Growth inhibition of foodborne pathogens and food spoilage organisms by select raw honeys

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1 International Journal of Food Microbiology 97 (2004) Growth inhibition of foodborne pathogens and food spoilage organisms by select raw honeys Melissa A. Mundo, Olga I. Padilla-Zakour*, Randy W. Worobo New York State Agricultural Experiment Station, Department of Food Science and Technology, Cornell University, 630 W. North St., Geneva, NY 14456, USA Received 1 October 2003; received in revised form 22 March 2004; accepted 30 March 2004 Abstract Twenty-seven honey samples from different floral sources and geographical locations were evaluated for their ability to inhibit the growth of seven food spoilage organisms (Alcaligenes faecalis, Aspergillus niger, Bacillus stearothermophilus, Geotrichum candidum, Lactobacillus acidophilus, Penicillium expansum, Pseudomonas fluorescens) and five foodborne pathogens (Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica Ser. Typhimurium, and Staphylococcus aureus) using an overlay inhibition assay. They were also tested for specific activity against S. aureus 9144 and B. stearothermophilus using the equivalent percent phenol test a well diffusion assay corresponding to a dilute phenol standard curve. Honey inhibited bacterial growth due to high sugar concentration (reduced water activity), hydrogen peroxide generation, and proteinaceous compounds present in the honey. Some antibacterial activity was due to other unidentified components. The ability of honey to inhibit the growth of microorganisms varies widely, and could not be attributed to a specific floral source or demographic region produced in this study. Antibacterially active samples in this study included Montana buckwheat, tarweed, manuka, melaleuca, and saw palmetto. Furthermore, the bacteria were not uniformly affected by honey. Varying sensitivities to the antimicrobial properties were observed with four strains of S. aureus thus emphasizing the variability in the antibacterial effect of honey samples. Mold growth was not inhibited by any of the honeys tested. B. stearothermophilus, a heat-resistant spoilage bacteria, was shown to be highly sensitive to honey in both the overlay and well diffusion assays; other sensitive bacteria included A. faecalis and L. acidophilus. Non-peroxide antibacterial activity was observed in both assays; the highest instance was observed in the specific activity assay against B. stearothermophilus. Further research could indicate whether honey has potential as a preservative in minimally processed foods. D 2004 Elsevier B.V. All rights reserved. Keywords: Honey; Antimicrobial activity; Foodborne pathogens; Food spoilage organisms 1. Introduction Honey has been used as a topical and gastrointestinal remedy for thousands of years, and has recently * Corresponding author. Tel.: ; fax: address: oip1@cornell.edu (O.I. Padilla-Zakour). gained recognition from the medical field (Stomfay- Stitz, 1960; Zumla and Lulat, 1989). The growth of many microorganisms associated with disease or infection is inhibited by honey (Molan, 1992a,b). Research (Willix et al., 1992; Cooper et al., 2002) suggests that honey is effective in vitro against wound-infecting bacteria including Escherichia coli, Staphylococcus aureus, and Salmonella enterica Ser /$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi: /j.ijfoodmicro

2 2 M.A. Mundo et al. / International Journal of Food Microbiology 97 (2004) 1 8 Typhimurium. The antimicrobial activity of honey is mainly credited to its acidity, osmolarity, and enzymatic generation of hydrogen peroxide via glucose oxidase (Dustmann, 1979; Molan, 1992a). Additional honey components, such as aromatic acids or phenolic compounds, may also contribute to the overall antimicrobial activity (Weston, 1999). Molds, yeasts, and bacterial spores can be present in honey at low levels, but vegetative bacteria generally are not found (Snowdon and Cliver, 1996). However, vegetative bacteria have been demonstrated to survive once they are introduced into honey (Tysset and Durland, 1976). Nonetheless, honey generally contains a low microbial load and has a long shelf-life. Due to its antimicrobial properties, honey may serve as a natural food preservative. Previous research has demonstrated preservative power of honey by reducing enzymatic browning of fruits (Oszmianski and Lee, 1990; Chen et al., 2000) and preventing lipid oxidation in meat (McKibben and Engeseth, 2002). This work confirmed the antioxidant capacity of some honeys, but limited research is available describing the antimicrobial activity of honey against foodborne microorganisms. Recently, Taormina