–1·94||Stead (1993), Tuncel and Nergiz (1993) and Merkl et al. (2010)|
In analogy to other weak organic acids, benzoic acid and hydroxybenzoic acids exert antimicrobial activity by diffusion of the undissociated acid across the membrane, resulting in acidification of the cytoplasm and, eventually, cell death (Herald and Davidson 1983; Ramos-Nino et al. 1996; Phan et al. 2002; Campos et al. 2009). Consequently, the pKa and the lipophilicity were proposed to determine the solubility of phenolic acids in bacterial membranes and thus their antimicrobial activity (Herald and Davidson 1983; Ramos-Nino et al. 1996; Campos et al. 2009). Hydroxybenzoic acids and hydroxycinnamic acids are weak organic acids but differ in their lipophilicity. Factors that affect the lipophilicity of phenolic acids include pH, which determines the charge of the carboxyl group, ring substitutions (hydroxyl and methoxy groups) and the saturation of the side chain of cinnamic acids.
A decrease in the pH increased the antibacterial activity of phenolic acids. The same trend has been reported for hydroxycinnamic acids and benzoic acid (Otto and Conn 1944; Herald and Davidson 1983; Wen et al. 2003; Almajano et al. 2007). The concentration of undissociated, more lipophilic phenolic acids increases with decreasing pH. The activity of undissociated phenolic acids is higher compared to dissociated phenolic acids, because they are more soluble in the cytoplasmic membrane (Ramos-Nino et al. 1996). However, our results demonstrate that dissociation of phenolic acids does not fully account for the effect of pH on their activity. The pH also had a strong effect on the MIC of phenolic acids when these were calculated on the basis of the undissociated portion (data not shown). Thus, dissociation is not the only factor responsible for their antibacterial activity.
The number of hydroxyl groups altered the antibacterial activity of hydroxybenzoic acids but did not affect the activity of hydroxycinnamic acids. Likewise, an increase in the lipophilicity by substitution of hydroxyl groups with methoxy groups increased the activity of hydroxybenzoic acids, but not of hydroxycinnamic acids. This result contrasts previous estimations that 80% of the antibacterial activity of phenolic acids is determined by their pKa and lipophilicity (Herald and Davidson 1983; Ramos-Nino et al. 1996). Hydroxycinnamic acids are more lipophilic than hydroxybenzoic acids because of their unsaturated chain (Campos et al. 2003). It is thus possible that their lipophilicity is less affected by substitutions of the aromatic ring. However, the double bond of the side chain, which is the main difference in the structure of hydroxybenzoic and hydroxycinnamic acids, likely contributes to the antibacterial activity of hydroxycinnamic acids.
The reduction of the double bond of hydroxycinnamic acids substantially decreased the antibacterial activity against LAB. This unexpected result further confirms that the double bond of hydroxycinnamic acids plays an important role in their mode of action. The reduction of the double bond, which strongly affects their antibacterial activity, has only a minor effect on the lipophilicity of the overall molecule. In contrast, the number of hydroxyl groups did not affect the antibacterial activity of hydroxycinnamic acids but has a more pronounced effect on the lipophilicity. Decarboxylation of protocatechuic acid decreased the antibacterial activity against some indicator strains (L. fermentum FUA3168, E. coli AW1.7 and B. subtilis FAD110). However, the MIC against other lactobacilli (L. plantarum TMW 1.460, L. hammesii DSM13681 and L. reuteri FUA3168) remained unchanged. The role of the carboxylic group in the activity of protocatechuic acid was thus not more pronounced than the role of hydroxyl groups.
LAB exhibit a strong strain-to-strain variation with respect to their tolerance to phenolic acids (Campos et al. 2003). Because the antibacterial activity of phenolic acid metabolites was generally lower when compared to the original substrates (Fig. 3), this variability likely relates to the strain-specific metabolism. In keeping with prior observations, lactobacilli metabolized phenolic acids by strain-specific decarboxylation and/or reduction (Van Beek and Priest 2000; De las Rivas et al. 2009; Svensson et al. 2010). Lactobacillus plantarum TMW 1.460 and L. fermentum FUA3165 produced decarboxylases and reductases. However, metabolism of L. fermentum FUA3165 differed from L. plantarum TMW 1.460 as the former strain also reduced caffeic and p-coumaric acids. L. hammesii DSM13681 only produced decarboxylases. Chlorogenic acid was hydrolysed by L. reuteri FUA3165 and L. fermentum FUA3165 to produce caffeic acid, indicating esterase activity of these two strains. Caffeic acid was also identified in supernatants of other strains, but the low conversion of chlorogenic acid by these strains may be attributable to factors other than enzyme activity. Among LAB, chlorogenic acid esterase activity was previously shown only for Lactobacillus gasseri (Coteau et al. 2001). Lactobacillus reuteri FUA3168 had the highest esterase activity among the strains tested in this work and converted more than 50% of the caffeic acid. This strain or its esterase could serve as suitable catalyst for the enzymatic conversion of chlorogenic acid and other phenolic acid esters for food and pharmaceutical purposes.
Although the assay systems for the determination of tolerance to phenolic acids and phenolic acid metabolism differed, L. plantarum TMW1.460, the strain with the highest metabolic activity towards phenolic acids, was also the most tolerant strain. Lactobacillus reuteri FUA3168 was the most sensitive among the four LAB strains tested in this work (Fig. 3; data not shown) and also exhibited the lowest metabolic activity towards phenolic acids. Lactobacillus fermentum FUA3165 and L. hammesii DSM13681 exhibited intermediate sensitivity and intermediate potential for the metabolism of phenolic acids. This relationship between metabolic capacity and sensitivity to phenolic acids further indicates that metabolism of phenolic acids by LAB contributes to their detoxification.
In conclusion, the antibacterial mode of action of hydroxybenzoic and hydroxycinnamic acids differs. The antibacterial activity of hydroxybenzoic acids decreases with an increasing number of hydroxyl groups and is thus primarily correlated with their lipophilicity. The antibacterial activity of hydroxycinnamic acids, particularly their activity against lactobacilli, depends to a much lesser extent on the substitutions of the aromatic ring with hydroxyl or methoxy groups but is strongly dependent on the double bond of the side chain. LAB metabolism of phenolic acids by decarboxylation and/or reduction thus likely is primarily a mechanism for detoxification of noxious compounds encountered by LAB in plant substrates. This knowledge on structure–function relationships of antibacterial phenolic acids facilitates the selection of phenolic acids or plant extracts containing phenolic acids for use as food preservatives as well as the selection of starter cultures for the fermentation of substrates that are rich in phenolic acids, such as sorghum or olives.