This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and

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1 This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier s archiving and manuscript policies are encouraged to visit:

2 Food Chemistry 126 (2011) Contents lists available at ScienceDirect Food Chemistry journal homepage: Storage-induced chemical changes in active components of honey de-regulate its antibacterial activity Katrina Brudzynski, Linda Kim Brock University, St. Catharines, Ontario, Canada L2S 3A1 article info abstract Article history: Received 6 August 2010 Received in revised form 19 October 2010 Accepted 25 November 2010 Available online 2 December 2010 Keywords: Honey antibacterial activity Storage Escherichia coli Bacillus subtilis MIC 90 Colour UV-absorbing compounds Melanoidins To elucidate reasons for the observed variability in the antibacterial activity of honeys, we analysed a causal relationship between (a) honey floral sources and the activity and (b) the effect of honey storage on stability of compounds conferring this activity. Honeys from diverse floral sources were screened against Escherichia coli (ATCC 14948) and Bacillus subtilis (ATCC 6633) using the broth microdilution method. Among active honeys, 37% originated from buckwheat, 18% from clover and 12% from blueberry, indicating that these floral sources produced phytochemical(s) that inhibited bacterial growth. The stability of the putative phytochemical(s) was analysed in active honeys (MIC % v/v) by measuring the activity every 3 6 months for a period of 1 3 years. A sharp decline in activity against both bacteria was observed in the first 3 6 months of storage. The decline coincided with major changes in chemical composition of honeys which included a significant change in colour (p < ), extremely significant change in concentration of UV-absorbing compounds (p < ) and appearance of melanoidins. While these changes reduced E. coli sensitivity to honey, it rendered B. subtilis completely insensitive. Thus, the data indicates that the presence of phytochemical(s) conferring the antibacterial activity is sensitive to storage. The de-regulation of the antibacterial activity with the concomitant appearance of melanoidins suggests that the active phytochemical components might be sequestered into melanoidin aggregates, losing their function. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. 1. Introduction Honey has been shown to efficiently inhibit bacterial growth in vitro and in vivo. This functional property has been found in a variety of honeys of diverse botanical origin and from a variety of geographical locations (Allen, Molan, & Reid, 1991; Ceyhan & Ugur, 2001; Lusby, Coombes, & Wilkinson, 2005; Mundo, Padilla-Zakour, & Worobo, 2004; Wilkinson & Cavanagh, 2005). The first laboratory and clinical studies on antibacterial activity were performed on a specific variety of honey, manuka honey, originated from Leptospermum scoparium and ericoides of New Zealand and Australia. These studies uncovered the unexpectedly potent antibacterial activity of Leptospermum spp. honeys and allowed for establishment a spectrum of bacteria that were sensitive to their action (Blair, Cokcetin, Harry, & Carter, 2009; Cooper, Molan, & Harding, 2002; Molan, 1992a, 1992b; Willix, Molan, & Harfoot, 1992). The research initiated worldwide search for other honey varieties which display antibacterial activity, specifically among types of ethnic honeys Corresponding author. Address: Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St. Catharines, Ontario, Canada L2S 3A1. Tel.: x5035; fax: address: kbrudzynski@brocku.ca (K. Brudzynski). including Indian jambhul (Syzygium cumini) honey (Subrahmanyam et al., 2001), Malaysian tualang (Tan et al., 2009) and gelam honeys (Aljadi, Kamaruddin, Jamal, & Yassim, 2000), Turkish honey from Anatolia (Kucuk et al., 2007) and honeys from different geographical regions such as North America and Europe (Brudzynski, 2006; Miorin, Levy, Custodio, Bretz, & Marcucci, 2003; Mundo et al., 2004; Taormina, Niemira, & Beuchat, 2001). Most of honey varieties showed either no activity or activities below that observed in honeys originated from Leptospermum spp. The intrinsic characteristic of honeys, e.g. high osmolarity, low water activity, low ph, production of hydrogen peroxide, although involved in antibacterial action, are common properties for all honeys and could not explain the variability in activity between honeys. From surveys of antibacterial activity in different honeys, it became clear that a phytochemical composition of honeys was responsible for the degree of bacteriostatic and bactericidal action (Allen et al., 1991; Ceyhan & Ugur, 2001; Lusby et al., 2005; Mundo et al., 2004; Wilkinson & Cavanagh, In the early studies, candidates for the active principal components were search among polyphenols; phenolic acids (Aljadi & Yusoff, 2003), and their derivatives (methyl syringate) (Russell, Molan, Wilkins, & Holland, 1990; Weston, Brocklebank, & Lu, 2000), aromatic acids and flavonoids (Bogdanov, 1997), hydrogen peroxide (Bang, Bunting, & Molan, 2003; Brudzynski, 2006;) and /$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi: /j.foodchem

