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In vitro antimicrobial activity of pistachio (Pistacia vera L.) polyphenols

Carlo Bisignano, Angela Filocamo, Richard M. Faulks, Giuseppina Mandalari
DOI: http://dx.doi.org/10.1111/1574-6968.12091 62-67 First published online: 1 April 2013

Abstract

We investigated the antimicrobial properties of polyphenol-rich fractions derived from raw shelled and roasted salted pistachios. American Type Culture Collection (ATCC), food and clinical isolates, of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Pseudomonas mirabilis), Gram-positive bacteria (Listeria monocytogenes, Enterococcus hirae, Enterococcus faecium, Bacillus subtilis, Staphylococcus epidermidis, Staphylococcus aureus), the yeasts Candida albicans and Candida parapsilosis and the fungus Aspergillus niger were used. Pistachio extracts were active against Gram-positive bacteria with a bactericidal effect observed against L. monocytogenes (ATCC strains and food isolates), S. aureus and MRSA clinical isolates. Extracts from raw shelled pistachios were more active than those from roasted salted pistachios. The bactericidal activity of pistachio extracts could be used to help control the growth of some microorganisms in foods to improve safety and may find application as a topical treatment for S. aureus.

Keywords
  • antimicrobials
  • clinical isolates
  • MRSA
  • Listeria monocytogenes
  • bactericidal

Introduction

A number of studies have shown beneficial effects of pistachio consumption on cardiovascular disease (CVD) risk factors, such as lipids, endothelial function, inflammation, blood pressure and oxidative status (Gebauer et al., 2008; Kay et al., 2010; Sari et al., 2010; Zhang et al., 2010; West et al., 2012). Phytochemicals previously identified from pistachios include phytosterols, fatty acids, lutein and tocopherols (USDA 2007; Philips et al., 2005; Wu & Prior, 2005). Other health-promoting compounds present in pistachios are polyphenols, which have been shown to be protective agents against cancer and CVD (Knekt et al., 2002; Liu, 2004). Catechins are particularly effective in CVD prevention and in decreasing LDL oxidation (Velayutham et al., 2008).

The antibacterial properties of plant-derived compounds have also been attracting attention, mainly in view of the increased antibiotic resistance both in hospitals and in community-acquired infections (Moellering, 2006; Gould, 2008). Several studies reported the antibacterial activity of flavonoid-rich natural products, including propolis (Koru et al., 2007), nuts (Mandalari et al., 2010a,b), Juniperus extracts (Marino et al., 2010; Miceli et al., 2011), grape seeds (Adamez et al., 2012) and Citrus plants (Mandalari et al., 2007). The identification of new compounds with bactericidal rather than bacteriostatic effect has gained understandable interest, together with the several recent reports of flavonoids increasing the activity of antibiotics through synergistic interactions (Bernal et al., 2009; D'Arrigo et al., 2010; Eumkeb et al., 2010). Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) are amongst the most significant Gram-positive pathogens. As several MRSA strains have become multidrug-resistant endemic pathogens, novel therapies are needed to treat these widespread infections. Therefore, the use of natural antimicrobials could play a positive role in reducing the rates of infection.

Consumer demand for natural and minimally processed foods is also driving the demand for natural compounds with antimicrobial properties, and a number of aromatic plant oils have been discussed for use as food preservatives (Hammer et al., 1999; Dorman & Deans, 2000; Burt, 2004).

In the present study, we evaluated the antimicrobial effect of raw and roasted pistachio extracts rich in polyphenols against Gram-negative bacteria, Gram-positive bacteria (ATCC strains, food and clinical isolates), yeasts and the fungus Aspergillus niger.

Material and methods

Materials

Natural raw shelled pistachios (Pistacia vera L.) (NP) and roasted salted pistachio (RP) kernels from Setton Pistachio, Terra Bella, California, were kindly provided by the Western Pistachio Growers, now known as American Pistachio Growers (CA, USA).

