Investigating C-4 Sugar Contamination of Manuka Honey and Other

Feb 26, 2014 - ... to New Zealand honey, due to the long-standing high reputation of the ... The net effect resulted in a more negative δ13C protein ...
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Investigating C‑4 Sugar Contamination of Manuka Honey and Other New Zealand Honey Varieties Using Carbon Isotopes Karyne M. Rogers,* Mike Sim, Simon Stewart, Andy Phillips, Jannine Cooper, Cedric Douance, Rebecca Pyne, and Pam Rogers National Isotope Centre, GNS Science, 30 Gracefield Road, Lower Hutt 5040, New Zealand ABSTRACT: Carbon isotopes (δ13C honey and δ13C protein) and apparent C-4 sugar contents of 1023 New Zealand honeys from 15 different floral types were analyzed to investigate which New Zealand honey is prone to failing the AOAC 998.12 C-4 sugar test and evaluate the occurrence of false-positive results. Of the 333 honey samples that exceeded the 7% C-4 sugar threshold, 324 samples of these were New Zealand manuka honey (Leptospermum scoparium, 97.2% of all fails found in the study). Three monofloral honeys (ling, kamahi, and tawari) had nine samples (2.8% of all fails found in the study) with apparent C-4 sugars exceeding 7%. All other floral types analyzed did not display C-4 sugar fails. False-positive results were found to occur for higher activity New Zealand manuka honey with a methylglyoxal content >250 mg/kg or a nonperoxide activity >10+, and for some ling, kamahi and tawari honeys. Recommendations for future interpretation of the AOAC 998.12 C-4 sugar method are proposed. KEYWORDS: New Zealand, honey, manuka, Leptospermum scoparium, methylglyoxal, apparent C-4 sugars, 5-hydroxymethylfurfural, dihydroxyacetone, AOAC 998.12, adulteration, carbon isotope, bioactive

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INTRODUCTION New Zealand has a number of distinctive native floral honeys, of which manuka (Leptospermum scoparium) honey is the best known due to its unique antibacterial properties, making it highly sought after in international markets. Depending on annual nectar flow conditions, around 10000−12000 tonnes of honey is produced each year in New Zealand, with between 6000 and 8000 tonnes of predominantly manuka honey available for export. Since 2010, New Zealand honey has been scrutinized more closely for cane sugar adulteration by international border testing agencies using the Association of Official Analytical Chemists (AOAC) 998.12 method. 1 Historically, the AOAC 998.12 method detects large-scale addition (>7%) of C-4 sugar (specifically cane sugar or highfructose corn syrups (HFCS)) to honey to stretch and improve honey yields.2,3 In New Zealand, cane sugar is the only commercially available sugar fed to bees by beekeepers and is usually given as a mixture of water and cane sugar, although globally C-4 (cane or HFCS) and C-3 sugars (beet, rice, manioc, or wheat, among others) are used.3−6 Apparent C-4 sugars are traditionally detected using stable carbon isotope ratio analysis (SCIRA) because of the isotopic discrimination between C-4 cane sugar syrup (ca. −10‰) and C-3 plant nectar (ca. −24.0 to −27.0‰). The carbon isotope value of the honey is compared with its corresponding protein, providing an internal comparison (AOAC 998.12 C-4 sugar method).1,7 The AOAC 998.12 method sets prescriptive limits for apparent C-4 sugar detection at 7%, not because this is an acceptable level but because it is deemed to be the lowest reliably detectable level using SCIRA. Detection of C-3 sugar adulteration requires more sophisticated analytical techniques including liquid chromatography combined with SCIRA as C-3 sugar carbon isotope values are often indistinguishable from honey and are difficult to detect.4,8,9 © 2014 American Chemical Society

In New Zealand, honey is routinely screened before export for apparent C-4 sugars using the AOAC 998.12 (repetitive wash) method using SCIRA as part of product verification. Until 2010, AOAC 998.12 method of C-4 sugar testing was not rigorously applied to New Zealand honey, due to the longstanding high reputation of the industry. However, with increasing fraud occurring globally due to growing honey prices, many international border agencies began to rigorously test all imported honey products. In the past three years, routine testing by importing authorities has resulted in some New Zealand honey failing C-4 sugar tests at overseas borders, even though the honey was purported to be genuine by the producers. When these New Zealand fails were first investigated, concerns were raised that the AOAC 998.12 method was generating “false-positive” caused by insoluble material (such as pollen or dust) contained in manuka honey being coextracted with protein and affecting its δ13C protein value.10 Studies showed that manuka honey with higher pollen contents (>500,000 grains/10 g honey) was found to have more negative δ13C protein values than the protein extracted from the same honey after pollen was removed.10 Analysis of the isolated pollen component from manuka honey confirmed that it had more negative δ13C values than the protein. This excess pollen was originally suggested as the main cause of artificially enhanced apparent C-4 sugar contents in manuka honey by contaminating the extracted protein. The net effect resulted in a more negative δ13C protein value from the contaminating pollen, consequently increasing the Δ(δ13C Received: Revised: Accepted: Published: 2605

