Available online at www.sciencedirect.

com

LWT - Food Science and Technology 41 (2008) 1834e1841 www.elsevier.com/locate/lwt

Antibacterial effects of American cranberry (Vaccinium macrocarpon) concentrate on foodborne pathogens

Vivian Chi-Hua Wu*, Xujian Qiu, Alfred Bushway, Laura Harper
Department of Food Science and Human Nutrition, University of Maine, 5735 Hitchner Hall, Orono, ME 04469-5735, USA Received 13 September 2007; received in revised form 20 December 2007; accepted 2 January 2008

Abstract Antibacterial effects of American cranberry (Vaccinium macrocarpon) concentrate on foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus in vitro were investigated. Cranberry concentrate at various concentrations was prepared in distilled water (DW) or Brain Heart Infusion (BHI) broth. Pathogens were inoculated in each sample and incubated at 21 and 4  C for 0, 1, 5, 7, and 24 h (DW samples) and 0, 1, 3, and 5 days (BHI samples). Transmission electron microscopy (TEM) was used to study the effects of cranberry concentrate on cellular structure of pathogens. DW results showed that S. Typhimurium and L. monocytogenes were reduced to non-detectable levels at 5 h in 100 ml/ml treatment at 21 and 4  C. At 24 h, no target pathogens were detected from the 100 ml/ml treatment. BHI data indicated that the 100 ml/ml treatment reduced the four pathogens by 3e8 log CFU/ml compared with the control on Day 5 at 21 and 4  C. TEM revealed damage to the bacterial cell walls and membranes. Cranberry concentrate has antibacterial effects on the four foodborne pathogens. Based on potential health benets and proven antimicrobial effects, American cranberry concentrate may have dual applications as a food preservative. 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: American cranberry concentrate; Vaccinium macrocarpon; Antibacterial effects; Foodborne pathogens

1. Introduction In the United States, the major foodborne pathogens include Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus. E. coli O157:H7 is an emerging foodborne pathogen which can cause hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), and thrombocytopenic purpura (TTP). Approximately 73,000 cases of E. coli O157:H7 infection occur annually in the Untied States resulting in more than 61 deaths according to the US CDC (2005). L. monocytogenes can cause listeriosis, which usually affects pregnant women, immunocompromised individuals, and the elderly (Jay, Loessner, & Golden, 2005). S. Typhimurium is one of the major strains causing salmonellosis. S. aureus can excrete an exotoxin which causes

* Corresponding author. Tel.: 1 20 75 813 101; fax: 1 20 75 811 636. E-mail address: vivian.wu@umit.maine.edu (V.C.-H. Wu).

staphylococcal food poisoning, another common foodborne disease (Jay et al., 2005). Addition of chemical preservatives is a conventional method of enhancing food safety. However, consumers today are increasingly concerned about chemical residues in food and tend to choose natural, healthful, and safe food (Gould, 1996). Consequently, the demand for natural preservatives and development of healthy antimicrobial compounds is on the rise. Berry fruits are rich sources of bioactive compounds, such as phenolics and organic acids, which may hold antimicrobial activities (Davidson, 1993; Puupponen-Pimia et al., 2001; Rauha et al., 2000). Puupponen-Pimia et al. (2001) studied an timicrobial properties of pure phenolic compounds and phenolic extracts from Finnish berries against probiotic bacteria and other intestinal bacteria; the authors found lactic acid bacteria (LAB) were more resistant than the other bacteria (generic E. coli, Salmonella spp.) to phenolic compounds. Rauha et al. (2000) studied antimicrobial effects of Finnish plant extracts

0023-6438/$34.00 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2008.01.001

