Food Chemistry 192 (2016) 1006–1014

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Fluorescence markers in some New Zealand honeys Jessie Bong a, Kerry M. Loomes a, Ralf C. Schlothauer a,b, Jonathan M. Stephens a,b,⇑ a b

School of Biological Sciences and Institute for Innovation in Biotechnology, University of Auckland, PB92019 Auckland, New Zealand Comvita NZ Limited, Wilson South Road, Paengaroa, PB1, Te Puke, New Zealand

a r t i c l e

i n f o

Article history: Received 24 March 2015 Received in revised form 21 July 2015 Accepted 23 July 2015 Available online 26 July 2015 Keywords: Fluorescence Manuka (Leptospermum scoparium) Kanuka (Kunzea ericoides) Honey Nectar

a b s t r a c t The fluorescence characteristics of various New Zealand honeys were investigated to establish if this technique might detect signatures unique to manuka (Leptospermum scoparium) and kanuka (Kunzea ericoides) honeys. We found unique fluorescence profiles for these honeys which distinguished them from other New Zealand honey floral types. Two excitation–emission (ex–em) marker wavelengths each for manuka and kanuka honeys were identified; manuka honey at 270–365 (MM1) and 330–470 (MM2) nm and kanuka honey at 275–305 (KM1) and 445–525 (KM2) nm. Dilution of manuka and kanuka honeys with other honey types that did not possess these fluorescence profiles resulted in a proportional reduction in fluorescence signal of the honeys at the marker wavelengths. By comparison, rewarewa (Knightia excelsa), kamahi (Weinmannia racemosa), and clover (Trifolium spp.) honeys did not exhibit unique fluorescence patterns. These findings suggests that a fluorescence-based screening approach has potential utility for determining the monoflorality status of manuka and kanuka honeys. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Honey is a complex and supersaturated sugar solution comprising approximately 80% sugars and a unique combination of other compounds suspended in water (White & Doner, 1980). The sugar component of honey is comprised principally of the monosaccharides, fructose and glucose. The non-sugar proportion comprises a range of plant- or bee-derived compounds such as organic acids, proteins, amino acids, enzymes, pollen, pigments, mineral salts, wax, and plant secondary metabolites (Anklam, 1998; White & Doner, 1980). The chemical composition of honey varies between honey floral types but may also be influenced by geographical origin, climate (Anklam, 1998), honey processing and age (Stephens et al., 2015). A honey can be monofloral or polyfloral in origin depending on whether it is derived from one or several plant species. According to international food standards, in order for a honey to be labelled with floral origin, it must originate wholly or predominantly from a particular floral source and display the corresponding organoleptic, physico-chemical, and microscopic properties (Codex Alimentarius Commission., 2001). It is generally accepted that honey produced in a natural environment containing mixed plant species is never

⇑ Corresponding author at: School of Biological Sciences and Institute for Innovation in Biotechnology, University of Auckland, PB92019 Auckland, New Zealand. E-mail address: [email protected] (J.M. Stephens). http://dx.doi.org/10.1016/j.foodchem.2015.07.118 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

monofloral as it is impossible to control honey bee behaviour in the forage field (Winston, 1987). It is therefore difficult to produce scientifically pure monofloral honeys. The co-existence of numerous floral species that produce surplus nectar in the same geographical region and flower together are exemplified by the Leptospermum scoparium and Kunzea ericoides populations in New Zealand (Stephens et al., 2010). The floral origin of honey is a major determinant of premium value. Monofloral honeys typically command a higher value than polyfloral honeys as they exhibit distinct flavour and quality attributes that are not present to the same extent in the polyfloral types. Certain monofloral honey types also have a greater retail value than others. The New Zealand manuka (L. scoparium) and Yemen sidr (Ziziphus spina-christi) honeys are examples of honeys traded at a premium worldwide due to their reported health benefits. Where a particular floral source commands a higher market value, an incentive exists to attribute that nectar source over others. Consumer expectation for true-to-label honeys as well as a concern over the authenticity of New Zealand premium honey products have identified a need for reliable and reproducible methods for determining honey monoflorality. The current standard reference method to ascertain honey floral types is melissopalynology based on microscopic identification and quantification of pollen composition (Jones & Bryant, 1992; Louveaux, Maurizio, & Vorwohl, 1978). However, some pollen grains are difficult to identify accurately, and in the case of New Zealand manuka (L. scoparium) and kanuka (K. ericoides) honeys,