October 24, 2013 January 31, 2014 February 25, 2014 February 26, 2014 dx.doi.org/10.1021/jf404766f | J. Agric. Food Chem. 2014, 62, 2605−2614

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honey − δ13C protein) value, which should be ≤1 in unadulterated samples. However, a recent international laboratory ring test undertaken to assess this potential modification11 found that filtering and/or centrifuging of honey prior to flocculation to remove insoluble material reduced the apparent C-4 sugar content not only of unadulterated honey but also of intentionally adulterated honeys (up to 15% C-4 sugar) to within acceptable limits, so the proposed filtration modification was not recommended. More recently, instances have also arisen when pollen filtration did not resolve the anomalously high apparent C-4 sugar content of honey (up to 15%), yet the honey was not derived from hives that had undergone sugar feeding, supporting the theory that some other mechanism was also contributing to more negative δ13C protein values and generating false-positive AOAC 998.12 results.11 This study was undertaken to investigate the cause of apparent C-4 sugar fails in New Zealand honey to address increasing concerns raised by border testing agencies in importing countries such as China, the United States of America, Hong Kong, and Europe. The stable carbon isotopes of 1023 New Zealand honeys and their corresponding proteins were analyzed, and apparent C-4 sugars were quantified. Honey floral types prone to AOAC 998.12 failure were investigated to understand which types of fails were real (where the honey is adulterated or contaminated by C-4 sugar) or false positive (contaminated via some mechanism other than sugar), and the mechanisms that may cause false-positive results were explored.

sucrose should not be present above 5 g/100 g of honey and in some exceptional cases 10 or 15 g/100g of honey (such as ling and lavender). Pollen Contamination (False-Positive Fail). Protein is extracted from honey as an internal standard; AOAC criteria require Δ(δ13C honey − δ13C protein) ≤ 1. The δ13C protein value may be contaminated by insoluble material (i.e., excess pollen from diverse sources, dust particles, or other hydrophobic components). However, when using the repetitive wash method, the AOAC 998.12 method does not remove this material and it is coextracted with the protein during flocculation. This anthropogenic pollen or dust has been shown to have a different carbon isotope value (ca. −28‰), shifting the final δ13C protein value by up to 1‰ and affecting the calculation of apparent C-4 sugars.10 In New Zealand honey, significant levels of pollen can occur in honey (in excess of 14,000,000 grains/10 g; personal observation by Rogers). At levels where pollen is >500,000 grains/10 g honey, contamination of protein by concurrently extracted pollen can result in false-positive results.10 High pollen levels can be introduced into honey due to the robust extraction methods used to dislodge thixotropic honey (such as manuka). Frames often need manual scraping followed by high-speed centrifuging to remove honey. This procedure may dislodge large pockets of pollen stored in the frames, in contrast to gentle “prick and drain” methods employed for runnier honeys, which generally retain the comb structure and associated pollen within the frame. Supplementary Protein Feeding (False-Positive and Genuine Fails). Supplementary protein feeding can affect δ13C protein values10 and contaminate nectar-derived protein sources. Common protein supplements include yeast (−24‰), soy flour (−25 to −27.5‰), and pea flour (−28‰),14−17 which have a range of isotopic values and are similar to the range of honey protein values found in this study (−24.3 to −28.2‰). Although the protein supplements are usually consumed by bees, it is possible small amounts are regurgitated and stored in collections boxes, similar to bee bread and pollen pockets, contributing foreign protein to the honey. Fed at inappropriate times, supplementary feeds might also cause genuine fails due to the practice of mixing sugar syrups with the protein feed to increase palatability and consumption by bees. Issues with using yeast as a protein source were also observed in this study, where honey was more prone to fermentation over time, especially with higher moisture content honey (19−21%) and higher storage temperatures (>22 °C). Hives fed either yeast or sugar syrup inverted with yeast had a tendency to show δ13C honey values that were more positive (−23 to −24.5‰), even if sugar feeding had occurred sparingly (personal observation by Rogers). Brood Box Contribution (Genuine Fails). C-4 sugar fails may also arise if honey extraction occurs from the brood box. The brood box is where bees store pollen and/or bee bread (key protein sources) as well as nectar and/or sugar syrup, which are converted to honey (key carbohydrate sources) to provide stored sustenance and ensure survival of the colony during food shortages or bad weather. In spring, if bees have filled brood box frames with unconsumed honey, and there is no further room for brood expansion, beekeepers may exchange filled frames for empty frames to allow continued brood expansion. The extraction of honey from brood box frames is generally unacceptable in good beekeeping practices due to the