V.C.-H. Wu et al. / LWT - Food Science and Technology 41 (2008) 1834e1841

1835

containing avonoids and other phenolic compounds on microorganisms and found that avone, quercetin and naringenin were effective in inhibiting the growth of the organisms. Vaccinium macrocarpon, American cranberry, has signicant commercial value and offers important health benets such as preventing urinary tract infections (Howell, 2002; Vattem, Lin, Ghaedian, & Shetty, 2005). Phenolic phytochemicals in the cranberries are now known to have potential for inhibition of development and progression of cancer and cardiovascular diseases (Reed, 2002; Vattem et al., 2005). Increasing interest in the health benets of various berries has led to investigation of their antimicrobial activity. However, there are no available reports on antibacterial activity of American cranberry (V. macrocarpon) concentrate against commonly occurring foodborne pathogens such as E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus. The study of the antibacterial effects of American cranberries and resulting concurrent development of natural food-grade antimicrobials have the potential not only to replace synthetic food additives, but to provide an additional layer of consumer protection beyond physical and conventional treatment methods (hurdle technology). As a long-term strategy, additional benets of American cranberries if identied may include dual function effect of both preservation and delivering functional phytochemicals with health benets. The objectives of the present research were to study (1) the antibacterial properties, including the bactericidal and suppressive effects, of American cranberry (V. macrocarpon) concentrate on E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus in vitro and (2) effects of American cranberry concentrate on cellular structure of foodborne pathogens. 2. Materials and methods 2.1. Culture preparation Four commonly occurring genera of foodborne pathogens, two strains of each including E. coli O157:H7 (ATCC 35150 and 43888), L. monocytogenes (ATCC 49594 and 7644), S. Typhimurium (ATCC 14028 and 13311), and S. aureus (ATCC 25923 and 4012) were used in the experiments. The cultures were tested for purity by Gram reactions, commercial diagnostic kits, and growth on selective media. E. coli O157:H7 was tested using API 20E (BioMerieux, Hazelwood, MO, USA) and RIM latex agglutination test (Remel, Lenexa, KS, USA). S. Typhimurium was tested by API 20E. BBL Crystal Gram-Positive ID system (Becton Dickinson, Sparks, MD, USA) was used for L. monocytogenes and S. aureus. Cultures were kept under refrigeration (4  C) as stock cultures and transferred weekly to maintain viability. Each culture was grown in 9.0 ml Brain Heart Infusion broth (BHI, Difco Laboratories, Detroit, MI, USA) and incubated at 37  C for 24 h. After incubation, 1 ml of each culture broth was transferred to sterilized 250-ml polycarbonate centrifuge bottles (Nalgene, Rochester, NY, USA) containing 99 ml of BHI broth, and the bottles were incubated at 37  C

for 24 h. Cells were harvested by centrifugation (Model JA-14, International Equipment Co., Needham Heights, MA, USA) at 15,300g for 20 min at 4  C. After centrifugation, the pellet was suspended in 1 mg/ml peptone water (Difco) to serve as culture suspensions. Two strain suspensions of each pathogen were combined with equal populations as a cocktail mixture for each pathogen. 2.2. Media Thin agar layer (TAL) plates, a recovery method, were used to enumerate both injured and healthy bacterial cells. The TAL plates were made by overlaying a total of 14 ml of tryptic soy agar (TSA, Difco), onto a prepoured and solidied selective medium in a petri dish (100 15 mm) (Wu & Fung, 2001; Wu, Fung, & Kang, 2001; Wu, Fung, Kang, & Thompson, 2001). The pathogen-specic agar was MacConkey sorbitol agar (MSA, Difco) for E. coli O157:H7, modied oxford agar (MOX, Difco) for L. monocytogenes, xylose lysine desoxycholate agar (XLD, Difco) for S. Typhimurium, and Baird-Parker agar (BP, Difco) for S. aureus. 2.3. Antibacterial effects of American cranberry concentrate on foodborne pathogens American cranberry concentrate (V. macrocarpon) was obtained from Ocean Spray Inc. (Lakeville-Middleboro, MA, USA), and sterilized by passage through a sterile ltration apparatus (lter size: 0.45 mm, Nalgene, Rochester, NY, USA.). The concentrate was then refrigerated at 4  C. Cranberry concentrate was prepared at ve concentration levels (0, 25, 50, 75, and 100 ml/ml) in 100 ml of sterilized distilled water (DW) in order to study its bactericidal effect with limited nutrients, and in 100 ml of BHI broth to gauge the suppressive effect. Both DW and BHI at each concentration level were inoculated with 1 ml of 6 log CFU/ml of each pathogen suspension to achieve an initial inoculum level of approximately 4 log CFU/ml, which provides a wide range for observation of bacterial reductions. Each sample was mixed well by vortexing, then incubated at 21 and 4  C for 0, 1, 5, 7, and 24 h (DW samples) and 0, 1, 3, and 5 days (BHI samples). At each sampling time, the samples were serially diluted with 1 mg/ml sterile peptone water and the appropriate dilution was spiral plated onto the pathogen-specic TAL plates using a Spiral Plater (Autoplate 4000, Spiral Biotech, Bethesda, MD, USA). After incubation at 37  C for 24e36 h, viable cells were determined. 2.4. Measurement of total phenolics The total phenolics of the cranberry concentrate were determined as gallic acid equivalents by FolineCiocalteu method (Waterhouse, 2004) with absorbance measured at 765 nm. The phenolic content was then calculated using actual absorbance versus a gallic acid standard curve.

