Article pubs.acs.org/JAFC
The Unique Manuka Effect: Why New Zealand Manuka Honey Fails the AOAC 998.12 C‑4 Sugar Method Karyne M. Rogers,*,† Megan Grainger,§ and Merilyn Manley-Harris§ †
National Isotope Centre, GNS Science, 30 Gracefield Road, Lower Hutt, New Zealand Department of Chemistry, University of Waikato, Hamilton, New Zealand
§
ABSTRACT: Conversion of dihydroxyacteone (DHA) to methylglyoxal (MGO) has been shown to be the key mechanism for the growth in “apparent” C-4 sugar content in nonperoxide activity (NPA) manuka honey. This reaction is enhanced by heating and storage time and is demonstrated for the first time in clover honey adulterated with DHA purchased from a chemical supplier and in manuka honey containing naturally occurring DHA and MGO. After heating at 37 °C for 83 days, pure clover honey with no added DHA has the same apparent C-4 sugar content as at t = 0 days. The same clover honey adulterated with synthetic DHA added at t = 0 days and heated at 37 °C over the same time scale shows a change in apparent C-4 sugars from 2.8 to 5.0%. Four NPA manuka honeys heated over longer periods show an increase in apparent C-4 sugars of up to 280% after 241 days. This study strongly suggests that a protein fractionation effect occurs in the conversion of DHA to MGO in higher NPA manuka honey, rendering the remaining δ13C protein value more negative and falsely indicating C-4 sugar addition when using the AOAC 998.12 method. KEYWORDS: New Zealand, manuka, Leptospermum scoparium, methylglyoxal, apparent C-4 sugars, 5-hydroxymethylfurfural, dihydroxyacetone, AOAC 998.12, adulteration, carbon isotope, honey
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INTRODUCTION New Zealand manuka honey commands a premium price worldwide because of its nonperoxide antibacterial activity (NPA).1 The NPA is principally due to the presence of unusually high levels of methylglyoxal (MGO).2,3 MGO has been shown to derive from dihydroxyacetone (DHA) in the nectar of the manuka flower.4 Immature honeys contain large amounts of DHA and little MGO; during the course of maturation and storage, DHA is chemically converted to MGO, although this conversion is not stochiometric.4 The price for which manuka honey may be sold is directly proportional to the level of NPA and therefore proportional to the MGO content. To maximize MGO content in honey, beekeepers may resort to storage for protracted periods and/or warming (although it is generally accepted that warming of honey is not a good manufacturing practice and may affect honey quality parameters regulated in the Codex Alimentarius Standard).5 When temperature-controlled storage is not available, stored honeys may be subject to fluctuations in temperature due to climatic variation. During storage of honey and subsequent shelf life 5(hydroxymethyl)-2-furaldehyde (hydroxymethylfurfural or HMF) also forms chemically from sugars, principally fructose, and the formation may be accelerated by heat.6 HMF is regulated and, with certain specified exceptions, should not exceed 40 mg kg−1.5 Recently, sugar adulteration concerns have arisen because the AOAC 998.12 C-4 sugar method (a test for illicit addition of C4 cane sugar)7 has shown the frequent occurrence of apparent C-4 sugars in manuka honey. It has been shown that 70% of manuka honeys (n = 220 samples) with MGO > 263 mg kg−1 (NPA > 10+) tested in a survey failed the AOAC 998.12 C-4 sugar method8 and contained apparent C-4 sugars up to 15%. © 2014 American Chemical Society
In contrast, manuka honeys (n = 156 samples) with MGO < 250 mg kg−1 (NPA < 10+) gave only 11% failure. These fails were proposed to actually be false-positive results, the result of some unknown mechanism within the honey.8,9 False-positive results occurred when genuine honey exceeded the 7% threshold for apparent C-4 sugars because of a negative shift in the δ13C protein rather than a positive shift in the δ13C honey; the latter is usually attributed to C-4 sugar adulteration.10−12 Previously false-positive results reported in manuka honey have been attributed to pollen contamination of the extracted protein;13 however, even after filtration and centrifugation to remove pollen were applied, genuine manuka honey samples were still shown to exceed 7% C-4 sugars.9 This study reports for the first time the effects of storage temperature and storage time (aging and maturation) on apparent C-4 sugar, DHA, MGO, and HMF contents of clover honey with prior addition of DHA and of bioactive manuka honey to investigate why higher NPA honey has high apparent C-4 sugars.