et al. (2001) found that both peroxide and non-peroxide components in honey affected the growth of six food pathogens. However, little is known about the ability of honey to inhibit growth of food spoilage organisms. If honey can slow or stop the growth of spoilage organisms or food pathogens, then its incorporation into foods as a preservative can be explored. Methods to test the efficacy of the antimicrobial activity of honey usually feature different concentrations of honey either incorporated into the agar or placed in wells for a well diffusion assay (Molan, 1992a). Because exposure to heat using the former method may damage the enzyme responsible for hydrogen peroxide production in honey, the latter method was employed. Tests were performed against five food pathogens and seven spoilage organisms to determine the broad spectrum of the antimicrobial activity of honey. Overlaying the microorganisms over the honey-containing wells was done instead of seeding the agar so that the microbes were in an early growth phase and growth inhibition was more easily visible. Additionally, the equivalent percent phenol test, a well diffusion assay used by other honey researchers to assay antibacterial activity, was performed against S. aureus ATCC 9144 and B. stearothermophilus for comparison between the two different methods. 2. Materials and methods 2.1. Honey samples Twenty-six honey samples were provided by the US National Honey Board (NHB), Colorado, which received samples of raw honey from beekeepers in different regions of the United States. Table 1 lists the honey samples by floral source, state, and year produced. Active manuka honey, which exhibits antibacterial activity against S. aureus, was purchased and used as a positive control (Comvita Active 10+, New Zealand; imported by Frontier Products, CA). Samples were kept refrigerated in the dark in airtight containers. Table 1 Floral source, state and year of production of 26 honey samples Floral source State produced Year Alfalfa Unknown 1999 Blackberry WA 2000 Blackberry a WA 2001 Black button sage CA 1998 Blueberry Unknown 2000 Buckwheat China 2000 Buckwheat MT 2000 Christmas berry FL 1998 Cotton CA 1999 Gallberry FL 1998 Horsemint H TX 1999 Horsemint I TX 2001 Horsemint J TX 2001 Horsemint S TX 2001 Knotweed WA 2001 Lavender WA 2001 Maple WA 2000 Melaleuca FL 2000 Orange blossom CA 1999 Rabbit bush CA 2001 Red sumac AL 2000 Sage CA 2000 Saw palmetto FL 1999 Soybean MS 1999 Sunflower Unknown 1998 Tarweed CA 2000

3 M.A. Mundo et al. / International Journal of Food Microbiology 97 (2004) Microbes used and growth conditions Five food pathogens and seven food spoilage organisms were used in this study. The optimum growth media and incubation temperature as suggested by ATCC were used. The growth media used were commercially prepared and rehydrated according to the manufacturer s instructions (Difco, Detroit, MI). Table 2 indicates the media, temperature, and classification (spoilage or pathogenic) of each organism used Overlay inhibition assay for broad spectrum inhibitory activity The method of Ahn and Stiles (1990) was followed. Honey samples (0.1 g of undiluted honey) were placed in duplicate 10-mm diameter wells cut into the agar plates and allowed to diffuse. Plates were overlaid with 12 ml of 0.75% agar inoculated with 200 Al of 24-h culture (bacteria) or spore suspension (molds). Plates were incubated (overnight for bacteria, 3 days for molds) and examined for the presence of inhibition zones. A positive sample was defined as one with a visible inhibition zone greater than 1 mm surrounding the diffusion well. Positive samples were serially diluted two-fold and 200 Al of the various dilutions were placed in agar diffusion wells in order to ascertain the level of inhibition. The reciprocal of the highest dilution showing inhibition was deemed the minimum inhibitory concentration. On the periphery of the diffusion wells containing antibacterially active honeys, 5 Al of catalase (10 mg/ml), proteinase K (10 mg/ml), and a-chymotrypsin (25 mg/ml) (Sigma, St. Louis, MO) were spotted to determine if the inhibition was caused by hydrogen peroxide or a proteinaceous substance. Loss of inhibition zones in close proximity to the enzyme spots indicated sensitivity of the antimicrobial activity to the particular enzyme. The plates were then overlaid, incubated, and examined as described above. Assays were performed in duplicate at least twice. Inhibition zones caused by high sugar content were determined by measuring inhibition zones caused by high fructose corn syrup, which contains about the same sugar content (80j Brix) as honey. Corn syrup samples were tested with and without