1836

V.C.-H. Wu et al. / LWT - Food Science and Technology 41 (2008) 1834e1841

2.5. Transmission electron microscopy analysis The four pathogens were incubated in BHI broth (Difco) individually at 37  C for 18 h (108 CFU/ml). Cultures were centrifuged at 3750 g (Beckman Coulter Inc., Fullerton, CA, USA) for 10 min at 4  C. The cell pellets were rinsed with sterile 8.5 mg/ml saline (Fisher Chemicals, Fair Lawn, NJ, USA) twice. Samples were then resuspended in sterile 8.5 mg/ml saline with and without 5 ml/ml cranberry concentrate. Samples were rested for 20 min at room temperature (21  C). One millilitre aliquots of each were centrifuged at 9000 g in microfuge (Beckman Coulter Inc.) for 5 min. The procedures for xation, dehydration, inltration, embedding, trimming, sectioning, and photography of the samples for TEM according to the standard procedures (Bozzola & Russell, 1999) were carried out at Electron Microscopy Laboratory at University of Maine. 2.6. pH effect analysis An acidic solution was made by combination of citric acid (61 mg/g), malic acid (51 mg/g), and quinic acid (65 mg/g) based on the cranberry juice composition analysis by Hong and Wrolstad (1986). This acidic solution was ltered and stored at 4  C. The acidic solution was prepared at two concentration levels (25 and 90 ml/ml) in BHI which have pH values equal to those of 25 (pH 4.7) and 100 ml/ml (pH 3.6) of cranberry concentrate in BHI, respectively. One millilitre of E. coli O157:H7 mixture was inoculated in BHI samples (acidic solutions at 25 and 90 ml/ml and

cranberry concentrate at 25 and 100 ml/ml) to reach initial inoculum level of approximately 5 log CFU/ml. The samples were kept at 21  C. Pathogen counts were determined at 0 and 24 h using TAL plates. 2.7. Statistical analysis All experiments were repeated three times. Bacterial populations were reported as log CFU/ml. The experimental design used was Randomized Complete Blocks (RCB). Analysis of variance (ANOVA) was performed on cell counts using the SAS General Linear Models (GLM) procedure with SAS software 8.0 (Statistical Analysis System. Inst. Inc., Cary, NC, USA). Signicance of differences was dened as P < 0.05. Differences among treatments were examined for the level of signicance by least signicant difference (LSD). 3. Results 3.1. Bactericidal effects of American cranberry concentrate in sterile distilled water E. coli O157:H7 was less sensitive to the antimicrobial effects of American cranberry concentrate than other pathogens in water at 21  C (Table 1). The 75 and 100 ml/ml treatments resulted in 0.8 log and 3 log CFU/ml reductions respectively, when compared to the control at 24 h. All treatments signicantly reduced E. coli O157:H7 compared to the control, starting from 0 h at 4  C (Table 2).

Table 1 Effect of American cranberry concentrate (Vaccinium macrocarpon) on microbial growth of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus in sterilized distilled water at 21  C for 0, 1, 5, 7, 24 h Target species Concentration (ml/ml) Viable cell counts (log CFU/ml)a 0h E. coli O157:H7 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 4.30 0.36a 4.35 0.13a 4.23 0.14a 3.87 0.76a 4.09 0.26a
b

1h 4.18 0.37a 4.38 0.09a 4.27 0.20a 4.08 0.21ab 3.67 0.43b 4.66 0.30a eb eb eb eb 3.98 0.88a 3.57 0.54a 3.00 0.38a 1.43 0.75b ec 4.81 0.06a 2.37 0.84b 2.98 0.51b 2.13 0.05b 1.95 0.82b

5h 4.07 0.08a 4.35 0.08a 4.20 0.13a 3.78 0.43ab 3.07 0.65b 4.67 0.02a eb eb eb eb 3.97 0.55a 2.78 0.55b 2.36 0.25b ec ec 4.71 0.11a 1.67 0.06b 1.36 0.39bc 1.26 0.24bc 1.00 0.00c

7h 4.08 0.20ab 4.38 0.07a 4.14 0.23ab 3.51 0.62b 2.58 0.72c 4.75 0.13a eb eb eb eb 3.80 0.89a 2.41 0.72b 1.42 0.39b ec ec 4.65 0.07a eb 1.10 0.17b eb eb

24 h 3.03 0.19ab 4.18 0.26a 3.36 0.47ab 2.19 0.86b ec 4.58 0.18a eb eb eb eb 3.76 0.82a 2.06 0.08b ec ec ec 4.47 0.18a eb eb eb eb

L. monocytogenes

4.63 0.11a eb eb eb eb 4.12 0.78a 4.01 0.68b 3.68 0.57b 2.76 0.57c 1.10 0.17d 4.70 0.16a 3.30 0.58ab 3.35 0.40ab 3.15 0.70b 2.63 0.84b

S. Typhimurium

S. aureus

e Means < 1 log CFU/ml (below detection limit). a The experiments were repeated three times, and data are expressed as mean standard deviation. b Within the same column, means with different letters for the same species are signicantly different (P < 0.05).