3 1156 K. Brudzynski, L. Kim / Food Chemistry 126 (2011) recently, the Maillard reaction products (Brudzynski & Miotto, 2010a, 2010b). The Maillard reactions between amino groups of amino acids/proteins and reducing sugars, leads to formation of, among other molecules, a-dicarbonyl compounds such as glyoxal and methylglyoxal (Adams et al., 2008; Mavric, Wittmann, Barth, & Henle, 2008; Stephens et al., 2010). The observed strong relationship between the concentration of methylglyoxal and antibacterial activity of honeys of Leptospermum spp. origin pointed to this molecule as being the potential active principle for manuka honey. However, variability in the antibacterial activity that has been observed among honeys can not be solely related to the differences in the phytochemical composition. For example, differences in the antibacterial activity were observed within manuka honeys, ranging from totally inactive to highly active honeys, although these honeys all derived from the same Leptospermum spp. Thus, other factors and/or chemical processes might play a role in a final antibacterial potency. It has been long recognised that seasonal variations, post-harvest handling of honeys and their storage conditions influence quality of honey. In this study we assessed contribution of two factors to the variability in the antibacterial activity of Canadian honeys: botanical origin of honey and storage time. 2. Materials and methods 2.1. Honeys Honeys were donated by beekeepers and included both commercial (pasteurised) and apiary (raw) samples. Over 174 samples of honey were collected from the following Canadian provinces: Ontario-, Manitoba-, Alberta-, British Columbia during the seasons. Upon arrival at the laboratory, honey samples were assigned a number and analysed for colour, Brix, and ph. Honey samples were divided into portions and stored at room temperature in the dark Determination of honey colour Honey colour was determined spectrophotometrically by measuring the net absorbance at (A 560 A 720 )(Huidobro & Simal, 1984) Determination of Maillard reaction content The melanoidin content was assessed spectrophotometrically as net absorbance at (A 450 A 720 )(Ramonaityte, Keriene, Adams, Tehrani, & De Kimpe, 2009) using Ultrospec 3100 Pro, GE Healthcare. The melanoidin content was in absorption units (au) Determination of the content of UV-absorbing compounds in honey The quantitative and qualitative data on the levels of UVabsorbing compounds were obtained from the absorbance spectrum profiles of honey scanned at wavelengths nm. Honeys were two-fold serially diluted with water and the concentration of UV-absorbing compounds was determined from the area-under the curve (AUC) for each dilution. Absorbencies of the honey dilutions were analysed in 1 cm path-length quartz cells using a spectrophotometer Ultrospec 3100 Pro equipped with SWIFT II wavescan software. The concentrations were expressed in arbitrary units as the AUC units Antibacterial activity Standard strains of Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 14948) were purchased from Ward s Natural Science Ltd. (St. Catharines, ON, Canada) Preparation of working inoculum Each of the bacterial strains was inoculated into Mueller Hinton Broth (MHB)(Difco Laboratories) and incubated overnight in a shaking water bath at 37 C. Overnight culture were diluted with the broth to the equivalent of 0.5 MacFarland standard (approximately.10 8 cfu/ml) measured spectrophotometrically at A 600 nm Antibacterial assay The antibacterial activity of honeys was determined using a broth microdilution assay in a 96-well microplate format. Serial two-fold dilutions of honey were prepared by mixing 110 ul of honey with 110 ul of inoculated broth (10 6 cfu/ml final concentrations for each of microorganisms) and transferring from row A to row H of a microplate. Row G contained only inoculum and served as a positive control. Row H contained sterile MHB and served as a blank. After overnight incubation of plates at 37 C in a shaking waterbath, bacterial growth was measured at A 595 nm using the Synergy HT multi-detection microplate reader (Synergy HT, Bio-Tek Instruments, Winooski, Vt). The contribution of colour of honeys to the