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Keywords:

  • Aliphatic aldehyde;
  • α,β-Unsaturation;
  • Olive;
  • Antibacterial activity;
  • Chain length

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. References

In the present paper we report the ‘in vitro’ activity of eight aliphatic long-chain aldehydes from olive flavor (hexanal, nonanal, (E)-2-hexenal, (E)-2-eptenal, (E)-2-octenal, (E)-2-nonenal, (E)-2-decenal and (E,E)-2,4-decadienal) against a number of standard and freshly isolated bacterial strains that may be causal agents of human intestinal and respiratory tract infections. The saturated aldehydes characterized in the present study do not exhibit significant antibacterial activity, while the α,β-unsaturated aldehydes have a broad antimicrobial spectrum and show similar activity against Gram-positive and Gram-negative microorganisms. The effectiveness of the aldehydes under investigation seems to depend not only on the presence of the α,β-double bond, but also on the chain length from the enal group and on the microorganism tested.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. References

The fruit and leaves of the olive (Olea europaea L.) contain a series of compounds that represent multichemical mechanisms of defence against microbe and insect attacks [1]. One type of defence is represented by bitter molecules, such as oleuropein and ligstroside (and, perhaps, their metabolites), which are present in large amounts in the olive fruit. Another mechanism is represented by the water-insoluble triterpene, oleanolic acid, which constitutes a crystalline barrier coating the surface of the olive leaf (but not that of the fruit). A third mechanism might be represented by several aliphatic saturated and unsaturated aldehydes, produced by enzymatic cleavage of unsaturated fatty acids when plants undergo microbial attack [1,2].

Many researches have demonstrated that the above-mentioned biocompounds are able to inhibit or delay the rate of growth of a range of bacteria and microfungi, so that they might be used as alternative food additives or in integrated pest management programs [3–7]. However, few data are reported in literature concerning the possible employment of O. europaea biocompounds as antimicrobial agents against human pathogenic bacteria [8,9].

Recently a series of aliphatic long-chain aldehydes from olive flavor have been demonstrated to exhibit a noticeable activity against several food-borne microfungal and bacterial strains [10]. In the present paper, we report the ‘in vitro’ activity of some of these aldehydes against a number of standard and isolated bacterial strains that may be causal agents of human intestinal and respiratory tract infections.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. References

2.1Drugs used

Hexanal, nonanal, (E)-2-hexenal, (E)-2-eptenal, (E)-2-octenal, (E)-2-nonenal, (E)-2-decenal and (E,E)-2,4-decadienal (Fig. 1) were purchased from Fluka (Milan, Italy). Ampicillin and erythromycin were purchased from Sigma Chemical Co. (Milan, Italy); working solutions were prepared according to procedures of the National Committee for Clinical Laboratory Standards [11]. All culture media and supplements were obtained from Oxoid (Unipath s.p.a., Milan, Italy).

image

Figure 1. Chemical structure of (1) hexanal, (2) nonanal, (3) (E)-2-hexenal, (4) (E)-2-eptenal, (5) (E)-2-octenal, (6) (E)-2-nonenal, (7) (E)-2-decenal, (8) (E,E)-2,4-decadienal.

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2.2Preparation of test solutions

Stock solutions of each aldehyde (10 mg ml−1) to be tested were prepared in dimethylsulfoxide (DMSO) and then diluted in standard buffer at least 1:10 (v/v). Further dilutions (1:1, v/v) were prepared to give final concentrations ranging from 0.015 to 500 μg ml−1. Stock solutions of ampicillin and erythromycin (1 mg ml−1) were prepared in DMSO and then diluted in standard buffer (1:1, v/v); further dilutions were prepared to give final concentrations ranging from 0.0076 to 125 μg ml−1. In order to avoid any effect of the solvent, the DMSO concentration in the test solution was well below the levels which showed an antibacterial effect.