V.C.-H. Wu et al. / LWT - Food Science and Technology 41 (2008) 1834e1841

1837

Table 2 Effect of American cranberry concentrate (Vaccinium macrocarpon) on microbial growth of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus in sterilized distilled water at 4  C for 0, 1, 5, 7, 24 h Target species Concentration (ml/ml) Viable cell counts (log CFU/ml)a 0h E. coli O157:H7 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 4.32 0.11a 3.38 0.65b 3.25 0.69bc 2.82 0.85cd 2.53 1.13d 4.62 0.05a eb eb eb eb 4.50 0.09a 4.03 0.04ab 3.67 0.46b 2.44 0.25c ed 4.66 0.08a 3.74 0.16ab 3.13 0.55b 3.06 0.55b 2.56 0.49b
b

1h 4.21 0.18a 3.49 0.48b 2.99 0.76c 2.48 1.21d 2.39 1.22d 4.57 0.11a eb eb eb eb 4.17 0.31a 3.55 0.45a 3.28 0.23ab 2.28 0.53b 1.15 0.21c 4.63 0.19a 3.14 0.84b 3.10 0.53b 2.98 0.54b 2.72 0.61b

5h 4.13 0.39a 3.05 0.98b 2.65 1.24c 2.20 1.48c 1.87 1.51c 4.64 0.03a eb eb eb eb 4.17 0.13a 3.45 0.01a 3.11 0.11a 1.02 1.18b eb 4.60 0.06a 3.02 0.66b 2.87 0.17b 2.90 0.69b 2.75 0.83b

7h 4.10 0.31a 3.30 0.75b 2.22 1.62b 2.12 1.55b 1.78 1.36b 4.68 0.16a eb eb eb eb 4.28 0.01a 3.25 0.11b 2.63 0.31b 1.45 0.64c ed 4.50 0.25a 2.91 0.40ab 2.47 0.08b 2.09 1.16b 2.53 0.69b

24 h 3.76 0.57a 2.09 1.89b 1.79 1.37b 1.43 0.74b ec 4.60 0.19a eb eb eb eb 4.02 0.04a 2.30 0.06b ec ec ec 4.65 0.09a 1.26 0.24b eb eb eb

L. monocytogenes

S. Typhimurium

S. aureus

e Means < 1 log CFU/ml (below detection limit). a The experiments were repeated three times, and data are expressed as mean standard deviation. b Within the same column, means with different letters for the same species are signicantly different (P < 0.05).

L. monocytogenes was the most sensitive to the cranberry concentrate among the pathogens tested when limited nutrients were present in the environment. L. monocytogenes could not be detected even immediately after inoculation in water at either 21 or 4  C (Tables 1 and 2). The mechanism of the sufcient inhibition needs further investigation. S. Typhimurium and S. aureus were also sensitive to the cranberry concentrate at both 21 and 4  C. At 24 h, a signicant reduction of S. Typhimurium was observed in the 25 ml/ ml cranberry-concentrate treatments, and no S. Typhimurium was detected in the 50, 75, and 100 ml/ml cranberry-concentrate treatments (Tables 1 and 2). At 24 h, no S. aureus was detected from the 50, 75, and 100 ml/ml treatments at 21  C (Table 1). After 24 h at 4  C, there were 3e4 log CFU/ml signicant differences (P < 0.05) for S. aureus between the cranberry concentrate treatments and the control (Table 2). 3.2. Suppressive effects of American cranberry concentrate in BHI For E. coli O157:H7 at 21  C (Table 3), signicant reductions (P < 0.05) began on Day 1 in all treatments. There was no E. coli O157:H7 detected on Day 5 from the 100 ml/ml treatment at 21  C. Although at 4  C (Table 4), E. coli O157:H7 counts remained similarly from Days 0 to 5 in the control, the 75 and 100 ml/ml treatments exhibited signicantly decreased cell numbers (P < 0.05) beginning on Day 3. For L. monocytogenes at 21  C, all treatments demonstrated inhibitory effects with 3e5.3 log CFU/ml differences when