Pistachio polyphenol-rich extracts

Pistachio polyphenolic extracts were prepared and analysed as previously reported (Mandalari et al., 2010b, 2013). Briefly, NP or RP (10 g) were extracted five times with n-hexane (100 mL) under constant agitation (2 h) to remove lipids. After filtration, the residues were mixed with 100 mL of methanol/HCl 0.1% (v/v), extracted and centrifuged. The pellets were extracted four more times. All methanolic fractions were combined and evaporated, after which the residues were dissolved in distilled water (40 mL) and extracted five times with ethyl acetate (40 mL). The organic phases were combined, dried with Na2SO4 for 20 min and evaporated under vacuum. Analyses were performed on a Shimadzu Prominence HPLC system using an Ascentis Express C18 column (150 × 4.6 mm; 2.7 µm, Ascentis Express, Supelco, Bellefonte, USA).

Microbial strains and culture conditions

The following strains were used for the antimicrobial testing and were obtained from the University of Messina's in-house culture collection (Messina, Italy): Gram-negative bacteria, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Pseudomonas mirabilis (wild-type strain), P. aeruginosa (wild-type strain), Gram-positive bacteria, Listeria monocytogenes (ATCC 7644 and ATCC 1392), Enterococcus hirae ATCC 10541, Enterococcus faecium ATCC 8459, Bacillus subtilis ATCC 6633, S. aureus ATCC 6538P, S. epidermidis ATCC 12228, MRSA ATCC 43300, 21 food isolates of L. monocytogenes belonging to serotypes 1/2a (14 strains) and 1/2b (7 strains), 44 clinical isolates of S. aureus (of which 9 were MR and 3 were methicillin, ampicillin, penicillin G and imipenem resistant) obtained from specimens of skin infections and surgical infections, yeasts (Candida albicans ATCC 10231, Candida parapsilosis ATCC 29947) and the fungus A. niger ATCC 16404.

Cultures for antimicrobial activity tests were grown in Mueller–Hinton Broth (MHB, Oxoid, CM0405) at 37 °C (24 h) for bacteria and Sabouraud Liquid Medium (SLM, Oxoid, CM0147) at 30 °C (48 h) for the yeasts. For solid media, 1.5% (w/v) agar (Difco) was added. Aspergillus niger was grown in Sabouraud Dextrose Agar at 30 °C for 7 days. A cell suspension was then transferred to 10 mL of polysorbate 80 (0.05% w/v) solution, and conidiospores were detached from the culture surface. The suspension was transferred into a conical flask, gently shaken for 1 min and then filtered through a fritted filter. Microscopic examination under 400× magnification was carried out immediately after the preparation to show the absence of mycelial fragments and spore germination.

Antimicrobial testing

The minimum inhibitory concentrations (MICs), the minimum bactericidal concentrations (MBCs) and the minimum fungicidal concentration (MFC) of NP and RP polyphenolic-rich extracts were determined by the broth microdilution method, according to CLSI (2008). The MICs were also performed in the Bioscreen C (Labsystems Oy, Helsinki, Finland) for all strains as previously reported (D'Arrigo et al., 2010).

All experiments were performed in triplicate on three independent days. A number of positive and negative controls with selected antibiotics (ampicillin and fluconazole) and solvent (DMSO) were included in each assay.

Results

Flavonoids and phenolic acids in pistachios

As previously reported (Mandalari et al., 2013), the major compounds identified in the pistachio samples were gallic acid, (+)-catechin and isoquercetin (Fig. 1).

Flavonoids and phenolic acids in natural raw shelled (NP, black bars) and roasted salted (RP, white bars) pistachios. Values are expressed as mg per 100 g n = 3, SD < 5%. Legend: 1: gallic acid; 2: protocatechuic acid; 3: chlorogenic acid; 4: catechin; 5: epicatechin; 6: eriodictyol-7-O-glucoside; 7: quercetin-3-O-rutinoside; 8: isoquercetin; 9: daidzein; 10: eriodictyol; 11: luteolin.

Slightly higher total amounts of polyphenols were observed in NP (6.7 mg per 100 g) compared with RP (6.0 mg per 100 g). However, hydroxybenzoic acids and chlorogenic acid were higher in the roasted pistachios. As reported by other authors (Ballistreri et al., 2009), the lower polyphenolic content in RP may be due to roasting. Catechin and epicatechin were present at more than double concentrations in NP compared with RP, and isoquercetin concentration was 1.75 times higher in NP.