2.3Bacterial isolates

A total of 11 American Type Culture Collection (ATCC) standard strains were used: Haemophilus influenzae ATCC 9006, Moraxella catarrhalis ATCC 10541, Escherichia coli ATCC 10538, Staphylococcus epidermidis ATCC 7540, Streptococcus pyogenes ATCC 12348, Staphylococcus aureus ATCC 6538, Streptococcus pneumoniae ATCC 7070, Salmonella enteritidis ATCC 6017, Salmonella typhi ATCC 7521, Bacillus cereus ATCC 10876, Listeria monocytogenes ATCC 7646. Furthermore, we employed 89 strains isolated from the upper respiratory tract in humans (H. influenzaeβ-lactamase+, four strains; H. influenzaeβ-lactamase, eight strains; M. catarrhalisβ-lactamase+, five strains; M. catarrhalisβ-lactamase, seven strains; S. pyogenes erythromycin S, 11 strains; S. pyogenes erythromycin R, 15 strains; S. pneumoniae, nine strains; S. aureus methicillin S, 18 strains; S. aureus methicillin R, 11 strains) and three strains isolated from contaminated food (S. aureus toxin B producer, one strain; S. enteritidis, one strain; L. monocytogenes, one strain). All these strains were identified by conventional procedures (confirmed by API system, Bio Merieux).

An inoculum of each bacterial strain was suspended in 3 ml tryptone soy broth and incubated overnight at 37°C. The turbidity of overnight cultures was adjusted to match that of a 0.5 McFarland standard (ca. 108 colony forming units (CFU) ml−1).

2.4Susceptibility test

The drugs were tested by the disc-diffusion method [12]. Briefly, 0.01 ml of the 0.5 McFarland standard was diluted 1:10 in sterile 0.85% saline and was spread on sterile Muller–Hinton agar plates, after which 6-mm diameter discs, impregnated with 500 μg of the drug to be tested, were placed on the plates. Ampicillin and erythromycin (100 μg) were used as control drugs. The plates were incubated for 24 h at 37°C under aerobic conditions and then the diameter of the inhibition zone around each disc measured and recorded.

If active in the disc-diffusion test (inhibition zone ≥10 mm), the drugs were evaluated to determine minimal inhibitory concentration (MIC) values, by the broth microdilution test [13]. A portion of the 0.5 McFarland standard was diluted from 1:20 to 1:100, resulting in final inoculum concentrations from 1×105 to 5×105 CFU ml−1; the appropriate amount of the inoculum was chosen according to the characteristics of the bacterial strain tested [11]. The plates were incubated at 37°C for 18 h. MIC is defined as the lowest drug concentration that inhibits the visible growth after 18 h incubation. All determinations were carried out in triplicate.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. References

Despite the wide availability of clinically useful antimicrobial drugs, several arguments (limited antimicrobial spectrum and serious side-effects of major antibiotics, increasing clinical resistance of previously sensitive microorganisms, emergence of previously uncommon infections) plead in favor of the development of new molecules exhibiting antibacterial activity.

We have demonstrated previously the broad activity of two phenolic compounds from O. europaea, oleuropein and hydroxytyrosol, against several human pathogens [8]. In our on-going search for new antimicrobic plant molecules, the antibacterial properties of eight long-chain aldehydes from olive flavor have been investigated.

The MICs reported in Tables 1 and 2 are evidence of the antimicrobial activity of the bioaldehydes against ATCC and clinically isolated strains. Since hexanal, nonanal and (E)-2-octenal appeared almost inactive against all ATCC bacterial strains (data not shown), the susceptibility of clinically isolated bacterial strains to (E)-2-eptenal, (E)-2-nonenal, (E)-2-decenal and (E,E)-2,4-decadienal was tested. MIC values ranged between 0.48 and 250 μg ml−1 for ATCC strains and between 1.9 and 250 μg ml−1 for clinical isolates; the lowest values were for (E,E)-2,4-decadienal. Furthermore, when (E)-2-eptenal, (E)-2-nonenal, (E)-2-decenal and (E,E)-2,4-decadienal were tested together (ratio=1:1:1:1) against ATCC and clinically isolated microbial strains, a strong synergic effect was observed; in fact, MIC values were lower than those calculated when the same aldehydes were used alone, ranging between 0.48 and 31.25 μg ml−1 for ATCC strains and between 0.48 and 3.9 μg ml−1 for clinical isolates.