1007

J. Bong et al. / Food Chemistry 192 (2016) 1006–1014

the pollens of these species are virtually indistinguishable in a honey medium due to their close resemblance (Moar, 1985). Furthermore, pollen count also does not always accurately represent nectar contribution of a particular floral species in honey because the floral structure of certain plant species, such as the New Zealand rewarewa (Knightia excelsa), allows honey bees to collect nectar without transferring the pollen grains (Moar, 1985). Physico-chemical and sensory analysis are routinely used in conjunction with melissopalynology for characterisation of honey floral origin (Bogdanov, Ruoff, & Persano Oddo, 2004; Piana et al., 2004). Some honey types are also characterised by the presence of unique chemical compounds enabling chemical fingerprinting to determine floral origin. Manuka honey, for instance, is characterised by the presence of the dihydroxyacetone (DHA) and methylglyoxal (MGO) which are unique to the Leptospermum genus (Stephens et al., 2010; Windsor, Pappalardo, Brooks, Williams, & Manley-Harris, 2012) and also elevated concentration of the phenolic compound 2-methoxybenzoic acid (Beitlich, Koelling-Speer, Oelschlaegel, & Speer, 2014; Senanayake, 2006; Stephens et al., 2010). More recently, a novel glycoside of methyl syringate, leptosperin, has been proposed as a potential chemical marker for manuka honey (Fearnley et al., 2012; Kato et al., 2012, 2014; Oelschlaegel et al., 2012). Kanuka honey appeared to be characterised by elevated concentrations of 4-methoxyphenyllactic acid and methyl syringate (Beitlich et al., 2014; Senanayake, 2006; Stephens et al., 2010). Pasture and other pale-coloured honey types, in contrast, contain low level of phenolic and polyphenolic compounds (Stephens et al., 2010; Tan, Holland, Wilkins, & Molan, 1988). Application of fluorescence spectroscopy in food analysis is becoming increasingly popular and has been demonstrated to be capable of characterising foods such as milk (Kulmyrzaev & Dufour, 2002; Kulmyrzaev, Levieux, & Dufour, 2005), cheeses (Karoui, Bosset, Mazerolles, Kulmyrzaev, & Dufour, 2005; Karoui et al., 2004), cereals (Karoui, Cartaud, & Dufour, 2006), and honeys (Aitkenhead, Rosendale, Schlothauer, & Stephens, 2014; Gebala & Przybylowski, 2010; Ghosh, Verma, Majumder, & Gupta, 2005;

Karoui, Dufour, Bosset, & De Baerdemaeker, 2007; Lenhardt, Bro, Zekovic´, Dramic´anin, & Dramic´anin, 2015; Ruoff et al., 2006). The fluorescence property of honey is attributed to the presence of phenolic and polyphenolic compounds (Aitkenhead et al., 2014; Gebala & Przybylowski, 2010; Ghosh et al., 2005; Karoui et al., 2007; Lenhardt et al., 2015; Ruoff et al., 2006), aromatic amino acids (Karoui et al., 2007; Lenhardt et al., 2015; Ruoff et al., 2006), and Maillard reaction products (Karoui et al., 2007; Lenhardt et al., 2015). Phenolic and polyphenolic compounds are good indicators of honey botanical and geographical origin (Andrade, Ferreres, & Amaral, 1997; Stephens et al., 2010; Tomás-Barberán, Martos, Ferreres, Radovic, & Anklam, 2001; Yao et al., 2003). In addition to the characterisation and classification of honeys, fluorescence spectroscopy could also potentially detect unique intrinsic fluorophores and their relative concentrations, and inform on the physico-chemical parameters of the honey matrix (Lenhardt et al., 2015). The distinctive phenolic and polyphenolic composition in New Zealand honeys, coupled to the high sensitivity of fluorescence spectroscopy, might therefore be expected to generate unique excitation–emission (ex–em) spectra identifiable to the individual honey types. Dilution of a honey by other honey floral types may result in a proportional change in the chemical composition of the honey and thus the fluorescence signal. We hypothesised that fluorescence arising from the unique chemical composition of New Zealand manuka and kanuka honeys might have utility to determine the relative floral contributions and therefore monoflorality. The aim of this study was to examine the fluorescence characteristics of New Zealand honeys and establish a fluorescence screening method for estimating their monoflorality based on fluorescence profiles. Instead of generating full spectra for honeys, this study selectively identified unique ex–em wavelength pairs to screen a range of high-value New Zealand honeys. This selective approach greatly reduced the complexity of the scanning technique by isolating and targeting only characteristic ex–em wavelengths. Consequently, the analysis time was minimised making it more suitable for commercial applications that often involve screening a large number of samples.

Table 1 Representative New Zealand honeys, propolis, and nectar samples. Sample

Honey/nectar

Geographic origin

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Manuka (Leptospermum scoparium) Manuka (L. scoparium) Manuka (L. scoparium) Manuka (L. scoparium) Manuka (L. scoparium) Manuka (L. scoparium) Manuka (L. scoparium) Manuka (L. scoparium) Kanuka (Kunzea ericoides) Kanuka (K. ericoides) Kanuka (K. ericoides) Kanuka (K. ericoides) Kanuka (K. ericoides) Rewarewa (Knightia excelsa) Rewarewa (K. excelsa) Kamahi (Weinmannia racemosa) Kamahi (W. racemosa) Clover (Trifolium spp.) Clover (Trifolium spp.) Clover (Trifolium spp.) Propolis (botanical source varies) Nectar (L. scoparium) Nectar (L. scoparium) Nectar (L. scoparium) Nectar (L. scoparium) Nectar (L. scoparium)

Northland Northland Northland Waikato Wetlands Waikato Wetlands East Coast East Coast Central North Island Northland Northland Northland Northland Waikato Wetlands Bay of Plenty, North Island North Island South Island North Island South Island South Island North Island New Zealand Collected, Bay of Plenty, North Collected, Bay of Plenty, North Collected, Bay of Plenty, North Collected, Bay of Plenty, North Collected, Bay of Plenty, North