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MATERIALS AND METHODS
Chemicals and Solvents. MGO (40% w/w), DHA (97%), and HMF (99%) were supplied by Sigma-Aldrich. o-(2,3,4,5,6-Pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA, 99+%) was supplied by Alfa Aesar. Hydroxyacetone (HA, ≥ 90%) was from Aldrich. Type I water (distilled and deionized) was obtained using a Barnstead E-pure system (18.0 MΩ·cm). HPLC grade acetonitrile (ACN) was obtained from Honeywell, Burdick and Jackson or Merck. HPLC grade methanol was supplied by Scharlau. Received: Revised: Accepted: Published: 2615
October 24, 2013 December 10, 2013 January 21, 2014 January 21, 2014 dx.doi.org/10.1021/jf404767b | J. Agric. Food Chem. 2014, 62, 2615−2622
Journal of Agricultural and Food Chemistry
Article
Table 1. Honey and Protein Carbon Isotopes, Apparent C-4 Sugar Content, DHA Remaining, MGO and HMF Contents, and DHA/MGO Ratio of Honey Inoculated with Various Levels of DHA Incubated at 37 °C for 83 Daysa DHA dosage (mg kg−1)
δ13C honey (‰)
δ13C protein (‰)
apparent C-4 sugars (%)
DHA (mg kg−1)
MGO (mg kg−1)
HMF (mg kg−1)
DHA/MGO
0 (t = 0) 0 (t = 83) 250 500 1000 2000 4000 8000
−26.3 −26.3 −26.4 −26.3 −26.4 −26.4 −26.4 −26.3
−26.8 −26.8 −26.9 −26.9 −27.0 −27.1 −27.2 −27.3
2.8 2.8 2.8 3.0 3.3 3.6 3.9 5.0
0.0 0.0 105.9 290.4 544.0 1119.4 2196.5 4127.3
35.9 39.1 87.4 140.9 249.2 477.6 893.4 1649.3
64.0 86.0 94.6 86.7 95.1 92.9 98.5 102.2
0 0 1.2 2.1 2.2 2.3 2.5 2.5
a
Values are reported as an average of three replicates. SDs are within 0.1‰ for isotopes and percentage relative SD of DHA, MGO, and HMF is
The Unique Manuka Effect: Why New Zealand Manuka Honey Fails the AOAC 998.12 C‑4 Sugar Method Karyne M. Rogers,*,† Megan Grainger,§ and Merilyn Manley-Harris§ †
National Isotope Centre, GNS Science, 30 Gracefield Road, Lower Hutt, New Zealand Department of Chemistry, University of Waikato, Hamilton, New Zealand
§
ABSTRACT: Conversion of dihydroxyacteone (DHA) to methylglyoxal (MGO) has been shown to be the key mechanism for the growth in “apparent” C-4 sugar content in nonperoxide activity (NPA) manuka honey. This reaction is enhanced by heating and storage time and is demonstrated for the first time in clover honey adulterated with DHA purchased from a chemical supplier and in manuka honey containing naturally occurring DHA and MGO. After heating at 37 °C for 83 days, pure clover honey with no added DHA has the same apparent C-4 sugar content as at t = 0 days. The same clover honey adulterated with synthetic DHA added at t = 0 days and heated at 37 °C over the same time scale shows a change in apparent C-4 sugars from 2.8 to 5.0%. Four NPA manuka honeys heated over longer periods show an increase in apparent C-4 sugars of up to 280% after 241 days. This study strongly suggests that a protein fractionation effect occurs in the conversion of DHA to MGO in higher NPA manuka honey, rendering the remaining δ13C protein value more negative and falsely indicating C-4 sugar addition when using the AOAC 998.12 method. KEYWORDS: New Zealand, manuka, Leptospermum scoparium, methylglyoxal, apparent C-4 sugars, 5-hydroxymethylfurfural, dihydroxyacetone, AOAC 998.12, adulteration, carbon isotope, honey
■
INTRODUCTION New Zealand manuka honey commands a premium price worldwide because of its nonperoxide antibacterial activity (NPA).1 The NPA is principally due to the presence of unusually high levels of methylglyoxal (MGO).2,3 MGO has been shown to derive from dihydroxyacetone (DHA) in the nectar of the manuka flower.4 Immature honeys contain large amounts of DHA and little MGO; during the course of maturation and storage, DHA is chemically converted to MGO, although this conversion is not stochiometric.4 The price for which manuka honey may be sold is directly proportional to the level of NPA and therefore