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BACKGROUND Pure and authentic honey derived solely from unique nectar sources should theoretically have similar δ13C honey and δ13C protein values as bees do not substantially fractionate or alter the carbon isotopes of nectar. Previous research has raised the possibility of false-positive AOAC 998.12 C-4 sugar results9,10,12 via pollen and other insoluble material coextracted with the protein. However, there are several different mechanisms to explain the failure of honey tested under the AOAC 998.12 method. Excess Cane Sugar Contamination (Genuine Fail). Sugar syrups are fed to bees as a supplementary carbohydrate source in times of winter nectar dearth or to prevent starvation during poor weather. Sugar syrups are also used to retain bees in specific areas such as orchards or fields to provide pollination services or to boost hives into breeding mode several weeks prior to nectar flow. However, supplementary syrup feeding also risks overuse, resulting in bees storing excess sugar syrups into brood boxes, which can later be uncapped and redistributed into collection boxes by the bees as they make more room for brood. Cane sugar (a C-4 plant, with a more positive carbon isotope value than C-3 plants) is the sole bulk sugar product available in New Zealand for commercial beekeepers. Addition or mixing of C-4 sugar syrups (which has a stable carbon isotope or δ13C value of ∼ −10‰) with plant nectar will shift the δ13C honey isotope values to more positive isotope values than the δ13C values of available C-3 plant nectar sources, which range from −24 to −27‰. After-harvest additions of noninverted cane sugar can be readily detected using sugar chemical profiles that measure sucrose content, as there is no opportunity for the sucrose to undergo a reaction with invertase (a bee digestive enzyme), which breaks the sucrose molecule into monosaccharides of fructose and glucose. Codex13 prescriptively sets limits whereby 2606

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risk of contamination from winter or prenectar flow hive treatments (i.e., for varroa and other common diseases) and supplementary feeding of sugar or protein. Unknown (Non C-4 Sugar) Mechanism (False-Positive Fail). New Zealand honey samples exceeding 7% apparent C-4 sugars continue to occur; however, in most instances none of the above causes were cited by participating beekeepers as being possible. Until this study, an unknown mechanism (which is unrelated to C-4 sugar adulteration) has been suspected as being responsible for these failed tests.10,11 This research aims to investigate and understand false-positive results that were not caused by protein feeding or excess pollen levels in the honey.

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where Rs is the isotopic ratio measured for the sample and Rref that of the international standards. The δ13C value is relative to the international Vienna Pee Dee Belemnite (VPDB) standard. Results are expressed in δ (‰) versus the specific reference, and analytical precision is within ±0.2‰ for carbon. Carbon isotopes of the whole honey were compared with their extracted protein and assessed for apparent C-4 cane sugar content using eq 1:

apparent C‐4 sugar (%) = 100 × [δ13C(protein) − δ13C(whole honey)] /[δ13C(protein) − (− 9.7)]

The acceptance criteria state that pure honey generally yields a value of apparent C-4 sugars of ≤7%. Some unusual varieties may slightly exceed the range, but will have δ13C values for honey that are in the normal range (more negative than −24.0‰).7 Other Analyses. Methylglyoxal (MGO) or NPA and hydroxymethylfurfural (HMF) measurements were supplied by some honey submitters whose honey had already been tested at Hills Laboratories or Eurofins according to their in-house commercial procedures. To complete data sets where MGO and NPA data were not available and perform checks on supplied values, samples were sent to Hills Laboratories for further testing. Due to several bioactive honey measurement systems within New Zealand,18 the NPA, UMF, and MGO bioactivity values were standardized and reported as NPA according to the UMF Honey Association conversion tool Web site (http://www.umf.org.nz/umf-trademark/methylglyoxal-npa-honeyconversion-calculator).

MATERIALS AND METHODS

AOAC 998.12 Method and “Apparent” C-4 Sugar Determination. Carbon isotopes (δ13C honey and δ13C protein) and apparent C-4 sugar contents of 1023 New Zealand honey samples (mainly manuka but 14 multifloral and other monofloral varieties also included) were analyzed at the Stable Isotope Laboratory at GNS Science, Lower Hutt, New Zealand, between January 2011 and December 2012. The honeys were harvested between December 2007 and March 2012 and were submitted by honey companies and beekeepers as part of quality assurance requirements to test for apparent C-4 sugars before export. Honey was submitted blind for testing, and further information was supplied after analysis based on organoleptic (i.e., flavor, odor, and color), physicochemical (i.e., conductivity and thixotropy), and/or pollen count as well as from observations by the beekeepers who target specific nectar flows. Antibacterial activity was also reported if available, including total peroxide activity (PA, caused by hydrogen peroxide which originates from the activity of the enzyme glucose oxidase in honey) or nonperoxide activity (NPA, related to a further amount of antibacterial activity attributed to other bioactive compounds such as methylglyoxal in the product).18,19 Activity analyses were carried out in commercial testing laboratories by Hills Laboratories, Hamilton, New Zealand, or Eurofins, Hamilton, New Zealand. Protein Extraction. Protein was extracted from honey using the AOAC 998.12 repetitive wash method whereby 4 mL of distilled H2O was added to 10−12 g of honey in a clear 50 mL centrifuge tube. The solution was mixed well and heated gently in a water bath to aid dissolution. Two milliliters of 10% sodium tungstate and 2 mL of 0.67 N H2SO4 (Sigma-Aldrich) were mixed together and immediately added to the honey solution. The mix was heated at 80 °C in a water bath until a visible protein flocculent formed with clear supernatant, with further 2 mL increments of 0.67 N H2SO4 added if no visible flocculent formed after 5 min. The centrifuge tube was filled with water and centrifuged for 5 min at 3500 rpm The supernatant was discarded and the pellet rinsed and centrifuged five times with 50 mL portions of water, thoroughly dispersing the pellet each time. Precipitated protein was dried in an oven overnight at 60 °C. Two manuka honey samples (stored at 4 °C) were routinely analyzed as standards with each batch (18 samples or less) of samples to ensure protein preparation consistency. Stable Isotope analysis. Approximately 0.5 mg of honey or protein was transferred into 6 × 4 mm tin capsules (IVAAnalysentechnik, Germany) and analyzed for stable carbon isotopes using a Eurovector elemental analyzer coupled to an Isoprime mass spectrometer in continuous flow mode (EA-IRMS). International and working reference standards (IAEA-CH6, leucine, EDTA, cane and beet sugars), the two honey standards (honey and protein), and blanks were included during each run for calibration. Isotopic ratios (13C/12C) are expressed as isotopic deviations δ defined as δ (‰) =