Island Island Island Island Island

1008

J. Bong et al. / Food Chemistry 192 (2016) 1006–1014

2. Materials and methods 2.1. Materials Honey, nectar, and propolis samples (Table 1) were kindly supplied by Comvita NZ Ltd. The honeys had been extracted and collected by manual scraping of the honey and wax on freshly capped frames followed by straining through a muslin cloth. These samples were approximately six-months old at collection. The florality status was established from floral-source field site analysis during collection, the presence of diagnostic compounds such as DHA and MGO, and phenolic composition. To prevent changes in chemical composition, the samples were stored at 4 °C until analysis. The nectars were obtained by direct sampling using micropipette (Stephens et al., 2010). In brief, nectar droplets secreted onto the surface of L. scoparium flowers were drawn up and dispensed into a 1.5 ml Eppendorf tube, and sampling was repeated on flowers of the same plant to obtain approximately 100 ll of nectar volume for each sample. To prevent fermentation, the samples were kept in a portable freezer unit during transportation from field site, and transferred to a freezer (20 °C) for storage until analysis. Propolis was supplied as dry propolis solids, and stored at 4 °C until analysis. 2.2. Chemical standards 1,3-Dihydroxyacetone (DHA, 97%) and methylglyoxal (MGO, 40% in H2O) were purchased from Sigma–Aldrich (St. Louis, MO, USA). 4-Methoxyphenyllactic acid was synthesised by Hangzhou Sage Chemical Co. Limited (Hangzhou, China). 2.3. Sample preparation Prior to analysis, all samples were equilibrated to room temperature. Honey samples were thoroughly mixed to ensure homogeneity. A subsample of the well-mixed honey was weighed out and diluted with distilled water to a 2% w/v testing concentration (Aitkenhead et al., 2014). Due to the high viscosity of honey and difficulty of accurately measuring a small amount of honey, the water volume was adjusted to achieve the desired honey concentration. Propolis solution was prepared by solubilising 10 mg dry propolis solids in 10 ml of 70% w/v ethanol, followed by a 200-fold dilution with distilled water to give a 5 mg/L propolis in 0.35% aqueous ethanol solution. Nectar samples were measured for sugar content using a handheld refractometer (Eclipse 45–03; Bellingham + Stanley Inc.) and diluted by normalisation to the sugar content relative to 2% w/v honey solution. An aliquot of each sample (100 ll) was pipetted into the microplate wells (Optiplate™-384, black) in duplicate unless otherwise specified. Plates containing honey samples were assessed promptly to avoid the effects of photo-bleaching and evaporative losses. Fresh honey samples were prepared daily as honeys, once diluted, are prone to fermentation at room temperature. 2.4. Fluorescence spectroscopy Fluorescence measurement was carried out in a commercial spectrofluorometer (Gemini EM Dual-Scanning Microplate Spectrofluorometer; Molecular Devices Inc.) coupled to an external computer equipped with SoftMaxÒ Pro Software. Instrumental settings for fluorescence measurements were set up using SoftMaxÒ Pro. A fluorescence top read was selected for better signal-to-noise ratio. Initial fluorescence screening used spectrum scans to establish the set of wavelengths unique to individual

honeys. Once wavelengths were determined, endpoint scans were used for targeted measurement of fluorescence at the identified ex–em wavelengths. Temperature control for microplate chamber was set to ambient laboratory temperature. Auto calibration was activated to enable automatic pre-read calibration for the instrument and the photomultiplier tube (PMT) sensitivity was set to automatic.

2.5. Spectrum analysis Table 1 lists the representative New Zealand honey, propolis, and nectar samples analysed to establish fingerprinting fluorescence baselines for the different New Zealand honey types. Spectral emission scans were carried out at excitation wavelengths ranging from 250 nm to 710 nm with a 20 nm increment at each subsequent scan. The emission wavelength was set to range from a wavelength 20 nm above the excitation wavelength to 850 nm with 20 nm incremental steps between readings. The resulting peaks were then refined to select for discrete ex–em wavelength pairs with the strongest signals. At the selected excitation wavelengths, emission scans of honey samples were carried out at 5 nm increments to select for the maximum emission wavelength. This process was followed by 5 nm incremental excitation scans at the resulting maximum emission wavelength to select for the maximum excitation wavelength.

2.6. Variability in fluorescence signal In order to ensure that the fluorometer responded in a similar manner between scans of different plates, a reference honey standard was incorporated into all scans. In this case, a well-homogenised monofloral manuka honey (sample 1 in Table 1) was selected as the reference honey. Two replicates were incorporated into each scan and the mean fluorescence was compared to determine fluorescence variability.

2.7. Blending experiment Using the candidate honeys selected from initial spectrum analysis, representative honeys for the different floral sources were constructed by combining selected honeys of the same floral origin in equal proportion. A dilution series was performed on manuka and kanuka honeys with honeys of other floral sources to examine the effect of honey monoflorality on fluorescence.

2.8. Supplementation of 4-methoxyphenyllactic acid into honey A 10 mg/ml stock solution 4-methoxyphenyllactic acid was prepared in distilled water, and diluted serially to the range of concentrations present in 4% w/v honey solutions. A 1:1 dilution was carried out in 4% w/v clover honey solutions that did not contain detectable level of endogenous 4-methoxyphenyllactic acid to obtain 2% w/v clover honey solutions with the corresponding range of supplemented 4-methoxyphenyllactic acid concentrations.

2.9. Statistical analysis Statistical analysis of data was performed using the software Graphpad Prism (Version 5.03). Correlation was analysed by regression analysis. Differences between correlations were determined by slope and intercept comparison.

