Cronobacter sakazakii reduction by blueberry proanthocyanidins

Highlights

• Blueberry polyphenols have antimicrobial effects against foodborne pathogens.

• Blueberry juice and polyphenols reduced Cronobacter sakazakii after 1 h at 37 °C.

• Scanning electron microscopy showed changes in morphology of treated strains.

Abstract

Blueberry juice and blueberry polyphenols reportedly have antimicrobial properties against foodborne pathogens, without much currently known on their effects against Cronobacter sakazakii. This study evaluated the antimicrobial effects of blueberry proanthocyanidins (PAC) and commercial blueberry juice (BJ) against two strains of C. sakazakii, ATCC 29004 and 29544. BJ (pH 2.8), blueberry PAC (5 mg/ml) and controls (phosphate buffered saline (PBS), pH 7.2, and malic acid pH 3.0) were mixed with equal volumes of washed overnight cultures of C. sakazakii and incubated for 30 min, 1 h, 3 h and 6 h at 37°C. Reductions of ∼1 and 1.50 log CFU/ml were obtained for strains 29004 and 29544, respectively after 30 min with BJ or blueberry PAC. Both C. sakazakii strains 29004 and 29544 were reduced to undetectable levels from 8.25 ± 0.12 log CFU/ml and 8.48 ± 0.03 log CFU/ml, respectively with BJ (pH 2.8) or blueberry PAC after 1 h, while malic acid (pH 3.0) showed ∼1.3 log CFU/ml reduction for both strains. Scanning electron microscopy studies showed differences in cell membrane morphology with clumping and formation of blebs of the treated strains compared to untreated controls. These results warrant further in vivo studies with blueberry bioactives to determine potential for preventing and treating C. sakazakii infections.

Introduction

Cronobacter sakazakii is a gram-negative, rod-shaped, opportunistic bacterial pathogen and a major cause of invasive infections in neonates. The clinical symptoms of Cronobacter infection include necrotizing enterocolitis, bacteremia, and meningitis, with reported case fatality rates of 50–80% (Healy et al., 2010). However, in addition to neonates, immunocompromised adults and elderly are also susceptible to Cronobacter infections. Cronobacter outbreaks have been associated with contaminated powdered infant formula as well as raw and processed foods of animal and plant origin including fresh, frozen, and ready-to-eat products (Friedemann, 2007). Based on the wide-range of reported Cronobacter-contaminated foods, Cronobacter remains a potential risk for food safety and numerous studies are being carried out towards developing improved strategies for inactivation.

Among the seven Cronobacter species, C. sakazakii has been associated with milk and milk products. Thermal inactivation methods for Cronobacter include ultra-high temperature (UHT) processing for milk and fluids, manothermosonication (MTS), ultraviolet light, x-ray irradiation, and high pressure processing (Arroyo et al., 2011, Gonzalez et al., 2006, Ha and Ha, 2011, Lee et al., 2006).

In addition to processing technologies and chemical inactivation methods, there is currently an increased trend towards research on natural products as antimicrobials. The advantages of naturally occurring antimicrobials include increased consumer acceptance and availability. Trans-cinnamaldehyde (TC), a component of cinnamon oil, at 38 mM and 750 μM was shown to cause 4.0 and 3.0 log CFU/ml reduction of C. sakazakii after 96 h, respectively, inhibiting biofilm formation on various surfaces at temperatures of 12 °C and 24 °C, with downregulation of biofilm associated genes (Amalaradjou and Venkitanarayanan, 2011). Caprylic acid at 30 mM was found to cause 7.8 log CFU/ml reduction of C. sakazakii after 60 min at 45 °C (Jang and Rhee, 2009). The minimum inhibitory concentrations (MIC) against C. sakazakii were 2 mg/ml for ethyl vanillin, 3 mg/ml for vanillin, and 0.8 mg/ml for vanillic acid (Yemis et al., 2011), while carvacrol and thymol were shown to have MICs of 0.0625 mmol/l at 37 °C (Lee and Jin, 2008).

Various natural extracts including polyphenols from fruits and berries are also being studied as potential antimicrobials for bacteria, viruses, and parasites (Al-Habib et al., 2010, Su and D'Souza, 2011, Su et al., 2010, Takeshita et al., 2009). Muscadine seed extracts were found to be effective against C. sakazakii strains Fec39 and MSDH resulting in complete reduction (6 log CFU/ml) within 1 h of treatment at 37 °C (Kim et al., 2009).

Blueberries are known to exhibit strong antioxidant capacity associated with phenolic acids, catechins (flavanols), and proanthocyanidins (condensed tannins) (Huang et al., 2012). Blueberry PACs have antimicrobial activity against Listeria monocytogenes, Helicobacter pylori, Salmonella Typhimurium, Escherichia coli and Candida albicans (Chatterjee et al., 2004, Lacombe et al., 2012, Ofek et al., 1996). Water and ethanol extracts of blueberries were shown cause 5.9 log CFU/ml reduction of L. monocytogenes after 24 h (Park et al., 2011).

Therefore, the objectives of this research were to (1) determine the antimicrobial effects of commercial blueberry juice (BJ) and 5 mg/ml blueberry proanthocyanidins (PAC) against two isolates of C. sakazakii over 6 h at 37°C and (2) determine the mode of action of blueberry PACs against C. sakazakii using Scanning Electron Microscopy.