compared with the control starting on Day 1 (Table 3). At 4  C (Table 4), signicant suppressive effects (P < 0.05) of all treatments were observed from Day 3 onward. In contrast to the control, all treatments had approximately 2.9e3.7 log CFU/ ml suppression of L. monocytogenes on Day 5 (Table 4). Treatments with concentrations higher than 25 ml/ml showed a strong effect on S. Typhimurium. No S. Typhimurium was detected in the 75 or 100 ml/ml treatments on Day 3 or 5 at 21 or 4  C (Tables 3 and 4). At 21  C (Table 3), all treatments signicantly suppressed S. aureus (P < 0.05) compared with the control on Days 1 and 3. By Day 5, the 25, 50, 75, and 100 ml/ml treatments signicantly suppressed S. aureus (P < 0.05) by approximately 2.3, 2.4, 3.8, and 5.2 log CFU/ml respectively, when compared to the control. At 4  C (Table 4), all treatments signicantly inhibited S. aureus (P < 0.05) on Days 3 and 5. 3.3. Observation of cellular damage by TEM Fig. 1 provides detailed images of the effects of American cranberry concentrate on cellular structure using transmission electron microscopy. A relatively low concentration of cranberry concentrate (5 ml/ml) was used in this treatment, because high concentration levels destroyed the cell structures making it difcult to observe cellular damage. Fig. 1a-ied-i shows that untreated cells of E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus (in sterile saline) had a uniform cellular structure with well-dened membranes and little debris in the cells surrounding environment. Exposure to

1838

V.C.-H. Wu et al. / LWT - Food Science and Technology 41 (2008) 1834e1841

Table 3 Effect of American cranberry concentrate (Vaccinium macrocarpon) on microbial growth of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus in sterilized BHI at 21  C for 0, 1, 3, 5 days Target species Concentration (ml/ml) Viable cell counts (log CFU/ml)a 0 day E. coli O157:H7 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 4.65 0.40a 4.52 0.36a 4.23 0.24a 4.40 0.48a 4.05 0.13a
b

1 day 8.60 0.10a 5.10 1.00b 3.70 0.87b 3.95 0.53b 3.62 1.40b 8.35 0.73a 4.71 0.08ab 4.63 0.28b 4.39 0.45b 2.96 0.05b 7.51 0.79a 4.93 0.30b 2.72 0.24c 1.18 0.31d ed 7.77 0.29a 4.77 0.10b 4.59 0.17b 4.55 0.10b 4.41 0.34b

3 days 8.58 0.24a 6.56 0.57ab 5.60 0.87bc 3.21 1.00cd 1.58 0.81d 8.04 0.27a 4.80 0.68b 5.02 0.58ab 2.83 0.07b 2.62 0.47b 7.88 0.69a 6.56 0.39b 3.10 0.50c ed ed 7.84 0.24a 4.96 0.49b 4.97 0.55b 4.27 0.06b 3.90 0.35b

5 days 8.85 0.32a 6.73 0.80a 5.66 0.11ab 2.99 0.65bc ec 8.26 0.63a 5.33 0.79bc 5.82 0.05b 3.32 1.37cd 2.53 0.48d 8.47 0.59a 6.97 0.26a 4.18 0.82b ec ec 7.89 0.26a 5.55 0.57b 5.52 0.46b 4.06 0.06c 2.66 0.51d

L. monocytogenes

4.60 0.05a 4.55 0.10a 4.53 0.25a 4.62 0.14a 3.82 1.32a 4.23 0.29a 4.84 0.76a 3.75 0.74a 3.46 0.73a 2.97 0.92a 4.68 0.09a 4.65 0.06a 4.67 0.18a 4.51 0.08a 4.51 0.03a

S. Typhimurium

S. aureus

e Means < 1 log CFU/ml (below detection limit). a The experiments were repeated three times, and data are expressed as mean standard deviation. b Within the same column, means with different letters for the same species are signicantly different (P < 0.05).

cranberry concentrate (5 ml/ml) resulted in morphological damage such as loss of the structural integrity of the wall, membrane and intracellular matrix (Fig. 1a-iied-ii). Cell deformation, breakage of cell wall and membrane, condensation

of cellular material, and presence of signicant amounts of ctyoplasmic material and membrane debris in the cells surrounding environment were observed from the damaged cells of E. coli O157:H7 and S. Typhimurium (Fig. 1a-ii and c-ii).

Table 4 Effect of American cranberry concentrate (Vaccinium macrocarpon) on microbial growth of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus in sterilized BHI at 4  C for 0, 1, 3, 5 days Target species Concentration (ml/ml) Viable cell counts (log CFU/ml)a 0 day E. coli O157:H7 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 4.69 0.75a 4.72 0.59a 4.50 0.57ab 4.01 0.67b 4.02 0.34b 4.66 0.16a 4.51 0.16a 4.59 0.16a 4.61 0.06a 4.51 0.00a 4.21 0.30a 4.20 0.23ab 4.28 0.28a 3.77 0.23bc 3.56 0.00c 4.64 0.13ab 4.68 0.08a 4.63 0.12ab 4.62 0.04ab 4.49 0.14b
b

1 day 4.05 0.25a 4.56 0.81a 3.72 0.10ab 3.81 0.59ab 2.63 0.21b 4.81 0.23a 4.58 0.18ab 4.55 0.03ab 4.58 0.19ab 4.30 0.06b 4.07 0.27a 4.17 0.24a 3.71 0.42a 2.09 0.55b ec 4.92 0.18a 4.63 0.08ab 4.54 0.07b 4.56 0.22ab 4.42 0.04b