J. Bong et al. / Food Chemistry 192 (2016) 1006–1014

1009

Fig. 1. (A and B) Emission spectra of Northland (—), Waikato Wetlands (—), and East Coast (- - -) manuka honeys, and Leptospermum scoparium nectar (    ) at 270 and 330 nm ex respectively. (C) Emission spectra of rewarewa (--), kamahi (--), clover (  ), and propolis (  ) at 270 nm ex. (D) Emission spectra of kanuka (- - -), rewarewa (--), kamahi (--), clover (  ), and propolis (  ) at 330 nm ex. (E) Emission spectra of kanuka honey (- - -) at 270 nm ex. (F) Emission spectra of Northland (—), Waikato Wetlands (—), and East Coast manuka (- - -), kanuka (- - -), rewarewa (--), kamahi (--), clover (  ), and propolis (  ) at 430 nm ex. (M = manuka specific peak, K = kanuka specific peak, L = L. scoparium nectar peak, X = non-diagnostic peak.) Data shows mean spectra from measurements performed in duplicate, except for nectar samples 25 and 26.

3. Results and discussion 3.1. Variability in fluorescence signal Examination of the reference honey demonstrated repeatability of this method. A total of 43 scans were completed in duplicate for this honey and the data revealed ±5% variability in fluorescence signal between scans, with a mean fluorescence of 11,359 ± 513 RFU (n = 2 replicates, ±1 standard deviation) at the manuka marker

wavelength identified in this study (270–365 nm ex–em). This variability was consistent throughout all scans, and was evident in smaller sample sets as well as larger sample sets. 3.2. Honey emission spectrum Initial fluorescence screening was performed on a range of New Zealand honeys, propolis, and nectars as listed in Table 1. The honey samples provided a good coverage of the major commercial

1010

J. Bong et al. / Food Chemistry 192 (2016) 1006–1014

honey production locations in New Zealand, and were employed as representative honey samples to establish fingerprinting fluorescence baselines for the different monofloral honey types. Analyses showed that manuka and kanuka honeys displayed distinct fluorescence patterns that could potentially distinguish them from each other as well as other honey types (Fig. 1). These distinctive signatures were identified as unique fluorescence peaks in the emission spectra. For a fluorescence emission peak to be diagnostic it must be exclusive to that honey and the peak size should be sufficiently large to be distinguishable from background noise interference. All the manuka honey samples exhibited a prominent fluorescence peak at 270–370 and 330–470 nm ex–em (Fig. 1A and B). The signal intensity at 270–370 nm ex–em was the strongest, and the Northland manuka honeys produced the greatest intensity at both wavelength pairs. At the 270–370 nm ex–em, the Waikato Wetlands manuka honeys exhibited greater fluorescence than the East Coast manuka honeys; whereas at the 330–470 nm ex–em, the reverse was observed. These findings suggested that there are distinct fluorophores contributing to the fluorescence intensities at these ex–em wavelengths and which could vary depending on the region. Examination of the L. scoparium nectars revealed identical fluorescence emission spectra to that of manuka honey with a peak at 270–370 and 330–470 nm ex–em (Fig. 1A and B). The mean fluorescence yield of L. scoparium nectar was greater than that of a monofloral manuka honey at the 270–370 nm ex–em wavelength pair. Variability was observed in the fluorescence intensity exhibited by the individual L. scoparium nectar samples at both ex–em wavelength pairs. However, nectars have been documented to differ greatly in their physico-chemical characteristics (Nicolson & Thornburg, 2007) and the natural ability of L. scoparium plants to produce nectars that give rise to honeys with different levels of non-peroxide antibacterial activity reinforces this finding (Stephens, 2006). Fig. 1C and D show the comparative lack of fluorescence from rewarewa, kamahi, and clover honeys at the wavelengths of the manuka diagnostic peaks. The kanuka honeys exhibited a peak at 270–310 nm ex–em (Fig. 1E) which was distinct from the manuka peak, and at 430–530 nm ex–em (Fig. 1F). There was a slight elevation in fluorescence spectra of the pasture and forest type honeys as well as propolis at the 270–330 nm ex–em (Fig. 1C) but this was clearly distinguishable from the kanuka peak. It was likely that this slight elevation at the 270–330 nm ex–em was present in the manuka and kanuka spectra as well, but was masked by the greater signal from the manuka and kanuka diagnostic peaks (Fig. 1C). Propolis was incorporated in this study as a source of flavonoids, and was found not to exhibit a significant fluorescence pattern (Fig. 1C–F). Flavonoids in honey may be propolis-derived and therefore are not suitable as markers for honey floral origin (Tomás-Barberán et al., 2001). As flavonoids can fluoresce (Ghosh et al., 2005) it was necessary to identify and distinguish these peaks, if any, and exclude them in predicting the monoflorality status of honeys. Instead of individual flavonoid standards, New Zealand propolis comprising a mixture of flavonoids was examined because flavonoids are often present in honeys as a mixture rather than a single component. The fluorescence spectra yielded by the presence of multiple compounds in the mixture may be different from those of the individual compounds (Lakowicz, 2006). However, our findings showed that there was an absence of detectable fluorescence in propolis examined in this study suggesting that any flavonoid contribution was negligible (Ghosh et al., 2005). The emission bands at 270–530, 330–650, and 430–850 nm ex–em observed in Fig. 1 (indicated by X) were non-diagnostic as they were present in the spectra of all honeys examined. Furthermore, these peaks were observed in all scans including