the addition of 3% hydrogen peroxide, and these inhibition zones were compared to 3% hydrogen peroxide in water controls and honey samples. Water activity of select 1- cm 2 sections of agar were measured using an Aqua- Lab CX-2 water activity meter (Decagon, Pullman, Table 2 Classification and optimal growth parameters for microorganisms used Microorganism Classification Media Incubation temperature (jc) Escherichia coli O157:H7 (ATCC 43889) Pathogenic Nutrient 37 Salmonella enterica Ser. Typhimurium a Pathogenic Nutrient 37 Bacillus cereus b Pathogenic Nutrient 30 Listeria monocytogenes a Pathogenic Brain heart infusion 37 Staphylococcus aureus (ATCC 8095, 9144, 25923; 1 c ) Pathogenic Nutrient (ATCC 8095); Tryptic soy (ATCC 25923, 9144); Brain heart infusion (1) Pseudomonas fluorescens (ATCC 11150) Spoilage Nutrient 25 Geotrichum candidum (ATCC 755) Spoilage Yeast mold 25 Penicillium expansum (ATCC 7861) Spoilage Malt extract 25 Aspergillus niger b Spoilage Potato dextrose 25 Alcaligenes faecalis (ATCC 8750) Spoilage Nutrient 37 Lactobacillus acidophilus d Spoilage Lactobacillus MRS 37 Bacillus stearothermophilus (ATCC 12980) Spoilage Nutrient 55 a From Dr. Kathryn Boor s collection, Cornell University, Ithaca, NY. b From Dr. Don Splittstoesser s collection, Cornell University, Geneva, NY. c A laboratory isolate from Cornell University Life Sciences, Ithaca, NY. d From Dr. John Stamer s collection, Cornell University, Geneva, NY. 37

4 4 M.A. Mundo et al. / International Journal of Food Microbiology 97 (2004) 1 8 WA). Agar was measured at distances 1 to 4 cm from the well after 100% honey had diffused into the agar Well diffusion assay for specific inhibitory activity Methods used for the well diffusion assay of honey in tryptic soy agar seeded with S. aureus ATCC 9144 were those described by Allen et al. (1991). Briefly, inhibition zones of 25% honey solutions diluted with water or water and catalase were compared to zones from dilute phenol solutions and reported as equivalent percent phenol as determined from the standard curve. The test was repeated replacing the indicator organism S. aureus 9144 with B. stearothermophilus since preliminary tests demonstrated high sensitivity to the antibacterial activity of honey. All experiments were performed in triplicate, and inhibition zones were measured twice per well at perpendicular angles, thus totaling six measurements per honey sample Statistical analysis Comparison of means was conducted using Analysis of Variance (ANOVA) and Least Significant Difference (LSD) at the 95% confidence level using Minitab software (Minitab version 10.51Xtra; State College, PA). 3. Results 3.1. Broad spectrum inhibition assay The antibacterial activity observed with the various honey samples was classified into four causative agents: inhibition due to high sugar concentration (reduced water activity), hydrogen peroxide formation, presence of proteinaceous antimicrobial compounds, or unidentified components. Table 3 summarizes the four types of inhibition observed against each bacteri- Table 3 Cause of inhibitory activity by honey against each bacteria Bacteria Inhibition by Inhibition by hydrogen peroxide Inhibition by proteinaceous Other inhibition c high sugar formation a compounds b Escherichia coli O157:H7 All samples Christmas berry, saw palmetto, Manuka None tarweed, MT buckwheat Salmonella enterica Ser. All samples None Manuka None Typhimurium Pseudomonas fluorescens None Tarweed, MT buckwheat None None Bacillus cereus None Tarweed, MT buckwheat Manuka None Listeria monocytogenes None Melaleuca, tarweed, MT buckwheat None None Staphylococcus aureus 1 None None None None S. aureus ATCC 8095 All samples Cotton, christmas berry, saw palmetto, None Manuka tarweed, MT buckwheat S. aureus ATCC 9144 None Melaleuca, saw palmetto, sunflower, Manuka None MT buckwheat, horsemint J S. aureus ATCC None Soybean, melaleuca, saw palmetto, None None sunflower, tarweed, MT buckwheat, manuka, rabbit bush Alcaligenes faecalis None Blueberry, tarweed, MT buckwheat, Soybean, manuka Knotweed rabbit bush Lactobacillus acidophilus None Soybean, melaleuca, christmas berry, None Manuka tarweed, MT buckwheat B. stearothermophilus None Chinese buckwheat, red sumac, melaleuca, cotton, christmas berry, saw palmetto, sunflower, tarweed, MT buckwheat, horsemint J, blackberry a Blueberry, black sage, alfalfa, blackberry Soybean, melaleuca, horsemint I, rabbit bush, horsemint S, knotweed a Inhibition reversed in the presence of catalase. b Inhibition reversed in the presence of a-chymotrypsin. Proteinase K did not affect inhibition in any honey sample tested. c Inhibition unaffected by catalase, a-chymotrypsin, and proteinase K.