absorption was corrected by subtracting the absorbance of wells before (zero time) and after overnight incubation. Statistical analysis and dose response curve were obtained using K4 software Determination of minimum inhibitory concentration (MIC) The absorbance readings obtained from the dose-response curve were used to construct growth inhibition profiles (GIPs) using the following formula: %growth inhibition ¼ A control A experimental A control 100 The minimal inhibitory concentrations (MIC 90 ) were determined from the growth inhibition profiles curves and represented the lowest concentration of the honeys that inhibited the bacterial growth by 90% as measured by the absorbance at A 595 nm Monitoring changes in honeys during storage Upon arrival to the laboratory, honeys were divided into portions and stored at room temperature (approximately 24 C) in a dark, dry place for 1 3 years. Every 3 6 months, a portion of honey was analysed for its antibacterial activity, honey colour, the content of UV absorbing compounds and the content of the Maillard reaction products Statistical analysis Analyses were performed using the statistical program Graph- Pad Instat version (GraphPad Software Inc.). Data were analysed using a one-way ANOVA with subsequent Tukey Kramer Multiple Comparison test or an unpaired t-test. Differences between means were considered to be significant at p < 0.05.

4 K. Brudzynski, L. Kim / Food Chemistry 126 (2011) Results 3.1. Large-scale screening of Canadian honeys for antibacterial activity The purpose of large-scale screening of Canadian honeys of different botanical origin was two-fold: (1) to provide evidence that one of the main factors responsible for the observed variability in the antibacterial activity is the botanical source of honey, i.e. the differences in the chemical composition of nectars and (2) to identify botanical sources which would provide honey with high antibacterial activity in a reproducible way. Active honeys selected from the pool of screened honeys were used in further experiments to monitor changes in their activity during their storage. To define the link between antibacterial activity and floral variety of honeys, 177 Canadian honeys were screened for their bacteriostatic activities against E. coli (ATTC 14948) and B. subtilis (ATTC 6633) using a broth microdilution assay. The growth inhibitory action of honeys against these two representatives of Gram-positive and Gram-negative bacteria varied from MIC 90 of % v/v (Fig. 1A and B). To eliminate the non-specific contribution of osmolarity to the antibacterial action of honey, the MIC 90 values of honeys were compared to those of an artificial honey, (38.4% fructose and 30.3% glucose dissolved in sterile water). Since the artificial honey inhibited bacterial growth at MIC 90 25% v/v, the obtained results indicated that the bacteriostatic effect observed in low and medium active honeys (almost 60% of tested honeys) might simply result from honey osmolarity (Fig. 1A and B). Among honeys possessing higher MIC 90 values than that of a sugar solution, were buckwheat (37%), sweet clover (18%), blueberry (12%) and wildflower honeys (10%) (Fig. 2). The floral source of honey appeared to be the dominant factor in providing honey of high antibacterial activity. Therefore, these varietal differences in the antibacterial activity were interpreted as being due to variability in the content of phytochemicals influencing this activity. Importantly, we observed the differences in sensitivities of B. subtilis (Gram-positive) and E. coli (Gram-negative) bacteria to different honeys. B. subtilis was consistently less sensitive to the growth inhibitory action of Canadian honeys (Fig. 1). These data support our previous observations on different susceptibility of these two bacteria to Canadian honeys (Brudzynski, 2006). In contrast, manuka honeys of Leptospermum spp. origin showed higher bacteriostatic action against Gram-positive bacteria (Cooper et al., 2002; Lusby et al., 2005; Willix et al., 1992). Thus, these results suggest that Canadian honeys originating from buckwheat, sweet clover, blueberry and wildflower possess phytochemical(s) that inhibited bacterial growth. The observed differences in bacterial susceptibilities to honey actions suggest that phytochemicals involved in anti-proliferative effects on B. subtilis and E. coli, either are of different chemical nature or they differ in the mechanism of their action. Fig. 1. (A and B) Variability in the antibacterial activity of Canadian honeys originated from different botanical sources. Red colour: MIC 90 against E. coli, black colour: MIC 90 against B. subtilis. (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this papar.)