the blank samples and was likely to be a background interference arising from reflection or autofluorescence of the wells. The emission band also showed a shift in emission pattern across the range of excitation wavelength examined and was characterised by an offset of 40 nm towards higher wavelengths at each subsequent scan. According to Lakowicz (2006), emission band shifts across varying excitation wavelengths are most probably due to interference and therefore, should not be interpreted as fluorescence arising from the sample. The position of a fluorescence peak on an emission spectrum is independent of the excitation wavelength provided the solvent is the same (Lakowicz, 2006). The unique fluorescence pattern exhibited by manuka and kanuka honeys and the absence of fluorescence from propolis suggested that manuka and kanuka honeys contain unique fluorophores that are unlikely to be propolis-derived flavonoids. The identical emission spectra exhibited by L. scoparium nectar and manuka honey strongly suggested that the fluorophores giving rise to fluorescence in manuka honey are most probably nectar-derived. It is possible that the fluorophores in kanuka honeys are also associated with the floral source, however examination of K. ericoides nectars would be necessary for confirmation. 3.3. Characteristic marker wavelengths for manuka and kanuka honeys In order to establish the discrete ex–em wavelengths that could be used as markers for manuka and kanuka honeys, the diagnostic peaks for these honeys were resolved by 5 nm increment excitation and emission scans. This experiment successfully identified four ex–em wavelength pairs specific to manuka and kanuka honeys; the former honey at 270–365 and 330–470 nm ex–em designated as MM1 and MM2 respectively, and the latter honey at 275–305 and 445–525 nm ex–em designated as KM1 and KM2 respectively (Table 2). It was also established in this study that the absence of signal would indicate honey types other than manuka and kanuka honeys. 3.4. Fluorescence and honey monoflorality The relationship between honey monoflorality on fluorescence was examined in manuka and kanuka honeys using the best candidate monofloral honeys selected from the initial spectrum analysis. The use of highly monofloral honeys in this study was considered essential to produce the most accurate and representative fluorescence profiles. The correlation between fluorescence and honey dilution was determined by fluorescence scanning at the established marker wavelengths. Honey monoflorality in this experiment was expressed in terms of percentage honey content (%) whereby a higher percentage of a given honey represents a higher content contribution from the nominal honey. There was a strong correlation between fluorescence and Northland, Waikato Wetlands, and East Coast manuka content at both manuka marker wavelength pairs, MM1 (270–365 nm ex–em) and MM2 (330–470 nm ex–em). The dilution of a particular manuka honey with kanuka, rewarewa, kamahi, and clover honeys at these marker wavelengths resulted in a decrease in Table 2 Summary of fluorescence marker wavelengths for New Zealand manuka and kanuka honeys. Marker wavelengths are expressed as excitation–emission (ex–em) wavelength pairs. Honey

Marker wavelength (nm)

Nomenclature

Manuka

270–365 330–470

MM1 MM2

Kanuka

275–305 445–525

KM1 KM2

J. Bong et al. / Food Chemistry 192 (2016) 1006–1014

Fig. 2. (A) Dilution of Northland manuka honey by kanuka (d), clover (h), and kamahi (N) honeys at MM1 (270–365 nm ex–em). (B) Combined data for dilution of Northland, Waikato Wetlands, and East Coast manuka honeys () by kanuka, clover, kamahi, and rewarewa honeys at MM1; ycombined = 88.0x + 1594.2. (C) Correlation between fluorescence and Northland (), Waikato Wetlands (.), and East Coast (j) manuka honeys at MM2 (330–470 nm ex–em); yNorthland = 31.0x + 466.5, yWaikato Wetlands = 16.2x + 384.6, yEast Coast = 26.2x + 338.2. Dilution of all manuka honeys by kanuka, clover, kamahi, and rewarewa honeys resulted in a proportional decrease in fluorescence signal irrespective of the dilutant honey type. Data shows mean ± standard error mean from measurements performed in duplicate.

1011

Fig. 3. (A) Dilution of kanuka honey by Northland (d), Waikato Wetlands (h), and East Coast manuka (N), clover (s), and kamahi () honeys at KM1 (275–305 nm ex–em). The dilutant honey type did not have any effect on the correlation between fluorescence and kanuka content. (B and C) Illustrate dilution of kanuka honeys by manuka, clover, kamahi, and rewarewa honeys combined () at KM1 and KM2 (445–525 nm ex–em) respectively; yKM1 = 52.1x + 1390.6, yKM2 = 3.7x + 34.9. Data shows mean ± standard error mean from measurements performed in duplicate.