Table 1.  Antimicrobial activity of some aldehydes of the olive flavor on ATCC standard bacterial strains
Strains (ATCC)MIC (μg ml−1)
 (4)(6)(7)(8)(4+6+7+8)(E)(A)
  1. (4) (E)-2-eptenal; (6) (E)-2-nonenal; (7) (E)-2-decenal; (8) (E,E)-2,4-decadienal; (E) erythromycin; (A) ampicillin.

S. aureus (6538)1251252507.83.90.240.12
E. coli (10538)25012550012531.250.48NA
S. epidermidis (7540)12562.52507.87.80.247.68
M. catarrhalis (10541)62.51251257.81.950.120.97
S. pneumoniae (70770)7.831.21251.90.481.950.48
S. pyogenes (12348)62.531.21253.91.950.240.03
H. influenzae (9006)7.87.815.61.90.481.951.95
Table 2.  Antimicrobial activity of some aldehydes of the olive flavor on (N) clinically isolated bacterial strains
Strains (number)MIC range (μg ml−1)
 (4)(6)(7)(8)(4+6+7+8)(E)(A)
  1. For each bacterial species, when the bacterial strains employed showed different susceptibility to the same antimicrobial agent, the lower value and the highest value of the MICs calculated (MIC range) are reported in the table. (4) (E)-2-eptenal; (6) (E)-2-nonenal; (7) (E)-2-decenal; (8) (E,E)-2,4-decadienal; (E) erythromycin; (A) ampicillin.

S. aureus methicillin S (18)31.25–12515.6–125125–2507.8–15.63.9–7.80.24–0.480.97–3.9
S. aureus methicillin R (11)15.6–250125125–50031.25–1251.95–7.83.9–15.6NA
M. catarrhalisβ-lactamase+ (5)15.6–12531.25–125125–2507.8–31.250.97–3.90.12–0.48NA
M. catarrhalisβ-lactamase (7)7.8–62.531.25–1251257.8–31.250.97–3.90.12–0.481.95–7.8
S. pneumoniae (9)0.48–7.87.8–31.2531.25–1251.9–7.80.48–0.971.95–3.900.48–3.9
S. pyogenes erythromycin S (11)3.9–62.515.6–62.562.5–1253.9–15.61.950.48–1.950.03–0.06
S. pyogenes erythromycin R (15)7.815.6–3.93.9–15.61.9–15.61.95NA0.015–0.06
H. influenzaeβ-lactamase+ (4)7.83.9–31.2531.251.95–3.90.48–0.973.9–31.25NA
H. influenzaeβ-lactamase (8)1.95–7.815.6–31.2531.251.95–15.60.48–0.973.9–7.81.95–7.8

Table 3 shows the MICs calculated against food-borne and ATCC microbial strains tested in our study. Aside from concerns about food quality degradation, these microorganisms may be causal agents of intestinal infections in humans. The findings were consistent with those described above: MICs ranged between 1.9 and 250 μg ml−1 and the lowest values were for (E,E)-2,4-decadienal. However, unlike previous experiments, (E)-2-hexenal appeared active also against these bacterial strains (both standard and food-isolated) and nonanal showed a significant activity against B. cereus and L. monocytogenes; only hexanal and (E)-2-octenal appeared inactive against all strains tested (data not shown).

Table 3.  Antimicrobial activity of some aldehydes of the olive flavor on some food-borne and ATCC bacterial strains
StrainsMIC (μg ml−1)
 (2)(3)(4)(6)(7)(8)
  1. (2) nonanal; (3) (E)-2-hexenal; (4) (E)-2-eptenal; (6) (E)-2-nonenal; (7) (E)-2-decenal; (8) (E,E)-2,4-decadienal.

  2. aFood-isolated.