3 days 4.75 0.97a 4.63 0.71a 3.90 0.54ab 2.81 0.19bc 2.37 1.08c 6.26 0.44a 4.58 0.08b 4.44 0.20b 4.38 0.18b 4.10 0.19b 3.94 0.59a 4.25 0.24a 3.03 0.44b ec ec 6.21 0.35a 4.63 0.01b 4.37 0.13bc 4.60 0.08b 2.56 0.37c

5 days 4.72 1.11a 4.42 0.87ab 3.36 0.90bc 2.58 0.63cd 1.64 1.55d 7.50 0.27a 4.62 0.10b 4.49 0.09b 4.35 0.33bc 3.82 0.23c 4.14 0.22a 4.44 0.14a 2.11 0.37b ec ec 7.44 0.23a 4.58 0.07b 4.39 0.37b 2.92 0.11b 2.92 1.44b

L. monocytogenes

S. Typhimurium

S. aureus

e Means < 1 log CFU/ml (below detection limit). a The experiments were repeated three times, and data are expressed as mean standard deviation. b Within the same column, means with different letters for the same species are signicantly different (P < 0.05).

V.C.-H. Wu et al. / LWT - Food Science and Technology 41 (2008) 1834e1841

1839

Fig. 1. Transmission electron microscopy micrographs of (a) Escherichia coli O157:H7 ( 34,000), (b) Listeria monocytogenes ( 34,000), (c) Salmonella Typhimurium ( 64,000), and (d) Staphylococcus aureus ( 34,000) cells from pure culture in (i) sterile 8.5 mg/ml saline as control and (ii) 5 ml/ml American cranberry concentrate (Vaccinium macrocarpon) as treatment.

1840

V.C.-H. Wu et al. / LWT - Food Science and Technology 41 (2008) 1834e1841 Table 6 pH of the media added with American cranberry concentrate (Vaccinium macrocarpon) Medium Concentrations of the cranberry concentrate (ml/ml) 0 25
a

Untreated cells of L. monocytogenes and S. aureus have thick cell walls. Cell wall damage and impaired cells surrounded by lysate material were observed when cells were treated with cranberry concentrate (Fig. 1b-ii and d-ii). 3.4. pH effect analysis Because the antibacterial effects of cranberry concentrate were easily observed when bacteria grew in a favorite environment, the pH effect analysis was conducted in BHI at 21  C. E. coli O157:H7, which is reportedly tolerant of acidic environments, was chosen as the representative bacterium for the pH effect study. Table 5 indicates that at the same pH level (pH 4.7), 25 ml/ml cranberry concentrate showed stronger antimicrobial effects (P < 0.05) than 25 ml/ml acidic solution on E. coli O157:H7 after 24 h. Cranberry concentrate of 100 ml/ml showed greater antibacterial effects (P < 0.05) on E. coli O157:H7 than 90 ml/ml acidic solution at the same pH level (pH 3.6) after 24 h. 4. Discussion In the present study, cranberry concentrate showed antibacterial effects on both Gram-positive (L. monocytogenes and S. aureus) and Gram-negative (E. coli O157:H7 and S. Typhimurium) bacteria. Gram-positive bacteria were less sensitive to the cranberry concentrate than the Gram-negative bacteria when nutrients are abundant in a suitable growth environment (21  C, BHI broth). Water combined with cranberry concentrate is a harsh environment for foodborne pathogens as few nutrients are available to support bacterial growth and the pH of cranberry-water treatments is lower than that in cranberry-BHI treatments (Table 6). The low pH of the cranberry concentrate plays an important role in inhibiting foodborne pathogens. However, our pH effect analysis indicated that at the same pH level, cranberry concentrate showed greater antibacterial effects than the acidic solution. In addition, no signicant differences (P > 0.05) were observed in the pH values among different concentrations of cranberry concentrate (25, 50, 75, and 100 ml/ml) in the water
Table 5 Survival of Escherichia coli O157:H7 in BHI broth at 21  C during 24 h under different treatments Treatments (ml/ml) in BHI Viable cell counts (log CFU/ml) 0h 0 ml/ml 25 ml/ml acidic solutionb 25 ml/ml cranberry concentrate 90 ml/ml acid solutionb 100 ml/ml cranberry concentrate 4.64 0.18aa 4.54 0.12a 4.66 0.12a 4.57 0.19a 4.55 0.14a 24 h 7.95 0.74a 5.88 0.78b 4.69 0.32c 2.84 0.61d 2.02 0.63e