(1)

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RESULTS AND DISCUSSION Which New Zealand Honey Varieties Are Prone to Exceeding 7% “Apparent” C-4 Sugars? Apparent C-4 sugar analysis of 1023 New Zealand honey samples showed that 690 samples had apparent C-4 sugars ≤7% (67% pass rate) and 333 samples had apparent C-4 sugars >7% (33% fail rate) (Figure 1). The apparent C-4 sugar content found in these honeys

Figure 1. Distribution of δ13C honey isotope values and apparent C-4 sugars for 1023 New Zealand honeys. Circles represent ≤7% apparent C-4 sugars, and crosses represent >7% apparent C-4 sugars. R2 = 0.81, y = 5.1585x + 136.55.

ranged between −5.4 and 26.9%; δ13C honey values ranged from −18.5 to −27.6‰, and δ13C protein values ranged from −24.3 to −28.2‰. The average δ13C honey and protein values of the 1023 New Zealand samples were −25.4 ± 0.8 and −26.3 ± 0.4‰, respectively (Table 1). The average δ13C honey and protein values of honeys with apparent C-4 sugars ≤7% were

R s − R ref × 1000 R ref 2607

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Table 1. Carbon Isotope Values of Honey and Protein, and Percent C-4 Sugar of 1023 New Zealand Multi- and Monofloral Honeys (Standard Deviations of Average Values Reported in Parentheses) honey type (no. of samples)

av

range

av

range

av

range

apparent %C-4

δ13C honey

δ13C honey

δ13C protein

δ13C protein

apparent

apparent

sugar failure rate

(‰)

%C-4 sugar

(‰)

blue borage and vipers bugloss (5) clover (67) dandelion (2) honeydew (15) kamahi (18) kanuka (19) ling (4) manuka (757)a manuka blend (6) multifloral (93) pohutukawa (3) rata (5) rewarewa (13) tawari (6) thyme (10)

−26.4 −26.0 −26.9 −26.2 −25.6 −26.1 −25.2 −25.1 −25.9 −26.1 −25.8 −26.1 −26.0 −24.0 −26.6

total honey (1023) manuka nectar (25) tawari nectar (6) honey with C-4 sugars 7% had average δ13C honey and protein values of −24.6 ± 0.9 and −26.4 ± 0.5‰, respectively. Most samples readily flocculated protein, with some manuka honey samples flocculating on warming, even before addition of the chemicals. It was noted that it was often difficult to flocculate protein from tawari and rata honey, suggesting they may contain lower levels of extractable protein. Due to the high overall failure rate (33%), honey samples were classified into 15 floral types to investigate how prone each floral type was to failure (Table 1). Four monofloral honey types (manuka, tawari, ling, and kamahi) had samples that exceeded 7% apparent C-4 sugars and failed acceptance criteria, whereas none of the other 11 New Zealand honey varieties exceeded 7% apparent C-4 sugars (Table 1). Comparison of the carbon isotope values of 690 honeys and their frequency of passes (apparent C-4 sugars ≤7%) to the 333 honeys and their frequency of fails (apparent C-4 sugars >7%) identifies three main regions of interest (Figure 2). In region 1, all honey samples passed and δ13C honey values ranged between −27.6 and −25.9‰; 250 samples were classed as genuine passes with no apparent C-4 sugar adulteration. In region 2, all samples failed the test (apart from a few tawari and multifloral samples) and δ13C honey values ranged between −24.7 and −18.5‰; 59 samples were classed as genuine fails which were attributed to apparent C-4 sugar contamination. In region 3, honey samples could either pass or fail the test and δ13C honey values were between −25.9 and −24.7‰; 440 samples passed, and 274 samples failed. Of interest are the mechanisms that allowed honey samples of similar florality (and hence nectar source) and bulk isotope composition to

Figure 2. Frequency distribution of 1023 New Zealand δ13C honey values. Region 3 represents honey samples that pass and those resulting in false positives. Several samples encompassed in the dashed box (more positive δ13C honey samples than those in region 3) are tawari, clover, and multifloral samples that had C-4 sugars ≤7%.

either pass or fail in region 3 and whether they were genuine fails or false-positive fails. The delineation of region 3 is made specifically for manuka honey, where all manuka honey samples with δ13C honey values more negative than −25.9‰ passed and all manuka honey samples with δ13C honey values more positive than −24.7‰ failed. All samples that failed in region 3 were manuka honey. In the past, honeys were not considered adulterated with C-4 sugars if their bulk isotope analysis (δ13C honey values) was more negative than −23.5 to −24.0‰.3,5 Even though 274 2608