5 M.A. Mundo et al. / International Journal of Food Microbiology 97 (2004) um. Most of the inhibition observed in this experiment was caused by hydrogen peroxide generation; the inhibition zones were reversed in the presence of catalase. Some activity was attributed to proteinaceous compounds, and the cause of other activity was not determined. The most active honey samples in this study were MT buckwheat, tarweed, manuka, saw palmetto, and melaleuca. B. stearothermophilus was the most sensitive microorganism to the antibacterial activity of honey in this experiment. A. faecalis, L. acidophilus, and S. aureus ATCC 25923, 8095, and 9144 were each moderately sensitive to the antimicrobial activity of honey. Table 4 describes the sensitivity of each bacterial strain to the type of honey used and its inhibitory concentration. E. coli, S. Typhimurium, and S. aureus ATCC 8095 each exhibited reduced growth due to the high osmolarity of honey. These plates showed both a broad inhibition zone of reduced growth, and a small inhibition zone with no growth. The water activity of the agar was also measured; most of the measurements were between and This range is lower than the minimum growth requirement for E. coli (0.96), Salmonella spp. (0.96), Psedomonas spp. (0.97), and Bacillus subtilis (0.95), but P. fluorescens, B. stearothermophilus, and B. cereus did not exhibit reduced growth (Jay, 1996). Furthermore, the minimum water activity necessary for S. aureus is 0.86, which is below the range observed (Molan, 1992a). Consequently, factors besides water activity must contribute to the reduced growth displayed by these bacteria Well diffusion assay for specific inhibitory activity Equivalent percent phenol values are reported in Table 5. Experimental data against S. aureus ATCC 9144 was compared to data provided by the National Honey Board (NHB) by an independent laboratory for the same test using the same samples. Positive results were compared to inhibition zones from dilute phenol solutions and assigned an equivalent percent phenol value. Of the 19 samples with an equivalent percent phenol number provided by the NHB, 12 samples also gave positive results in the experimental data trial. Knotweed honey showed no activity from the NHB Table 4 Bacterial sensitivity by type of honey and inhibitory concentration Bacteria Honey (inhibitory % concentration w/v) E. coli O157:H7 Christmas berry (100), saw palmetto (100), tarweed (100), MT buckwheat (100), manuka (50) S. enterica Ser. Manuka (50) Typhimurium A. faecalis Blueberry (100), soybean (100), tarweed (33), MT buckwheat (33), manuka (25), horsemint J (25), rabbit bush (100), knotweed (50) P. fluorescens Tarweed (100), MT buckwheat (50) L. acidophilus Soybean (100), melaleuca (50), christmas berry (100), saw palmetto (100), tarweed (50), MT buckwheat (100), manuka (100) L. monocytogenes Melaleuca (100), tarweed (100), MT buckwheat (100) B. cereus Tarweed (100), MT buckwheat (50), manuka (25) B. stearothermophilus Chinese buckwheat (33), blueberry (100), black sage (50), soybean (33), alfalfa (33), blackberry R (20), red sumac (50), melaleuca (50), cotton (33), christmas berry (50), saw palmetto (33), sunflower (17), tarweed (25), MT buckwheat (25), manuka (50), horsemint I (33), horsemint J (33), rabbit bush (33), horsemint S (50), knotweed (20), blackberry a (100) S. aureus ATCC 8095 Cotton (33), christmas berry (100), saw palmetto (50), tarweed (50), MT buckwheat (50), manuka (25) S. aureus ATCC 9144 Melaleuca (33), saw palmetto (100), sunflower (100), MT buckwheat (33), manuka (50), horsemint J (100) S. aureus ATCC Soybean (100), melaleuca (50), saw palmetto (50), sunflower (100), tarweed (33), MT buckwheat (33), manuka (50), rabbit bush (50) data but demonstrated activity in the experimental data trial. Horsemint honey samples I and J tested positively for non-peroxide antibacterial activity during both tests. Rabbit bush honey was reported to have non-peroxide antibacterial activity, but the experimental data did not confirm this. Manuka honey, used as a positive control in the experimental data trial, also displayed non-peroxide antibacterial activity against S. aureus ATCC 9144.