Antimicrobial activity of pistachio extracts

The MIC and MBC values of pistachio extracts against all the strains tested are shown in Table 1. Results of negative controls indicated the complete absence of inhibition of all the strains tested (data not shown). Analogue values of MICs were obtained with the broth microdilution method and in the Bioscreen C. Both NP and RP polyphenolic extracts were active against the Gram-positive bacteria, but were not active against any of the Gram-negative bacteria, the yeasts and the fungus tested in this study. NP was more effective than RP, the latter not showing any activity against B. subtilis, E. hirae, E. faecium and S. epidermidis. This effect could be explained by the higher amount of polyphenols present in NP compared with RP and the different qualitative composition of the two extracts, chlorogenic acid and daidzein, being present only in RP (Fig. 1).

View this table:
Table 1

MICs and MBCs of pistachio polyphenol-rich extracts against Gram-positive bacteria, Gram-negative bacteria, yeasts and fungus. Values are expressed as µg mL−1 and represent the mean of three determinations

NPRP
StrainMICMBCMICMBC
Gram-positive bacteriaE. hirae ATCC 10541500> 2000> 2000> 2000
E. faecium ATCC 8459500> 2000> 2000> 2000
B. subtilis ATCC 6633500> 2000> 2000> 2000
L. monocytogenes ATCC 764415.62100031.25> 2000
L. monocytogenes ATCC 139231.25200062.50> 2000
Staph. epidermidis ATCC 122281000> 2000> 2000> 2000
Staph. aureus ATCC 4330062.50> 2000125> 2000
Staph. aureus ATCC 6538P15.62200031.25> 2000
Gram-negative bacteriaPs. aeruginosa (wild-type)> 2000> 2000> 2000> 2000
Ps. mirabilis (wild-type)> 2000> 2000> 2000> 2000
Ps. aeruginosa ATCC 27853> 2000> 2000> 2000> 2000
E. coli ATCC 25922> 2000> 2000> 2000> 2000
YeastsC. parapilosis ATCC 29947> 2000> 2000> 2000> 2000
C. albicans ATCC 10231> 2000> 2000> 2000> 2000
FungusA. niger ATCC 16404> 2000> 2000> 2000> 2000

Staphylococcus aureus and L. monocytogenes were the most sensitive strains (complete inhibition achieved with a concentration of 15.62 µg mL1 NP), followed by B. subtilis, E. hirae and E. faecium (500 µg mL−1 NP). The effect of NP extract against L. monocytogenes ATCC 7644, L. monocytogenes ATCC 1392 and S. aureus ATCC 6538P was bactericidal at concentrations of 1000, 2000 and 2000 µg mL1, respectively, whereas a bacteriostatic effect was observed with NP on the other strains tested and with RP extract for L. monocytogenes and S. aureus. As previously shown (Filocamo et al., 2011), the MRSA ATCC 43300 was more resistant to both NP and RP extracts compared with S. aureus ATCC 6538 P (Table 1).

Table 2 reports the MICs and MBCs of NP and RP against the food isolates of L. monocytogenes. As observed with the reference strains, NP was more active than RP, and a concentration of 2000 µg mL−1 of NP was bactericidal for 50% of the isolates.

View this table:
Table 2

MIC and MBC of pistachio polyphenol-rich extracts against food isolates (n = 21) of L. monocytogenes. Values are expressed as µg mL−1

CompoundMIC 50MIC 90MBC 50MBC 90
NP62.501252000> 2000
RP125250> 2000> 2000

The MICs and MBCs of NP and RP extracts against the clinical isolates are reported in Tables 3 and 4. MIC values of 31.25 and 62.50 µg mL−1 of NP and RP, respectively, inhibited the growth of 50% of the methicillin-sensitive clinical isolates tested, whereas 62.50 and 125 µg mL−1 of NP and RP, respectively, inhibited the growth of 90% of the strains. A concentration of 2000 µg mL−1 of NP was bactericidal for 50% of the clinical isolates, whereas a bacteriostatic effect was observed when using RP (Table 3). Higher MIC values were obtained with the MR clinical isolates when using both NP and RP, with no differences in the bactericidal effect when compared with the methicillin-sensitive strains (Table 4).