S. enteritidis ATCC 6017NA1251252501257.8
S. enteritidisaNA250250500125125
S. typhi ATCC 7251NA12512525062.57.8
B. cereus ATCC 108767.87.87.815.67.81.9
L. monocytogenesa7.87.87.812531.21.9
L. monocytogenes ATCC 764662.562.562.512531.23.9
S. aureusa toxin B producerNA62.562.51251257.8

Among the Gram-positive bacteria tested, S. pneumoniae was the most susceptible; in the case of Gram-negative bacteria, H. influenzae was the most susceptible. However, all the active aldehydes tested showed similar activity against Gram-positive and Gram-negative microorganisms. This is a rather unique result, since most plant secondary metabolites show more potent activity against Gram-positive than Gram-negative bacteria [14]. Furthermore, our results confirm the antimicrobial activity of olive flavor aldehydes against S. aureus, a microorganism extensively studied due to its ability to produce enterotoxins and exceptionally resistant to a number of phytochemicals.

Aldehydes are often found as constituents of plant products [15] and may play an important role in the observed antimicrobial activity of plant aldehyde-containing material [16]. Their action, very likely due to an alteration in the function of membrane-associated proteins, seems to be exerted mainly at the cell surface [17,18]; for example, the activity of benzaldehydes against several bacterial strains (L. monocytogenes, S. enteritidis and Lactobacillus plantarum) does not depend on partitioning [17,19]. However, the capability to penetrate the outer layer of cells can help to explain the antimicrobial activity of some aldehydes, especially against Gram-negative bacteria [18].

Aldehydes are intrinsically very reactive compounds and readily react with biologically important nucleophile groups, because the side-chain is subjected to a variety of addition and condensation reactions. More particularly, α,β-unsaturated aldehydes are known to readily react with sulfhydryl, amino and hydroxyl groups; under physiological conditions the 1,4-addition seems to be the main reaction. Kubo and coworkers [10] have suggested that the carbon tail length influences the electronegativity of the aldehyde oxygen atom and consequently its interaction with the nucleophilic groups of the cell membrane; in fact, a greater electronegativity of the molecule would cause a greater incidence of intermolecular hydrogen bond formation with membrane nucleophilic groups and thus a significant disorder in the lipidic bilayer. Similarly, the antimicrobial activity of a series of long-chain alcohols was shown to depend on the alkyl chain length [20].

Our findings show that the saturated aldehydes, hexanal and nonanal, do not exhibit significant antibacterial activity; while some α,β-unsaturated, long-chain aldehydes have a broad antimicrobial spectrum. Moreover, the effectiveness of the aldehydes under investigation seems to depend not only on the presence of the α,β-double bond, but also on the chain length from the enal group and on the microorganism tested. The diunsaturated aldehyde (E,E)-2,4-decadienal appears to be more toxic to bacterial cells than the correspondent monounsaturated aldehyde (E)-2-decenal; but two double bonds in the cis configuration in the side-chain of 2,4-decadienal create more bends and shorten the length of the carbon tail.

The present data demonstrate that some α,β-unsaturated aldehydes of olive flavor may be good candidates for employment as antimicrobial agents against bacteria responsible for human gastrointestinal and respiratory tract infections. Particularly interesting is the synergic antibacterial effect observed when (E)-2-eptenal, (E)-2-nonenal, (E)-2-decenal and (E,E)-2,4-decadienal are employed in combination. In fact, the association of two or more antibacterial agents is generally recommended because it makes the development of resistance mechanisms less likely and enhances the total antibacterial activity. In addition, drugs from a regularly consumed edible plant and/or its processed products may be more effective than many non-natural compounds in the prevention of gastrointestinal infections. Thus one could speculate that dietary intake of olive aldehydes may lower the risk of bacterial infections particularly in the intestinal tract. However further studies are needed to elucidate the pharmacokinetic properties of these compounds and thus the possibility that they maintain their antibacterial activity ‘in vivo’.

Some of the aldehydes tested might find broad applications as food preservatives. The inhibitory activity of (2E)-alkenals against tyrosinase (the enzyme responsible for the unfavorable darkening observed in plant-derived foods and beverages) [21] might contribute to their potential employment in food industry. Olives and olive oil has proven its safety through many years of human use and consumption. This should be an additional advantage in the potential employment of olive aldehydes in human chemotherapy or as food preservatives, given that safety is a primary consideration for antimicrobial agents to be ingested by humans. Finally these olive α,β-unsaturated aldehydes might be good alternatives to other highly toxic disinfectants, such as glutaraldehyde [22], for hospital equipment.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. References
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