6 6 M.A. Mundo et al. / International Journal of Food Microbiology 97 (2004) 1 8 Table 5 Equivalent percent phenol values of the inhibitory activity of 25% honey solutions diluted with water or water and catalase against S. aureus 9144 or B. stearothermophilus Indicator organism S. aureus ATCC 9144 B. stearothermophilus Treatment a Water b Water Catalase Water Catalase Honey Maple N.D. c N.D. N.D F F 0.96 Chinese buckwheat N.D. N.D. N.D F F 0.86 Blueberry N.D. N.D. N.D F F 1.3 Lavender N.D. N.D. N.D F F 0.50 Knotweed N.D F 0.96 N.D F F 1.0 Blackberry a N.D. N.D. N.D F F 1.2 Horsemint S 3.44 N.D. N.D F F 1.3 Black sage 4.29 N.D. N.D F F 0.63 Orange 4.96 N.D. N.D F F 1.1 Horsemint J F F F F 2.3 Sage 5.57 N.D. N.D F F 1.4 Soybean 5.58 N.D. N.D F F 0.48 Blackberry R F 0.51 N.D F F 0.83 Alfalfa 6.23 N.D. N.D F F 0.33 Horsemint H 6.58 N.D. N.D F F 0.92 Horsemint I F F F F 1.0 Red sumac F 0.70 N.D F F 0.62 Melaleuca F 0.57 N.D F F 0.31 Gallberry F 0.23 N.D F 1.6 N.D. Cotton F 2.0 N.D F F 0.49 Christmas berry F.94 N.D F 1.8 N.D. Saw palmetto F 0.66 N.D F F 0.67 Sunflower F 0.96 N.D F F 0.85 Rabbit bush N.D. N.D F F 0.47 Tarweed F 2.2 N.D F F 0.23 MT buckwheat F 1.5 N.D F F 5.5 Manuka Not tested F F F F 4.6 a Honey samples (50%) were diluted with water or catalase (5600 units/ml) to make a 25% honey solution. This enabled differentiation between peroxide (water) and non-peroxide (catalase) antibacterial activity. b Data provided by the National Honey Board. c N.D.: No activity detected. In testing for peroxide and non-peroxide antibacterial activity against B. stearothermophilus, most honey samples demonstrated similar results for both tests. Only gallberry and christmas berry honey samples were rendered ineffective after exposure to catalase. 4. Discussion In the broad spectrum inhibition assay, each pathogen or food spoilage organism showed different sensitivity to the honey samples, ranging from highly sensitive (B. stearothermophilus) to unaffected (S. aureus 1, P. expansium, A. niger, G. candidum). Other researchers have also found differences in susceptibility of microorganisms to the antimicrobial activity of honey (Dustmann, 1979; Radwan et al., 1984; Allen et al., 1991; Willix et al., 1992; Bogdanov, 1997; Nzeako and Hamdi, 2000; Ceyhan and Ugur, 2001; Taormina et al., 2001). This variation could neither be attributed to the floral source or geographic region from which the honey was produced nor by the phylogenetic relatedness of the various indicator bacteria. In fact, the four horsemint samples demonstrated different antibacterial activity profiles though they came from the same flower and geographic region. However, mold growth was consistently uninhibited by any of the honey samples tested in this study.