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New Zealand honeys from region 3 (Figure 2) had δ13C honey values more negative than −24.7‰ (and could therefore be considered as unadulterated on the basis of traditional analyses), these honeys exceeded 7% apparent C-4 sugars and failed the original AOAC 998.12 acceptance criteria.1 As their δ13C honey values were well within the acceptable range, this suggested that a shift in the δ13C protein values might be affecting the outcome, rather than C-4 sugar adulteration. An assessment of the δ13C protein values of these 1023 honeys showed that the range of δ13C protein values of honey samples which passed the test (apparent C-4 sugars ≤7%, −24.9 to −27.1‰) is considerably narrower than the range of δ13C protein values that failed the test (apparent C-4 sugars >7%, −24.3 to −28.2‰) (Figure 3). The wide range of δ13C

pollen (protein source) within New Zealand’s honey-producing flora or a distinctive isotope fractionation process occurring in the protein. Characterization of New Zealand Monofloral Honey Using Carbon Isotopes and “Apparent” C-4 Sugar Content. Ling. Ling (or heather) honey has traditionally been identified by Codex13 as having “special properties” and is allowed higher sucrose contents than other honey (up to 10% w/v). All four ling samples in this study (Figure 4a) failed the AOAC 998.12 method, with apparent C-4 sugars ranging from 8.3 to 22.1%. Three samples (with apparent C-4 sugars between 8.3 and 9.0%) did not originate from hives that were sugar fed by the beekeepers, suggesting that these samples may be false-positive fails. Ling honey δ13C protein values (average δ13C ling protein value of −27.3‰) were up to 1.0‰ more negative than those of other honey proteins in this study (average δ13C total protein value of −26.3‰), whereas the average ling δ13C honey value was −25.2‰ and comparable with the average δ13C total honey value of −25.4‰. One ling sample (circled in Figure 4a) had δ13C honey and protein values of −24.1 and −28.2‰, respectively, which suggested a positive shift in the δ13C honey value, usually associated with C4 sugar contamination, but also indicated a significant negative shift of δ13C protein values. Kamahi. One kamahi honey (n = 18) failed the AOAC 998.12 method with an apparent C-4 sugar content of 13.2% (Figure 4b). This sample had a δ13C honey value of −24.5‰ and a δ13C protein value of −26.6‰. The Δ(δ13C honeyprotein) value of this sample was 2.2‰, which is much higher than those of the other kamahi samples in this study (−0.5 to 0.8‰), suggesting the fail was caused by either a significant honey or protein shift or both. This sample was the only sample to fail of 18 samples and was considered to be a genuine fail (most likely due to C-4 sugar) as its δ13C honey value (−24.5‰) was considerably more positive than the average

Figure 3. Frequency distribution of 1023 New Zealand δ13C protein values.

protein values found in these 1023 New Zealand honeys (up to 4‰) may suggest that there is either a diverse isotopic range of

Figure 4. Carbon isotope and C-4 sugar contents of 13 New Zealand mono- and multifloral honeys using AOAC 998.12 method. 2609

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Table 2. Breakdown of 757 Manuka Honey Samples Tested for Apparent C-4 Sugars into Pass (Region 1), Fail (Region 2), and Those That Have Pass or False-Positive Results (Region 3) no. of samples

av

range

av

range

range

δ13C honey

δ13C honey

δ13C protein

δ13C protein

apparent C-4 sugars

region

(n)

(‰)

SD

(‰)

SD

1 (pass) 2 (fail) 3 (false positive) 3 (pass) 3 (pass and false positive) 1 and 3 (genuine manuka honey) manuka nectar