proportional to the MGO content. To maximize MGO content in honey, beekeepers may resort to storage for protracted periods and/or warming (although it is generally accepted that warming of honey is not a good manufacturing practice and may affect honey quality parameters regulated in the Codex Alimentarius Standard).5 When temperature-controlled storage is not available, stored honeys may be subject to fluctuations in temperature due to climatic variation. During storage of honey and subsequent shelf life 5(hydroxymethyl)-2-furaldehyde (hydroxymethylfurfural or HMF) also forms chemically from sugars, principally fructose, and the formation may be accelerated by heat.6 HMF is regulated and, with certain specified exceptions, should not exceed 40 mg kg−1.5 Recently, sugar adulteration concerns have arisen because the AOAC 998.12 C-4 sugar method (a test for illicit addition of C4 cane sugar)7 has shown the frequent occurrence of apparent C-4 sugars in manuka honey. It has been shown that 70% of manuka honeys (n = 220 samples) with MGO > 263 mg kg−1 (NPA > 10+) tested in a survey failed the AOAC 998.12 C-4 sugar method8 and contained apparent C-4 sugars up to 15%. © 2014 American Chemical Society
In contrast, manuka honeys (n = 156 samples) with MGO < 250 mg kg−1 (NPA < 10+) gave only 11% failure. These fails were proposed to actually be false-positive results, the result of some unknown mechanism within the honey.8,9 False-positive results occurred when genuine honey exceeded the 7% threshold for apparent C-4 sugars because of a negative shift in the δ13C protein rather than a positive shift in the δ13C honey; the latter is usually attributed to C-4 sugar adulteration.10−12 Previously false-positive results reported in manuka honey have been attributed to pollen contamination of the extracted protein;13 however, even after filtration and centrifugation to remove pollen were applied, genuine manuka honey samples were still shown to exceed 7% C-4 sugars.9 This study reports for the first time the effects of storage temperature and storage time (aging and maturation) on apparent C-4 sugar, DHA, MGO, and HMF contents of clover honey with prior addition of DHA and of bioactive manuka honey to investigate why higher NPA honey has high apparent C-4 sugars.
■
MATERIALS AND METHODS
Chemicals and Solvents. MGO (40% w/w), DHA (97%), and HMF (99%) were supplied by Sigma-Aldrich. o-(2,3,4,5,6-Pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA, 99+%) was supplied by Alfa Aesar. Hydroxyacetone (HA, ≥ 90%) was from Aldrich. Type I water (distilled and deionized) was obtained using a Barnstead E-pure system (18.0 MΩ·cm). HPLC grade acetonitrile (ACN) was obtained from Honeywell, Burdick and Jackson or Merck. HPLC grade methanol was supplied by Scharlau. Received: Revised: Accepted: Published: 2615
October 24, 2013 December 10, 2013 January 21, 2014 January 21, 2014 dx.doi.org/10.1021/jf404767b | J. Agric. Food Chem. 2014, 62, 2615−2622
Journal of Agricultural and Food Chemistry
Article
Table 1. Honey and Protein Carbon Isotopes, Apparent C-4 Sugar Content, DHA Remaining, MGO and HMF Contents, and DHA/MGO Ratio of Honey Inoculated with Various Levels of DHA Incubated at 37 °C for 83 Daysa DHA dosage (mg kg−1)
δ13C honey (‰)
δ13C protein (‰)
apparent C-4 sugars (%)
DHA (mg kg−1)
MGO (mg kg−1)
HMF (mg kg−1)
DHA/MGO
0 (t = 0) 0 (t = 83) 250 500 1000 2000 4000 8000
−26.3 −26.3 −26.4 −26.3 −26.4 −26.4 −26.4 −26.3
−26.8 −26.8 −26.9 −26.9 −27.0 −27.1 −27.2 −27.3
2.8 2.8 2.8 3.0 3.3 3.6 3.9 5.0
0.0 0.0 105.9 290.4 544.0 1119.4 2196.5 4127.3
35.9 39.1 87.4 140.9 249.2 477.6 893.4 1649.3
64.0 86.0 94.6 86.7 95.1 92.9 98.5 102.2
0 0 1.2 2.1 2.2 2.3 2.5 2.5
a
Values are reported as an average of three replicates. SDs are within 0.1‰ for isotopes and percentage relative SD of DHA, MGO, and HMF is