1012

J. Bong et al. / Food Chemistry 192 (2016) 1006–1014

fluorescence intensity proportional to the extent of dilution irrespective of the dilutant honey type (p > 0.05). Fig. 2A illustrates the correlation between fluorescence and Northland manuka honey content at MM1. Similar observation was found for Waikato Wetlands and East Coast manuka honeys (data not shown). The fluorescence behaviour of manuka honey at MM1 was also consistent for the three regional areas examined (Fig. 2B). There were no statistically significant differences in the correlations between fluorescence and honey content for manuka honeys from these three regions at MM1 (p > 0.05) and therefore the data were combined. However, there appeared to be a regional influence on the fluorescence behaviour of manuka honeys at MM2 (Fig. 2C). The differences between individual correlations for Northland, Waikato Wetlands, and East Coast manuka honeys at MM2 were statistically significant (p < 0.0001). The establishment of a regional database for fluorescence behaviours of manuka honeys at this marker wavelength based on a larger sample size would be necessary to delineate these regional differences. As with manuka honey, the type of dilutant honey did not affect the correlation between fluorescence signal and kanuka content at both KM1 (275–305 nm ex–em) and KM2 (445–525 nm ex–em) (p > 0.05). Fig. 3A shows the effect of non-kanuka honey type dilution on kanuka fluorescence at KM1. The relationship between fluorescence and kanuka content at both KM1 and KM2 are summarised in Fig. 3B and C. In the case of manuka dilution with kanuka and vice versa, a decline in fluorescence yield at one honey type marker wavelengths resulted in a proportional intensification at the other honey type marker wavelengths. For instance, a pure Northland manuka honey diluted 50% with kanuka honey would result in half the honey fluorescence intensity at MM1 and MM2 but an increase in fluorescence at KM1 and KM2 relative to that of a 50% kanuka honey. The distinctive fluorescence patterns exhibited by diluted manuka and kanuka honeys, and the lack of fluorescence in rewarewa, kamahi, and clover honeys, formed the basis for a fluorescence-based determination of the monoflorality status of manuka and kanuka honeys. Currently, the principal initial determinant of monoflorality for the honey crop in New Zealand relies upon a hive site’s history, the surrounding vegetation, and the main nectar flow in relation to plant flowering. Despite beekeepers’ efforts to locate hives in sites that will yield monofloral honeys, it is not possible to control honey bee behaviour in the field and prevent collection of less desirable nectars. Our findings suggest that the use of fluorescence is limited to the determination of the amount of manuka and kanuka-derived nectar components in a honey. The proportion of nectar contribution from other New Zealand floral sources such as rewarewa, kamahi, and clover could only be established collectively as ‘‘contribution from other honey types’’. The individual contribution from these floral types could not be identified as they did not exhibit unique fluorescence characteristics. Therefore, the differentiation of New Zealand honeys based on current method was limited to manuka, kanuka, and other honey types. The fluorescence-based monoflorality determination method established in the present study is based on the presence of putative fluorophores unique to manuka and kanuka honeys. Dilution of manuka and kanuka honeys by other non-manuka and kanuka honey types resulted in a proportional change in fluorescence intensity at the established marker wavelengths. These findings show that fluorescence could be used potentially to determine the degree of honey monoflorality.

Fig. 4. (A) Correlation between 4-methoxyphenyllactic acid (4-MP) concentration in honey and fluorescence at KM1 (275–305 nm excitation–emission). (B) Fluorescence of 4-MP supplemented into a clover honey matrix (4) compared to fluorescence of endogenous 4-MP in honey (d) at KM1. Data shows mean ± standard error mean from measurements performed in duplicate. (C) Fluorescence emission spectra of a monofloral kanuka honey (—) and a clover honey supplemented with 4-methoxyphenyllactic acid chemical standard (  ) at 275 nm excitation. Both spectra were identical, with a peak at 305 nm emission corresponding to KM1 diagnostic peak (indicated by K). X indicates non-diagnostic peaks. Data shows mean spectra from measurements performed in duplicate.

J. Bong et al. / Food Chemistry 192 (2016) 1006–1014

3.5. Potential fluorophores We correlated fluorescence at the identified fluorescence marker wavelengths with concentrations of DHA, MGO, and phenolic compounds that have been reported in manuka and kanuka honeys. We subjected a set of 64 New Zealand honeys with known concentrations of DHA, MGO, 2-methoxybenzoic acid, methyl syringate, phenyllactic acid, 4-methoxyphenyllactic acid, 4-methoxybenzoic acid, and syringic acid to a fluorescence assay at both manuka (MM1 and MM2) and kanuka (KM1 and KM2) marker wavelengths. Although DHA and MGO appeared to be correlated with fluorescence at the manuka marker wavelengths MM1 and MM2 (data not shown), our fluorescence assay showed that these compounds in fact do not fluoresce. We also found no correlation between 2-methoxybenzoic acid, methyl syringate, phenyllactic acid, 4-methoxybenzoic acid, and syringic acid concentrations and fluorescence at all four marker wavelength pairs (data not shown). However, there was a strong linear correlation between 4-methoxyphenyllactic acid concentration and fluorescence at the kanuka marker wavelength KM1 (R2 = 0.9456, Fig. 4A), suggesting that 4-methoxyphenyllactic acid may contribute to fluorescence at this fluorescence marker wavelength. To examine the contribution of 4-methoxyphenyllactic acid in more detail, we supplemented a clover honey with 4-methoxyphenyllactic acid chemical standard within the concentration range we observed in various honeys (Fig. 4B). The clover honey in this case did not contain detectable level of endogenous 4-methoxyphenyllactic acid nor did it exhibit elevated fluorescence at the KM1 marker wavelength. The resulting fluorescence measurements showed a linear positive correlation between the supplemented 4-methoxyphenyllactic acid concentration and fluorescence at the KM1 marker wavelength. The signal intensity was also comparable to that observed for honeys with endogenous level of 4-methoxyphenyllactic acid (Fig. 4B), strongly suggesting that the KM1 fluorescence is substantially due to 4-methoxyphenyllactic acid. To further confirm that 4-methoxyphenyllactic may be the sole fluorophore responsible, we examined the fluorescence emission spectrum of a 4-methoxyphenyllactic acid chemical standard at 275 nm excitation. The spectral analysis of this compound in a clover honey matrix revealed a fluorescence emission spectrum identical to that of a monofloral kanuka honey (Fig. 4C). Thus both spectra displayed a peak with maximum emission at 305 nm corresponding to the KM1 diagnostic peak (indicated by K). These peaks appear as a shoulder peak on the spectrum due to an overlap with scattering light interference from the excitation wavelength. It is likely that this interference arose from the small Stoke shift of the fluorophore; namely the positions of the maximum excitation and emission wavelengths of the fluorophore on the fluorescence emission spectrum are too close together (Lakowicz, 2006).