161 113 211 272 483 644 25

−26.2 −24.0 −25.1 −25.5 −25.3 −25.4 −26.2

0.3 0.5 0.3 0.3 0.5 0.5 0.5

−25.9 −21.0 −24.7 −24.7 −24.7 −24.7 −25.7

(‰) −27.6 −24.7 −25.9 −25.9 −25.9 −27.6 −26.7

−26.3 −26.0 −26.7 −26.2 −26.5 −26.5 na

0.3 0.4 0.4 0.4 0.5 0.4

−24.7 −24.6 −25.8 −24.7 −24.7 −24.7 na

−27.1 −27.9 −27.7 −27.0 −27.7 −27.27

−3.9 to 6.8 10 to 26.9 7.0 to 16.7 −3.9 to 7.0 0 to 16.7 −3.9 to 16.7 na

total manuka

757

−25.1

0.5

−21.0 to −27.6

−26.3

0.5

−24.6 to −27.39

−3.9 to 26.9

δ13C kamahi honey value (−25.6 ± 0.6‰). It was not possible to discount that some other non C-4 sugar contribution may have also occurred due to the more negative δ13C protein value. Tawari. Four of six tawari honey samples failed the AOAC 998.12 method with apparent C-4 sugars ranging from 7.2 to 24.0% (Figure 4a). Two of these samples (circled in Figure 4a) were reported to have been sampled from hives where some C4 sugar feeding occurred prior to nectar collection (δ13C honey values of −21.8 and −23.0‰, respectively) and were considered to be genuine fails. The other two tawari honey samples, which had apparent C-4 sugars >7%, had no reported association with C-4 sugar feeding and may represent falsepositive results. Although only six samples of tawari honey were studied, the average δ13C tawari honey value (genuine samples) was −24.6‰ (0.8‰ more positive than the average δ13C honey values in this study), even though these four samples were reported by beekeepers to be genuine with no previous sugar feeding history. Six samples of tawari nectar originating from Gisborne (East Coast) were also collected and analyzed for their carbon isotopes. The average δ13C tawari nectar value was found to be −24.5 ± 0.5‰, corresponding to similar δ13C honey values found in tawari honey. Tawari honey was found to have more positive carbon isotope values than any other New Zealand honey floral variety in this study. These positive δ13C honey values are similar to other specialty honeys containing more positive isotope values found elsewhere globally such as citrus and mesquite honeys, which have δ13C honey values averaging −23.8‰.20 Clover and Multifloral. A large number of clover (n = 67) and multifloral (n = 93) honeys (sometimes called bush honey due to their mixed nectar source) (Figure 4b,c) were identified and analyzed. In New Zealand, these honeys are the next most popular export honeys after manuka honey. All clover and multifloral honey samples tested in this study had apparent C-4 sugars −24.7‰? Although some samples with δ13C honey values > −24.7‰ and apparent C-4 sugars >7% (considered to be C-4 sugar contaminated) were confirmed by beekeepers as originating from hives that had been fed some C-4 sugar syrup, other beekeepers stated that some samples included in this group originated from hives which had minimal C-4 sugar syrup feeding. There are several mechanisms other than C-4 sugar that can increase δ13C honey values to become more positive, such as a nectar contribution from another more positive floral source or supplementary protein feeding, which may artificially shift the δ13C honey values toward more positive values if the bee feed has more positive δ13C values and is incorporated into the honey instead of being completely consumed. The only floral type found in this study with substantially more positive isotope values than the average δ13C honey (−25.4‰) is tawari honey (average δ13C honey −24.0‰), although other types of honey with similar positive δ13C honey values are known.20 Tawari often flowers just prior to the manuka nectar flow, and brood can build up on this nectar before going directly onto manuka flowers. Tawari nectar has an average δ13C nectar value of −24.5 ± 0.5‰, and if combined with manuka nectar, it may be possible to isotopically shift the overall δ13C honey value to > −24.7‰, without the influence of any C-4 sugars. Other floral sources commonly used in spring built up before the manuka nectar flow are pollination crops such as apple and kiwifruit (minimal nectar) or nectar sources from willows and spring weeds. Analysis of a few commonly available pollen sources that may be consumed by bees prior to manuka nectar flow were undertaken during this study. Kiwifruit pollen had a δ13C pollen value of −24.2 ± 0.3‰, and willow and buttercup (a common weed) had δ13C pollen values of −23.0 ± 0.5‰. Other unknown mechanisms may occur if beekeepers feed sugar syrups or supplements that contain yeast. Further research on the consequential associated fractionation of sugars by yeast may explain more positive apparent C-4 sugar fails, although this study was not aimed at resolving how δ13C honey values could become more positive other than via C-4 sugar contamination. However, it will be an important area to consider in future years for New Zealand’s manuka industry if it wishes to eliminate sugar adulteration concerns completely. Why Does Manuka Honey Have False-Positive Fails Using the AOAC 998.12 C-4 Sugar Method? False-positive manuka honey (211 samples) from region 3 (where δ13C honey values were between −24.7 and −25.9‰ and δ13C protein values were between −25.8 and −27.2‰) had apparent C-4 sugars ranging from 7 to 15.0% with an average apparent C-4