50 2.8b 4.1c

75 2.8b 3.8d

100 2.7b 3.6d

DW BHI

6.5a 7.4a

3.2b 4.7b

a Values were means of three repeats and measured at the start of the experiment. Means in the same row with different letters (a, b, c, or d) indicate signicant differences (P < 0.05).

a The experiments were repeated three times, and data are expressed as mean standard deviation. Means in the same column with different letters (a, b, c, or d) indicate signicant differences (P < 0.05). b Acidic solutions of 25 and 90 ml/ml in BHI have the same pH value as American cranberry concentrate (Vaccinium macrocarpon) of 25 and 100 ml/ml in BHI, respectively.

study, while signicant pathogen reductions (P < 0.05) were observed at the higher cranberry concentrations (Table 6). This implies that other bioactive compounds such as phenolics in the cranberry concentrate may also contribute to the antimicrobial actions. The total phenolic content in the tested cranberry concentrate was 12.6 0.2 g/l (gallic acid equivalent). The total phenolic content of the various concentrations of cranberry concentrate tested (25, 50, 75, and 100 ml/ml) in 100 ml of medium were 31.5, 63, 94, and 126 mg, respectively. Vattem et al. (2005) suggested that phenolics with partial hydrophobicity could act efciently at the bacterial membraneewater interface by embedding in the membrane thereby impairing the cell membrane and the transport process. Earlier studies of cranberries natural resistance to deterioration (Clague & Fellers, 1934; Eschenbecher & Jost, 1977; Marwan & Nagelm, 1986) demonstrated that the suppressive effect of the berries is a complex interaction and that benzoic acid does not play the dominant role as rst hypothesized. Other plant substances including alkaloids, phenols, glycosides, steroids, essential oils, coumarins and tannins are believed to be involved. Marwan and Nagelm (1986) found that proanthocyanidins and avonols were the major microbial inhibitors on Saccharomyces bayanus and Pseudomonas uorescens. Exposure to low pH can cause sublethal injury to cell membranes, causing disruption of proton motive force owing to loss of H-ATPase (Lin, Labbe, & Shetty, 2004). This damage may make the bacteria more susceptible to the phenolic antimicrobial compounds in the cranberry concentrate. We hypothesize that both low pH and phenolics may contribute to the antibacterial activities of the American cranberry concentrate in the present study as indicated by damage to the cell walls and membranes observed through TEM study. The antimicrobial compounds in cranberry concentrate damaged the cell wall, cell membrane and induced cell lysis. Antimicrobial compounds enter the cells easily through these lesions which also facilitate leakage of the cell contents. These antimicrobials may even react with the bacterial DNA, eventually resulting in cell death. Puupponen-Pimia et al. (2001) previ ously hypothesized that phenolic compounds from Finnish berries could bind the outer membrane of the cells, disrupting the permeability barrier of the outer membrane in Gramnegative bacteria. Variations in cell wall structures between Gram-positive and Gram-negative bacteria may cause different damage when the bacteria are subjected to antimicrobial compounds (Puupponen-Pimia et al., 2001). The cell wall of Gram-positive bacteria contains much more peptidoglycan

V.C.-H. Wu et al. / LWT - Food Science and Technology 41 (2008) 1834e1841

1841

than that of Gram-negative cells, providing increased rigidity (Seltmann & Holst, 2002). This is reected in Fig. 1a-ied-i which shows that L. monocytogenes and S. aureus tend to be more rigid when compared with E. coli O157 and S. Typhimurium. L. monocytogenes and S. aureus cells were not easily destroyed by formation of lesions or channels on the cell wall. From the treated cell image of L. monocytogenes (Fig. 1bii), it seems that newly divided cells lack the clear cell wall structure of older cells. Microorganisms that are not growing or that have entered a stationary phase have been shown to be less susceptible to a wide variety of antimicrobial agents (Stewart, Mukherjee, & Ghannoum, 2004). Newly formed cells are in an active physiological state, dividing and multiplying quickly. The cell wall of these newly formed cells is likely easier to be destroyed than that of older cells. The cell wall of untreated S. aureus cells was clearly dened (Fig. 1d-i). However, the cell wall of treated cells of S. aureus was not easily discerned, indicating that the antimicrobial compounds could enter the cells easily while the substances inside the cells leaked out. 5. Conclusion In conclusion, the present study demonstrated that American cranberry (V. macrocarpon) concentrate has strong antibacterial effects against the foodborne pathogens, E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus in vitro. Temperature, pH, and other bioactive compounds of the cranberry concentrate inuenced or contributed to the antibacterial effects. Cranberry concentrate may be further investigated as a natural solution to the food industry by creating an additional barrier (hurdle technology) to inhibit the growth of foodborne pathogens while providing additional health benets (Puupponen-Pimia et al., 2001; Vattem et al., 2005). Acknowledgments This research was supported by Cranberry Institute and Wisconsin Cranberry Board, Inc., Maine Agricultural Center, and Maine Agricultural and Forest Experiment Station at the University of Maine with external publication number 2992. We thank Dr. Seth Tyler and Mr. Kelly Edwards for their support of our work at the facility of Electron Microscopy Laboratory at University of Maine. We thank Elizabeth Dodge for assistance with editing. References
Bozzola, J. J., & Russell, L. D. (1999). Electron microscopy: Principles and techniques for biologists. Sudbury, MA: Jones and Bartlett Publishing Co. pp. 17e146.