5 1158 K. Brudzynski, L. Kim / Food Chemistry 126 (2011) Fig. 2. Relationship between the level of antibacterial activity against E. coli and honey floral origin. Representation of honeys with MIC 90 values higher than that of a sugar solution. (MIC 90 < 25%) Reduction of antibacterial activity with honey storage The differences in the chemical compositions, i.e. the presence and concentration of active components, are not the only source of variability in antibacterial activity of honeys. We thought that the chemical stability of active components might be a critical factor for maintaining the activity at the same level during storage. To investigate this relationship, levels of the antibacterial activity of honeys were analysed every 3 6 months for a period of 1 3 years. The honeys used in this experiment included dark buckwheat honey #76 and #77, light buckwheat honey #23 and polyfloral honey #11. The initial antibacterial activity of these honeys was at MIC % v/v. As shown in Fig. 3, storage time had a detrimental effect on the activity. The antibacterial activity of honeys was rapidly reduced by 50% in the first 3 6 months of storage (Fig. 3). Further reduction of activity occurred more gradually over the period of months. These results demonstrated that the compounds responsible for growth inhibition were chemically unstable during storage. Significantly, the rate of the reduction of growth inhibitory activity of honeys against B. subtilis was consistently much faster than against E. coli. After three month of storage, B. subtilis cells were less responsive to growth inhibitors in honey than E. coli (Fig. 3). As in the cases of honey #11 (polyfloral) and #23 (light buckwheat), the growth inhibition of B. subtilis by honey was equal to that of the sugar solution (MIC 90 25% v/v) suggesting that the inhibitory effect of a hypothetical phytochemical was negligent after the first 3 6 months of storage. Thus, botanical origin of honey is a main source of variability in the antibacterial activity. However, the chemical instability of active components was the cause of the reduction of the activity over time in storage. These chemical changes regulated susceptibility of bacteria to honey, making E. coli less sensitive and B. subtilis insensitive to honey action Changes in honey colour, the UV absorbing compounds and Maillard reaction products/ melanoidins content with storage To shed some light on the chemical changes in honey during storage that affected honey antibacterial activity and bacterial susceptibility, we conducted a thorough investigation of changes in honey colour, content of UV absorbing compounds and the content of melanoidins (late-stage Maillard reaction products). Biological functions of honeys (antibacterial and antioxidant activities) have been frequently related to their colour. In a large number of reports, honeys of darker colour have been shown to possess higher antioxidant and antibacterial activities (Gheldof & Engeseth, 2002; Taormina et al., 2001; Martín, Hortigüela, Lozano, Cortina, & de Lorenzo Carretero, 2008; Brudzynski & Miotto, 2010a, 2010b). Colour of honeys results from the presence and concentration of compounds with several conjugated double bonds such as polyphenols, flavonoids, terpens, carotenoids that absorb light in visible range ( nm). In addition, Maillard reaction products (MRPs) contribute to the colour of honeys (Turkmen, Sari, Poyrazoglu, & Velioglu, 2006). The Maillard reaction or non-enzymatic browning is a common side effect of stored honeys. The late-stage MRPs have a characteristic absorption maximum between 420 and 450 nm. Polyphenols as well as intermediate- and late-stage MRPs also strongly absorb UV light in the range of A nm. In order to estimate quantitatively differences in the content of these groups of compounds in different honeys, we measured the changes which occurred between two time-points: at their arrival to the laboratory and after 2 years of storage. These parameters were analysed in selected honeys (Table 1) Changes in honey colour As shown in Fig. 4, a significant increase in honey colour, measured as net absorbance (A 560 A 720 ) was observed after two-year of storage (p < ). Relatively fewer differences in colour change were observed in light colour honeys (borage, fireweed, clover blend) as compared to darker honeys (pumpkin, sunflower, dandelion and blueberry) Changes in contents of UV absorbing compounds Given the difficulty in obtaining quick information on the differences in the chemical compositions of a large number of honey samples derived from different botanical sources, we explored applicability of UV spectroscopy for this purpose. The honey UV profile results from a summation of spectra from many different compounds, in which each of constituents contributes to the final UV