honey samples with apparent C-4 sugars below 7% were considered to be genuine passes, whereas 211 manuka honey samples with apparent C-4 sugars exceeding 7% were considered to be false-positive fails. The lower limit of δ13C honey values for manuka honey (region 3, −24.7‰) is determined by the analysis of pure manuka nectar sampled directly from Leptospermum (sp) flowers, which was found to have an average δ13C value of −26.2 ± 0.5‰ (n = 25) (Table 2). It is expected that δ13C protein and δ13C honey of authentic (non-C-4 sugar contaminated) manuka honeys would have similar honey isotope values as the nectar, because the bees do not alter the isotopic values. The average manuka nectar isotope value correlates well with the δ13C honey values of manuka honey, which passed the AOAC 998.12 method in region 1 and falsepositive samples from region 3, but may suggest that in some of the samples which are more variable (outliers) that other nectar sources (which contain more negative or positive carbon isotopes) may have a more significant contribution. Alternatively, there may be a small change in the δ13C honey value of some honeys with ripening and maturation. To ensure a statistical safety interval of 3 times the SD (i.e., a confidence level 99.7%), the lowest acceptable value (cutoff) for genuine (unadulterated) manuka δ13C honey value would be −24.7‰ (−26.2‰ − (−1.5‰) = −24.7‰) as suggested by the data in Figure 2. Moreover, of 757 manuka samples analyzed in this study, no manuka samples with δ13C honey value ≤ −24.7‰ (region 2) passed the AOAC 998.12 C-4 sugar method. False-positive samples from region 3 are also found to have more negative δ13C protein values (average −26.7 ‰) than those from region 2 (average −26.0 ‰, where none of the 757 screened manuka samples passed the AOAC 998.12 and C-4 sugar contamination is known to occur). Kanuka. Kanuka (Kunzea ecroides) honey (19 samples) were analyzed for carbon isotopes and apparent C-4 sugars (Table 1). Flowering concurrently with manuka, examples of pure kanuka honey are not frequently found, as most kanuka honey is collected with, and identified as, manuka honey. It is not possible to distinguish between the two species using pollen identification as they have identical pollen shape and size. In the few identified cases where kanuka nectar sources predominate, the honey is reported to be lighter in color, less intense in flavor, and lower in conductivity and to have little or no thixotrophy compared to manuka honey. Unlike manuka honey, kanuka honey is not reported to have a nonperoxide activity. Kanuka honey exhibits more negative δ13C honey values (average −26.1‰) than manuka (average −25.1‰), although δ13C protein values are similar (average −26.4 and −26.3‰, respectively). All 19 kanuka honeys had apparent C-4 sugars 12%. These samples are all classed as false positives because of a negative shift in δ13C protein values, which artifically increases the Δ(δ13C honey − δ13C protein) values to >1, whereas the δ13C honey isotope value are < − 24.7‰ and traditionally considered unadulterated by C-4 sugars. An assessment of protein isotope values of each region (Figure 2) showed that region 2 (failed samples) had average δ13C protein values of −26.0 ± 0.4‰, region 1 (passed samples) had average δ13C protein values of −26.3 ± 0.3‰, and region 3 (false-positive samples) had the most negative average δ13C protein values of −26.7 ± 0.5‰. A Δ(honeyprotein) shift of 0.4‰ (the difference between average δ13C protein values of passed and false-positive samples) equates to an extra 2.8% of C-4 sugar. A subset of 376 bioactive manuka honeys (Table 3) was selected from the 757 manuka samples, with NPA levels ranging from 4+ to 29.5+ to ensure a minimum level of floral purity (NPA content is considered to be unique to manuka honey and not yet reported in other honey types). From this group, 205 samples had apparent C-4 sugars ≤7% (55%), and 171 samples had apparent C-4 sugars >7% (45%), similar to the overall manuka population screened in this study. These 376 bioactive manuka honeys had δ13C honey values between −21.0 and −26.8‰, δ13C protein values between −24.5 and −27.9‰, and apparent C-4 sugars ranged from −3.9 to 24.5%. NPA was plotted against apparent C-4 sugar content, and the rate of failure (where apparent C-4 sugars exceed 7%) for high, medium, and low NPA samples was documented in Table 3. Higher NPA honey (17+ to 30+) had the highest apparent C-4 sugar fail rate at 81%, and medium NPA rated honey (10+ to 17+) failed at 61%, whereas only 11% of low NPA rated honey (4+ to 10+) had apparent C-4 sugars >7% (Figure 5a). High rates of failure for high NPA manuka honey were not consistent with excess sugar syrup feeding. High NPA manuka honeys were expected to have the highest nectar purity due to their higher concentrations of methylglyoxal (key active component in NPA manuka honey), which would become diluted if C-4 syrups were fed to bees. The predisposition of high NPA manuka honey to fail and have high apparent C-4 sugar content gives a strong indication toward the cause for false-positive results. Although the production of methylglyoxal (the compound responsible for high NPA) in manuka honey has been shown to be formed via a conversion pathway of dihydroxyacetone (DHA),18 the mechanisms and interactions with other compounds that may catalyze the reaction are poorly

understood. However, current knowledge suggests that temperature, pressure, humidity, and storage time also have critical effects on manuka honey quality and bioactivity.21 The issue now becomes one of understanding the effects chemical reactions, which may induce fractionation effects on honey or protein such as the conversion of DHA to methylglyoxal (the chemical reaction that increases NPA) resulting in an increase of the apparent C-4 sugars in higher NPA manuka honey. These same samples were compared again on the basis of a proposed δ13C manuka honey acceptance threshold of −24.7‰ (suggested by the upper limit of region 3 in this study), where all New Zealand manuka honey with δ13C honey values that are more negative than −24.7‰ are considered genuine, even if they pass or fail (Figure 5b). Of these samples, only manuka honey with apparent C-4 sugars −24.7‰. Medium (10+ to 17+) and low (4+ to 10+) NPA manuka honeys also had significantly reduced failure rates (34 and 3%, respectively) when the proposed carbon isotope threshold was used (Table 3). The failure rate of the entire subset of NPA manuka honey samples was reduced to 20% (where these failed samples had δ13C honey values ≥ −24.7‰). These remaining failed samples may have some C-4 sugar contamination, but currently there is insufficient evidence to provide a robust explanation for some other mechanism contributing toward more positive carbon isotope values. In the future, more research should be conducted to understand mixing of unusual New Zealand nectar sources (tawari with its average nectar values of −24.5‰ is a key example), and/or fractionation effects via some synergistic/reactive mechanism occurring within the honey, which can alter the carbon isotopes to become more positive. Why Do Some Other (Nonmanuka) Honey Varieties Fail? In this study, ling honey was found to have the most negative δ13C protein values (average −27.3‰). It is likely this thixotropic honey has a chemical reaction or mechanism occurring within the honey that affects its protein isotopic composition similar to manuka honey. Thixotropic honeys such as manuka and ling contain a higher protein content, primarily derived from plant proteins, rather than from bee enzymes typical of many nonthixotropic honeys,22 suggesting that there may be other thixotropic honey types from elsewhere around 2612