Clague, J. A., & Fellers, C. R. (1934). Relation of benzoic acid content and other constituents of cranberries to keep quality. Plant Physiology, 9, 631e636. Davidson, P. M. (1993). Parabens and phenolic compounds. In P. M. Davidson, & A. L. Branen (Eds.), Antimicrobials in foods (pp. 263e306). New York, NY: Marcel Dekker. Eschenbecher, F., & Jost, P. (1977). Research on inhibitors in cranberries. Acta Horticulturae, 61, 255e272. Gould, G. W. (1996). Industry perspectives on the use of natural antimicrobials and inhibitors for food applications. Journal of Food Protection, 59(Suppl.), 82e86. Hong, V., & Wrolstad, R. E. (1986). Cranberry juice composition. Journal of the Association of Ofcial Analytical Chemists, 69, 199e207. Howell, A. B. (2002). Cranberry proanthocyanidins and the maintenance of urinary tract health. Critical Reviews Food Science Nutrition, 42(Suppl. 3), 273e278. Jay, J. M., Loessner, M. J., & Golden, D. A. (2005). Modern food microbiology (7th ed.). New York, NY: Springer. pp. 545e634. Lin, Y. T., Labbe, R. G., & Shetty, K. (2004). Inhibition of Listeria monocytogenes in sh and meat systems by use of oregano and cranberry phytochemical synergies. Applied Environmental Microbiology, 70, 5672e5678. Marwan, A. G., & Nagelm, C. W. (1986). Microbial inhibitors of cranberries. Journal of Food Science, 51, 1009e1013. Puupponen-Pimia, R., Nohynek, L., Meier, C., Kahkonen, M., Heinonen, M., & Hopia, A., et al. (2001). Antimicrobial properties of phenolic compounds from berries. Journal of Applied Microbiology, 90, 494e507. Rauha, J. P., Remes, S., Heinonen, M., Hopia, A., Kahkonen, M., & Kujala, T., et al. (2000). Antimicrobial effects of Finnish plant extracts containing avonoids and other phenolic compounds. International Journal of Food Microbiology, 56, 3e12. Reed, J. (2002). Cranberry avonoids, atherosclerosis and cardiovascular health. Critical Reviews Food Science Nutrition, 42(Suppl. 3), 301e316. Seltmann, G., & Holst, O. (2002). The bacterial cell wall. Berlin, Heidelberg, Germany: Springer-Verlag. Stewart, P. S., Mukherjee, P. K., & Ghannoum, M. A. (2004). Biolm antimicrobial resistance. In M. A. Ghannoum, & G. A. OToole (Eds.), Microbial biolms (pp. 250e268). Washington, DC: ASM Press. US Centers for Disease Control and Prevention (US CDC). (2005). Escherichia coli O157:H7. http://www.cdc.gov/ncidod/dbmd/diseaseinfo/ escherichiacoli_g.htm Accessed 06.01.07. Vattem, D. A., Lin, Y. T., Ghaedian, R., & Shetty, R. K. (2005). Cranberry synergies for dietary management of Helicobacter pylori infections. Process Biochemistry, 40, 1583e1592. Waterhouse, A. L. (2004). Polyphenolics: determination of total phenoics. In R. E. Wrolstand, T. E. Acree, E. A. Decker, M. H. Penner, D. S. Reid, & S. J. Schwartz, et al. (Eds.), Handbook of food analytical chemistry, pigments, colorants, avors, texture, and bioactive food components (pp. 463e470). Indianapolis, IN: John Wiley & Sons, Inc. Wu, V. C. H., & Fung, D. Y. C. (2001). Evaluation of thin agar layer method for recovery of heat-injured foodborne pathogens. Journal of Food Science, 66, 580e583. Wu, V. C. H., Fung, D. Y. C., & Kang, D. H. (2001). Evaluation of thin agar layer method for recovery of cold-injured foodborne pathogens. Journal of Rapid Methods and Automation in Microbiology, 9, 11e25. Wu, V. C. H., Fung, D. Y. C., Kang, D. H., & Thompson, L. K. (2001). Evaluation of thin agar layer method for recovery of acid-injured foodborne pathogens. Journal of Food Protection, 64, 1067e1071.