7 M.A. Mundo et al. / International Journal of Food Microbiology 97 (2004) Molan (1992a) suggested that some honeys possess antifungal properties. White et al. (1963) suggested that bacteria are more susceptible to peroxide than molds and yeasts; other researchers have confirmed this (Molan, 1997; Nzeako and Hamdi, 2000; Ceyhan and Ugur, 2001). Results indicate that none of the honey samples tested impeded the growth of these three molds using an overlay inhibition method; changing one or more of these parameters may affect the inhibitory activity of honey against molds. Differences in the specific activity test between the experimental data and that provided by the NHB can be attributed to small differences in the experimental data trial during calibration and a loss of antibacterial activity over time. Hydrogen peroxide is generated by the enzyme glucose oxidase in honey as it converts glucose into gluconic acid (Schepartz and Subers, 1964). The enzyme remains inactive until honey is diluted, as the high sugar concentration prevents the enzyme from functioning (White et al., 1963). Unfortunately glucose oxidase is damaged by heat and light (White et al., 1963); Dustmann (1979) verified the loss of hydrogen peroxide generation from exposing honey to light for as little as ten minutes. Nonperoxide activity, on the other hand, is reported to be heat and light stable and remained active after 6 months of storage at room temperature (Bogdanov, 1984). Therefore, the higher equivalent percent phenol values could be evidence of the more stable nonperoxide antibacterial activity. However, since most of the antibacterial activity was eliminated with the addition of catalase, the higher values are most likely due to small differences in experimental procedures. Honey samples tested against B. stearothermophilus indicated substantially more non-peroxide antibacterial activity than shown against S. aureus ATCC Differences in results between the two indicator organisms may be attributed to increased diffusivity of antibacterial compounds due to the elevated incubation temperature of B. stearothermophilus. Conversely, the antibacterial compounds may be specific, with B. stearothermophilus demonstrating higher sensitivity to varying levels of antibacterial compounds present in the honey samples. These results concur with Allen et al. (1991) and Taormina et al. (2001) that non-peroxide components contribute to the antibacterial activity of honey. Weston (2000), however, suggests the non-peroxide activity detected in this method may actually be residual hydrogen peroxide activity. The causative agent of this non-peroxide activity remains evasive; future research may elucidate its identity. Specific honey samples were shown to be capable of inhibiting the growth of both spoilage microorganisms and foodborne pathogens and could be considered for use as a food preservative under appropriate conditions. Further research is needed to identify the efficacy of honey as an inhibitor of microbial growth in food systems. Acknowledgements This research was funded by the US National Honey Board and the New York State Agricultural Experiment Station. References Ahn, C., Stiles, M.E., Antibacterial activity of lactic acid bacteria isolated from vacuum-packed meats. Journal of Applied Bacteriology 69, Allen, K.L., Molan, P.C., Reid, G.M., A survey of the antibacterial activity of some New Zealand honeys. Journal of Pharmacy and Pharmacology 43, Bogdanov, S., Characterisation of antibacterial substances in honey. Lebensmittel-Wissenschaft und-technologie 17 (2), Bogdanov, S., Nature and origin of the antibacterial substances in honey. Lebensmittel-Wissenschaft und-technologie 30, Ceyhan, N., Ugur, A., Investigation of in vitro antimicrobial activity of honey. Rivista di Biologia 94, Chen, L., Mehta, A., Berenbaum, M., Zangerl, A., Engeseth, N., Honeys from different floral sources as inhibitors of enzymatic browning in fruit and vegetable homogenates. Journal of Agricultural and Food Chemistry 48, Cooper, R.A., Molan, P.C., Harding, K.G., The sensitivity to honey of Gram-positive cocci of clinical significance isolated from wounds. Journal of Applied Microbiology 93, Dustmann, J.H., Antibacterial effect of honey. Apiacta 14, Jay, J.M., Modern Food Microbiology. Chapman & Hall, New York. McKibben, J., Engeseth, N.J., Honey as a protective agent against lipid oxidation in ground turkey. Journal of Agricultural and Food Chemistry 50, Molan, P.C., 1992a. The antibacterial activity of honey: 1. The nature of the antibacterial activity. Bee World 73 (1), Molan, P.C., 1992b. The antibacterial activity of honey: 2. Variation

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