6 K. Brudzynski, L. Kim / Food Chemistry 126 (2011) Table 1 List of honeys, their plant sources and their initial colour. Honeys Plant source (Colour by absorption) A Visual colour 63 Fireweed Borage Clover blend Dandelion Blackberry Pumpkin Blueberry Fig. 3. Effect of honey storage on its antibacterial activity against E. coli and B. subtilis. Honeys: wildflower (#11), light buckwheat (#23), and two dark buckwheat honeys (#76, #77). Grey colour: MIC 90 against E. coli, black colour: MIC 90 against B. subtilis. absorbance profile. Honey scanning from 200 to 400 nm provided two characteristic patterns: complex, high absorbance UV spectra for medium and dark colour honeys, which are characterised by the high phenolic content, and simple two peak pattern for honeys of low phenolic content (manuscript in preparation). Due to limited transparencies of undiluted honeys, serial two-fold dilutions of each honey (2 to 64) were prepared and separately scanned. The total concentration of UV absorbing compounds was determined from the Area-Under-the Curve (AUC ) and arbitrarily expressed in AUC units. The linear relationship between the AUCs and the honey dilutions obtained for each honey indicated that there was a quantitative relationship between the absorbance and the concentration of UV absorbing compounds in honey samples. Therefore the honey solutions obeyed the Beer Lambert Law (Fig. 5). Comparison of AUCs of fresh and stored honeys showed an extremely significant increase in the content of UV absorbing compounds in the stored honeys (the two-tailed ANOVA, p < ) (Fig. 5). The amount of UV absorbing compounds doubled after the 2 years storage period and the rate of increase seemed to be fairly uniform to all honeys independent of their botanical source (mean 2.2 ± 0.17 SD, n = 28) (Fig. 5). The results point out to the chemical processes that spontaneously occurred during the 2 years storage. This process(s) apparently generated novel compounds of the high UV absorbance from the pool of existing substrates. Judging from the uniform two-fold maximal increase in the content of UV absorbing compounds, this storage-induced reaction possibly led to the complete substrate utilisation/exhaustion for a given honey MRPs/melanoidins content in stored honeys There is vast evidence in literature to demonstrate that the Maillard reaction and subsequent formation of melanoidins is a major cause of non-enzymatic browning in thermally processed

7 1160 K. Brudzynski, L. Kim / Food Chemistry 126 (2011) Fig. 4. Changes in the colour of honey after the 2-years storage period. List of honey varieties are included in Table 1. Fig. 5. Changes in the content of UV absorbing compounds in fresh honeys and after the 2-years storage period. List of honey varieties are included in Table 1. and stored foods. Browning of honey upon storage is a well known phenomenon that adversely affect honey quality and consumer acceptance. We therefore examined stored honeys for the presence of melanoidins. As shown in Fig. 6, all honeys absorbed at A 450 nm but to a differing degree. Lighter colour honeys showed a much lesser content of melanoidins than did darker honeys (Fig. 6). These results are in agreement with data of others in that the formation of the intermediate and advanced Maillard reaction products is concomitant with the appearance of compounds exhibiting a high UV absorbance and having characteristic absorbance maxima at A nm (for review Martins, Jongen, & van Boekel, 2001). Moreover, we have recently reported a functional link between the formation of melanoidins in unheated honeys and the antibacterial and antioxidant activities (Brudzynski & Miotto, 2010a, 2010b). Together, our data indicate that during storage honey undergoes marked chemical changes including a change to darker colours, increased production of UV absorbing compounds and appearance of melanoidins Changes in the antibacterial activity of stored honeys To determine whether the chemical changes observed in the above stored honeys affected their