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content of manuka honey with NPA > 10+ is discussed. Specifically, the ripening effects of heating and/or storage of higher bioactive manuka honey on apparent C4 sugars are investigated.21 To improve the interpretation of the AOAC 998.12 acceptance criteria for New Zealand manuka honey and avoid false-positive fails, the following amendment to the AOAC 998.12 method is suggested: Pure honey (free of corn or cane sugars) yields a value of “apparent” C-4 sugars of ≤7%. New Zealand’s manuka honey may exceed this value, but will have honey δ13C values which are in the normal range (more negative than −24.7‰) and an apparent C-4 sugar content no greater than 13.0% (this study and ref 20).

the globe that also behave in a similar fashion and will have a higher tendency to fail the AOAC 998.12 method. Tawari honey has also been found to have more positive carbon isotope values than all other New Zealand honey floral sources, where the average δ13C honey value was −24.5‰, and also prone to fail C-4 sugar tests. The consequence of a tawari nectar contribution to honey derived from other floral nectars may increase their failure rate, as the resulting δ13C honey values would shift to more positive values. Contamination of manuka honey by tawari nectar may be responsible for some manuka honey fails, especially when δ13C manuka honey values are > −25.1‰ (average manuka δ13C honey value), as concurrent flowering of the two species often takes place in the North Island of New Zealand, where much of the bioactive manuka honey is produced. Laboratory observations during AOAC 998.12 analysis have shown that tawari honey often contains relatively low levels of extractable protein, so the corresponding protein of a manuka/ tawari blend would not exhibit a similar positive shift, and it would appear that the sample contains excess C-4 sugars. An indication of this possible effect has also been observed by the presence of tawari pollen in some failed manuka honeys (personal observation by Rogers). Further studies using tawari and manuka plant-specific chemical markers would also confirm this interaction. Rewarewa and kamahi also have similar flowering times as manuka and kanuka, and it is possible that bees could collect these nectars concurrently with bioactive manuka and/or tawari nectar in sufficient quantities to cause a false-positive fail. In the future, examination of these honey varieties using specific chemical biomarkers, sugar profiles, or pollen counts may clarify mixing. In conclusion, specific New Zealand floral varieties (blue borage, clover, dandelion, honeydew, kanuka, manuka blend, multifloral, pohutukawa, rata, thyme, and vipers bugloss honeys) do not exceed 7% apparent C-4 sugar when tested using the AOAC 998.12 method. These honeys are not prone to false-positive results, nor would they show apparent C-4 sugar contamination unless excess C-4 sugar feeding has occurred. However, in some cases, sample numbers are not statistically significant, and further isotope characterization of some monofloral varieties would be complementary. It is expected that if beekeepers continue feeding minimal sugar and bees do not rob sugar from other nearby sources (up to a flight radius of 6 km, which is the effective foraging distance for a bee)23 and if nectar flows are strong and hive management practices are not significantly changed from one season to the next, then these specific floral types will continue to meet current AOAC 998.12 C-4 sugar method criteria. This study highlights that higher apparent C-4 sugar contents not only occur when the δ13C honey value becomes more positive (by addition of C-4 sugars or another undocumented effect) but also when the δ13C protein value becomes more negative. In the absence of C-4 sugar addition, false-positive results occur where the δ13C protein value becomes more negative during the honey ripening process and artificially elevates the apparent C-4 sugar content of honey. This research shows a strong tendency for manuka honey, particularly higher NPA rated manuka honey (NPA > 10+), which has undergone ripening, to have apparent C-4 sugars that exceed 7% and hence fail acceptance criteria for the AOAC 998.12 C-4 sugar method. Lesser studied honeys such as kamahi, tawari, and ling have also shown a tendency to fail the AOAC 998.12 method. In a subsequent paper, the relationship of apparent C-4 sugar

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AUTHOR INFORMATION

Corresponding Author

*(K.M.R.) Phone: +644 5704636. Fax: + 644 5704657. E-mail: [email protected]. Funding

We are grateful to AGMARDT, Ministry for Primary Industries and New Zealand honey industry cofunders for financial support of this project. Research has also been supported by GNS Science Direct Core Funding (DCF). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank numerous New Zealand beekeepers who contributed honey samples and generously shared information about their samples and production practices, with particular thanks to NZ Manuka Ltd. for NPA data. We also thank. Jonathan Stephens (Comvita New Zealand Ltd), Linda Newstrom-Lloyd (Trees for Bees), and Barry Foster (Beekeeper and National Beekeeping Association President 2012−2013) for supplying nectar and pollen samples.

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REFERENCES

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