4. Conclusion Using a closed set of honeys with known chemical composition, we demonstrated the potential utility of a fluorescence approach to determine the monoflorality status of New Zealand manuka and kanuka honeys. Both these honey types exhibited unique fluorescence profiles that distinguished them from each other and to other New Zealand honey types. Two marker wavelengths each for manuka and kanuka honeys were identified at 270–365 (MM1) and 330–470 nm ex–em (MM2), and 275–305 (KM1) and 445–525 nm ex–em (KM2), respectively. Dilution of manuka and kanuka honeys by other non-manuka and kanuka honey types resulted in a proportional change in

1013

fluorescence yield of the honeys at these unique marker wavelengths. It may therefore be possible to determine percentage nectar contribution of these floral species in a honey and effectively assign the honey with the predominant floral source type. Other New Zealand honeys including rewarewa, kamahi, and clover that are common floral contamination sources of manuka honeys did not exhibit distinct fluorescence patterns that could uniquely identify their nectar contribution in a honey. The proportion of nectar contribution from these floral sources could only be established collectively as ‘‘contribution from other honey types’’ through the absence of signal. While further validation studies are required, our findings open the possibility that fluorescence approach could provide a rapid screening method for determining the monoflorality status of manuka and kanuka honeys. We also showed that the fluorophores responsible for fluorescence of these honeys at the identified marker wavelengths were not propolis-derived flavonoids, and that 4-methoxyphenyllactic acid is likely the principal fluorophore responsible for KM1 fluorescence. In addition, the identical fluorescence spectra shared by both manuka honey and nectar strongly suggests that the fluorophore, or fluorophores, responsible for fluorescence at the manuka marker wavelengths are nectar-derived. Further work will focus on the identification of honey components responsible for fluorescence at these marker wavelengths. Disclosure statement Jonathan Stephens and Ralf Schlothauer are employees of Comvita NZ Ltd., Paengaroa, PB1, Te Puke, New Zealand. Acknowledgements This work was supported by TechNZ Fellowship funding from the New Zealand Ministry of Science and Innovation. We would like to thank Comvita NZ Limited for supplying the honey, nectar, and propolis samples. References Aitkenhead, C., Rosendale, D., Schlothauer, R. C., & Stephens, J. M. C. (2014). Method and apparatus that utilises fluorescence to determine plant or botanical origin characteristics of honey. Patent No. US 8759774 B2. Andrade, P., Ferreres, F., & Amaral, M. T. (1997). Analysis of honey phenolic acids by HPLC, its application to honey botanical characterisation. Journal of Liquid Chromatography and Related Technologies, 20(14), 2281–2288. Anklam, E. (1998). A review of the analytical methods to determine the geographical and botanical origin of honey. Food Chemistry, 63(1), 549–562. Beitlich, N., Koelling-Speer, I., Oelschlaegel, S., & Speer, K. (2014). Differentiation of manuka honey from kanuka honey and from jelly bush honey using HS-SPMEGC/MS and UHPLC–PDA-MS/MS. Journal of Agricultural and Food Chemistry, 62(27), 6435–6444. Bogdanov, S., Ruoff, K., & Persano Oddo, L. (2004). Physico-chemical methods for the characterisation of unifloral honeys: A review. Apidologie, 35, S4–S17. Codex Alimentarius Commission. (2001). Revised codex standards for honey. Codex Standard 12-1981, Rev. 2. Fearnley, L., Greenwood, D. R., Schmitz, M., Stephens, J. M., Schlothauer, R. C., & Loomes, K. M. (2012). Compositional analysis of manuka honeys by highresolution mass spectrometry: Identification of a manuka-enriched archetypal molecule. Food Chemistry, 132, 948–953. Gebala, S., & Przybylowski, P. (2010). Method for honey type authentication. Patent No. WO 2010/027286 A1. Ghosh, N., Verma, Y., Majumder, S. K., & Gupta, P. K. (2005). A fluorescence spectroscopic study of honey and cane sugar syrup. Food Science and Technology Research, 11(1), 59–62. Jones, G. D., & Bryant, V. M. (1992). Melissopalynology in the United States: A review and critique. Palynology, 16, 63–71. Karoui, R., Bosset, J.-O., Mazerolles, G., Kulmyrzaev, A., & Dufour, E. (2005). Monitoring the geographic origin of both experimental French Jura hard cheeses and Swiss Gruyere and L’Etivaz PDO cheeses using mid-infrared and fluorescence spectroscopies: A preliminary investigation. International Dairy Journal, 15, 275–286. Karoui, R., Cartaud, G., & Dufour, E. (2006). Front-face fluorescence spectroscopy as a rapid and non-destructive tool for differentiating various cereal products: A