antibacterial activity, we compared the MICs and growth inhibition profiles of fresh honeys to those obtained after 2 years in storage. The growth inhibition profiles of fresh honeys are presented in Fig. 7. A variation in the antibacterial activity with floral source was observed. Darker honeys (#65, #66, #67) exhibited antibacterial activity against both E. coli and B. subtilis at MIC %, which is definitively higher than that of a sugar solution. As seen in Fig. 7A and B, a serial dilution of honeys gradually reduced growth inhibitory action against E. coli. However, it caused an abrupt decrease in the inhibitory action against B. subtilis. The results provide additional strong evidence that the nature of compounds or mechanism of growth inhibition by honey is different against Gram-negative E. coli and Gram-positive B. subtilis. The same honeys analysed after 2 years in storage showed a drastic reduction of their antibacterial potencies. None of these

8 K. Brudzynski, L. Kim / Food Chemistry 126 (2011) Fig. 6. Melanoidin content in honeys after the 2-year storage period. L-liquid portion of honey, C-crystallised portion of honey. Labels A, C and D of honey #68 indicate three samples of different clover honeys. Fig. 7. Initial antibacterial activity of honeys #62 to #68. (A) Against E. coli and (B) B. subtilis. stored honeys inhibited bacterial growths with higher efficiency than a sugar solution (Fig. 8A and B). Moreover, higher dilutions of stored honeys (16 to 64) become stimulatory to the growth of B. subtilis. Indeed, the chemical changes induced by storage were detrimental to honey antibacterial activity. The results of this study provided new insight into factors and possible mechanisms that influenced the level of antibacterial activity of Canadian honeys

9 1162 K. Brudzynski, L. Kim / Food Chemistry 126 (2011) Fig. 8. Antibacterial activity of honeys #62 to #68 after the 2-years storage period. (A) Against E. coli, (B) against B. subtilis. and affected susceptibility of E. coli and B. subtilis to honey antibacterial action. There is scarce and sometimes contradictory literature on the levels of the antibacterial activity of honey during storage. Some studies reported that the exposure of honey to heat (White & Subers, 1964) or its prolonged storage resulted in loss of antibacterial activity (Radwan, El-Essawy, & Sarham, 1984) while in other studies, no correlation was found between the age of honey and the stability of the antibacterial activity (Allen et al., 1991; Rios, Novoa, & Vit, 2001). The phytochemical composition of honeys seemed to affect the antibacterial activity of honey much more significantly than storage conditions (Allen et al., 1991). Apparently, variability in the antibacterial activity might be related to the presence/concentration of active phytochemicals and their sensitivity to storage conditions. Our data show that the predominant active compound conferring bacteriostatic activity is a phytochemical(s) present in certain honey varieties. The chemical stability of the active phytochemical influenced honey functional efficacy. Storage-induced changes to the structure of active principal components coincided with partial loss of E. coli sensitivity and B. subtilis resistance to honey action. Thus, the chemical stability of the active compounds has been proposed here to be one of the main sources of variability in antibacterial activity. These findings will increase awareness of honey as a storage-sensitive product in such applications as a functional food and/or as an antibacterial agent that will prolong shelf- life of other food products. 4. Conclusions Our data indicates that the predominant active compound conferring bacteriostatic activity is a phytochemical(s) present in certain honey varieties such as buckwheat, sweet clover, blueberry and wildflower. The chemical instability of these active principal components played a major role in the reduction of the antibacterial activity of honey during storage. Storage-induced changes resulted in de-regulation of the antibacterial activity: a partial loss of E. coli sensitivity and B. subtilis resistance to honey action. The de-regulation of the antibacterial activity coincided with the formation of melanoidins, suggesting that compounds conferring antibacterial activity could be sequestered into melanoidin aggregates, losing their function.

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