View this table:
Table 3

MIC and MBC of pistachio polyphenol-rich extracts against clinical isolates (n = 35) of methicillin-sensitive Staphylococcus aureus. Values are expressed as µg mL−1

CompoundMIC 50MIC 90MBC 50MBC 90
NP31.2562.502000> 2000
RP62.50125> 2000> 2000
View this table:
Table 4

MIC and MBC of pistachio polyphenol-rich extracts against clinical isolates (n = 9) of methicillin-resistant Staphylococcus aureus (of which 3 were methicillin, ampicillin, penicillin G and imipenem resistant). Values are expressed as µg ml−1

CompoundMIC 50MIC 90MBC 50MBC 90
NP62.501252000> 2000
RP125250> 2000> 2000

Discussion

This article has demonstrated the bactericidal effect of polyphenol-rich fractions from pistachio against Gram-positive bacteria, including standard ATCC strains, food and clinical isolates. Other authors have reported on the antibacterial, antifungal and antiviral properties of Pistacia vera lipophilic extracts (Ozcelik et al., 2005) and on the antimicrobial and antiviral properties of pistachio essential oil (Alma et al., 2004; Loizzo et al., 2008). In the present study, extracts rich in polyphenols from raw shelled and RPs were active against Gram-positive bacteria, but were inactive against the Gram-negative bacteria, yeasts and the fungus tested. In our previous study (Mandalari et al., 2010a, we have shown that flavonoid-rich extracts from almond skins were active against the Gram-positive bacteria L. monocytogenes and S. aureus. Considerable advances in antimicrobial polyphenols research have occurred in the last decade with the identification of several new features that may improve the antibacterial properties of flavonoids, together with a better understanding of their structure–function relationship (Cushnie & Lamb, 2011). Although the main three mechanisms of the antibacterial activity of flavonoids may be attributable to cytoplasmic membrane damage, inhibition of nucleic acid synthesis and inhibition of energy metabolism, new studies suggest that flavonol, flavan-3-ols and flavolan classes damage the cytoplasmic membrane by generating hydrogen peroxide (Arakawa et al., 2004), whereas flavan-3-ols and isoflavones inhibit nucleic acid synthesis through topoisomerase and/or dihydrofolate reductase inhibition (Navarro-Martinez et al., 2005; Wang et al., 2010). Hydroxybenzoic acids (as gallic, protocatechuic), flavan-3-ols (as (+)-catechin) and flavonols (as isoquercetin) were the main identified polyphenols in the pistachio samples (Fig. 1). We have recently shown that polyphenols from raw shelled and RPs are bioaccessible in the upper gastrointestinal tract during simulated human digestion (Mandalari et al., 2013). Galloyl flavan-3-ols such as (−)-epicatechin gallate and catechins have been reported to modulate the MICs of β-lactam antibiotics against some strains of MRSA (Stapleton et al., 2004). (−)-Epicatechin gallate is also known to sensitise MRSA isolates to a range of β-lactam antibiotics (Yam et al., 1998; Hamilton-Miller & Shah, 2000).

In the present study, we have shown that pistachio polyphenols have bactericidal effects on S. aureus ATCC 6538P and MRSA strains. As previously stated by other authors (Al-Habib et al., 2010), the activity may be due to the cell wall or cell membrane disruption together with cell enlargement. The bactericidal activity of pistachio extracts against L. monocytogenes could be used to improve food safety, extend shelf life and improve food quality. The presence of L. monocytogenes in contaminated foods can cause listeriosis, a serious disease with a mortality rate of almost 30% (Painter & Slutsker, 2007). The antilisterial effects of grape seed extracts rich in proanthocyanidins have been recently reported in aqueous media (Bisha et al., 2010).

In terms of applications, the use of pistachio polyphenols could be tested in combination with antibiotics to identify new mechanisms of synergism and antibiotic-resistant modulating properties for the development of novel topical agents for the treatment of skin infections as well as for topical formulations.

In summary, the results of this study showed that polyphenols from pistachios are effective against a range of Gram-positive pathogens with a bactericidal activity on L. monocytogenes, S. aureus and MRSA strains. However, further studies are needed to understand the mechanisms responsible for these activities, and the obtained in vitro results need to be translated both in food and in vivo.

Acknowledgements

This research was funded by the Western Pistachio Growers (now known as the American Pistachio Growers; CA, USA). The authors declare no conflict of interest.

References

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