1014

J. Bong et al. / Food Chemistry 192 (2016) 1006–1014

preliminary investigation. Journal of Agricultural and Food Chemistry, 54, 2027–2034. Karoui, R., Dufour, E., Bosset, J.-O., & De Baerdemaeker, J. (2007). The use of frontface fluorescence spectroscopy to classify the botanical origin of honey samples produced in Switzerland. Food Chemistry, 101(1), 314–323. Karoui, R., Dufour, E., Pillonel, L., Picque, D., Cattenoz, T., & Bosset, J. (2004). Determining the geographic origin of Emmental cheeses produced during winter and summer using a technique based on the concatenation of MIR and fluorescence spectroscopic data. European Food and Research Technology, 219, 184–189. Kato, Y., Fujinaka, R., Ishisaka, A., Nitta, Y., Kitamoto, N., & Takimoto, Y. (2014). Plausible authentication of manuka honey and related products by measuring leptosperin with methyl syringate. Journal of Agricultural and Food Chemistry, 62(27), 6400–6407. Kato, Y., Umeda, N., Maeda, A., Matsumoto, D., Kitamoto, N., & Kikuzaki, H. (2012). Identification of a novel glycoside, leptosin, as a chemical marker of manuka honey. Journal of Agricultural and Food Chemistry, 60(13), 3418–3423. Kulmyrzaev, A., & Dufour, E. (2002). Determination of lactulose and furosine in milk using front-face fluorescence spectroscopy. Lait, 82, 725–735. Kulmyrzaev, A., Levieux, D., & Dufour, E. (2005). Front-face fluorescence spectroscopy allows the characterisation of mild heat treatments applied to milk. Relations with denaturation of milk proteins. Journal of Agricultural and Food Chemistry, 53, 502–507. Lakowicz, J. R. (2006). Principles of fluorescence spectroscopy (3rd ed.). USA: Springer. Lenhardt, L., Bro, R., Zekovic´, I., Dramic´anin, T., & Dramic´anin, M. D. (2015). Fluorescence spectroscopy coupled with PARAFAC and PLS DA for characterisation and classification of honey. Food Chemistry, 175, 284–291. Louveaux, J., Maurizio, A., & Vorwohl, G. (1978). Methods of melissopalynology. Bee World, 59, 139–157. Moar, N. T. (1985). Pollen analysis of New Zealand honey. New Zealand Journal of Agricultural Research, 28, 39–70. Nicolson, S. W., & Thornburg, R. W. (2007). Nectar chemistry. In S. W. Nicolson, M. Nepi, & E. Pacini (Eds.), Nectaries and nectar (pp. 215–248). Dordrecht, Netherlands: Springer. Oelschlaegel, S., Gruner, M., Wang, P.-N., Boettcher, A., Koelling-Speer, I., & Speer, K. (2012). Classification and characterisation of manuka honeys based on phenolic compounds and methylglyoxal. Journal of Agricultural and Food Chemistry, 60(29), 7237–7729.

Piana, M. L., Persano Oddo, L., Bentabol, A., Bruneau, E., Bogdanov, S., & Guyot Declerck, C. (2004). Sensory analysis applied to honey: State of the art. Apidologie, 35, S26–S37. Ruoff, K., Lüginbuhl, W., Künzli, R., Bogdanov, S., Bosset, J.-O., von der Ohe, K., et al. (2006). Authentication of the botanical and geographical origin of honey by front-face fluorescence spectroscopy. Journal of Agricultural and Food Chemistry, 54(18), 6858–6866. Senanayake, M. J. (2006). A chemical investigation of New Zealand unifloral honeys (unpublished Ph.D. thesis). Hamilton, New Zealand: University of Waikato. Stephens, J. M., Greenwood, D. R., Fearnley, L., Bong, J., Schlothauer, R. C., & Loomes, K. M. (2015). Honey production and compositional parameters. In V. R. Preedy (Ed.), Processing and impact on active components in food (pp. 675–680). London, United Kingdom: Academic Press. Stephens, J. M., Schlothauer, R. C., Morris, B. D., Yang, D., Fearnley, L., Greenwood, D. R., et al. (2010). Phenolic compounds and methylglyoxal in some New Zealand manuka and kanuka honeys. Food Chemistry, 120(1), 78–86. Stephens, J. M. (2006). The factors responsible for the varying levels of UMFÒ in manuka (Leptospermum scoparium) honey (unpublished Ph.D. thesis). Hamilton, New Zealand: University of Waikato. Tan, S.-T., Holland, P. T., Wilkins, A. L., & Molan, P. C. (1988). Extractives from New Zealand honeys. 1. White clover, manuka and kanuka unifloral honeys. Journal of Agricultural and Food Chemistry, 36, 453–460. Tomás-Barberán, F. A., Martos, I., Ferreres, F., Radovic, B. S., & Anklam, E. (2001). HPLC flavonoid profiles as markers for the botanical origin of European unifloral honeys. Journal of the Science of Food and Agriculture, 81(5), 485–496. White, J. W., & Doner, L. W. (1980). Honey composition and properties: Beekeeping in the United States. In Agriculture Handbook No. 335, Revised October, 8291. Windsor, S., Pappalardo, M., Brooks, P., Williams, S., & Manley-Harris, M. (2012). A convenient new analysis of dihydroxyacetone and methylglyoxal applied to Australian Leptospermum honeys. Journal of Pharmacognosy and Phytotherapy, 4(1), 6–11. Winston, M. L. (1987). The biology of the honey bee. London, England: Harvard University Press. Yao, L., Datta, N., Tomás-Barberán, F. A., Ferreres, F., Martos, I., & Singanusong, R. (2003). Flavonoids, phenolic acids and abscisic acid in Australian and New Zealand Leptospermum honeys. Food Chemistry, 81(2), 159–168.