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3/2015

Authenticity of Honey

Quality AssuranceTrainingAdvisoryOutsourcingAuditingCertificationInspectionTesting

Author: Dr. Lutz ElfleinIntertek Food Services GmbH Bremen

A Major Analytical Challenge

Contact us for more details:www.intertek.com/foodgermany@intertek.com

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ent Authenticity of honey

A Major Analytical Challenge

Our Author: Lutz Elflein,

Intertek Food Services GmbH Bremen, lutz.elflein@intertek.com

Honey is a natural and almost untreated food produced by the bees. the indus-trial processing for packaging is reduced

to only necessary treatments of the raw honeys, like careful warming for liquefaction, homogeni-zation and filtration in order to maintain the va-luable properties. therefore, honey is highly es-teemed by consumers as an authentic, naturally pure and healthy product. Honey is commercially offered in many varieties, e.g. as flower honey, forest or honeydew honey, or as monofloral ho-ney of a certain botanical origin like acacia, rape, sunflower, citrus, lavender or chestnut. Further-more, there are specialty and premium honeys, for example manuka honey from new Zealand which is famous for its high natural antibacterial activity. Honey is not only used in form of table honey (breakfast spread or sweetener for tea), but has also major importance as an ingredient in the food industry replacing plain sugar.

Food which was produced close to nature, organic food and natural food like honey are highly demanded by consumers due to the growing awareness for nature and the environ-mental and health aspects of food. thus, both the food industry and the consumers are wil-ling to spend more money for these products. For this reason, it is very important to control the raw and finished products very carefully in order to verify whether the labelled quality and purity is actually true. the production, tra-de and sale of honey are a globalized business. those countries showing the highest demand and consumption of honey can usually produce only a small amount of the required quantities. therefore, the majority has to be imported from other countries. according to the latest

statistics [1], germany imports approx. 83% of the honey from foreign countries, the import quota of the eu and uSa are 57% and 66%, re-spectively. the major export markets are asia (China, india, Vietnam, thailand, new Zealand, australia), South and Central america (argen-tina, Mexico, Brazil, uruguay, Chili, Cuba) and South and eastern europe (Spain, Hungary, Ro-mania, ukraine, Bulgaria, italy, Poland, France).

While the legal regulations for honey are very stringent in the eu [2], and although the related Codex alimentarius Standard of Honey [3] is ack-nowledged worldwide, the individual national re-gulations and controls all over the world are quite different and not yet harmonized. this leads to different quality levels, different consumer expec-tations and also different legal opinions regarding the marketability of honey. in the eu, honey can only be sold under the label “honey”, if the pro-duct consists of 100% honey. this is not always the case elsewhere, e.g. there are products on the market in the uSa labelled as “honey syrup” or “imitation honey”. these products either contain only a small portion of honey or no honey at all. also, there are different types of honey on the market in asia, e.g. the “traditional style” honey as it is defined by eu law, but also honey which is produced by feeding bees with sugar syrup. Such a product would not be marketable under the la-bel “honey” in the eu and would be considered as adulterated equal to honey which was delibe-rately diluted with sugar syrup.

the continuously growing demand for honey is in contrast to the stagnating or partly decrea-sing production volumes worldwide due to cli-mate changes and the impairment of bee health e.g. by the agricultural use of pesticides and the

overall increasing industrialization. this fact and the lack of internationally harmonized rules for honey makes honey adulteration, i.e. diluting ho-ney with cheaper sugar syrups, very attractive in order to increase the available volumes of honey and gain financial profit. the term often used in this context is economically motivated adultera-tion (eMa) [4, 5], because such adulterated ho-ney can be produced, offered and purchased at significantly lower costs, thus increasing the profit margins. additionally, the competition for the lowest (best) price in the retail market and the still partly existing consumer attitude “the che-aper the better” contribute to the currently low market value of honey (except premium honeys) which is not in line with the rather high produc-tion and trading prices.

as a consequence of the horse meat scandal, the eu Commission published a report about food fraud in December 2013, listing the ten most frequently adulterated food items [6]. according to this ranking, honey is in the sixth place. nu-merous cases of honey adulteration have been recorded in the recent years [7, 8]. Sourcing and trading authentic and high quality honey is a dif-ficult task for producers, importers, exporters, packers and dealers under these circumstances. in order to manage this, measures of many kinds are necessary along the supply chain. Besides au-dits and traceability programs an almost gapless analytical testing scheme is performed on each production batch including a broad spectrum of analytical methods. therefore, honey can be considered as one of the best controlled food products in general.

the authenticity assessment is one of the most crucial parts of honey testing besides quality para-

meters, residues and contaminants. authenticity of honey includes on the one hand the determi-nation of the geographical and botanical origin in order to verify the accurate labeling of the ho-ney type and correct origin declaration according to the legal requirements. On the other hand, it must be evaluated whether honey has been adul-terated with foreign sugars or whether honey was produced by excessive sugar feeding of the bees. the microscopic analysis of the pollen spec-trum is still the reference method for the determi-nation of the geographical and botanical origin. For this purpose, harmonized and standardized protocols are available [9,10]. However, for adul-teration detection such harmonized and standar-dized methods do not exist so far. Hereinafter, several analytical methods will be discussed which are routinely used nowadays for the assessment of honey adulteration.

Verification of honey authenticity is a complex analytical task. the determination of the common quality parameters like sugar spectrum (fructose, glucose, sucrose, maltose, turanose, melezitose, erlose and other minor sugars), enzyme activities (diastase, invertase), hydroxymethylfurfural (HMF), prolin content, pollen spectrum and sensory may give hints at possible manipulation or substantiate them, but are usually not sensitive enough to de-tect and prove admixtures of foreign sugars to ho-ney. the reason is that honey is a natural product showing large compositional variations depending on the geographical origin, the botanical type and environmental factors which complicate the defi-nition of exact product specifications and the dis-tinction from non-authentic products. additionally, retail honeys are often commercial blends of vario-us geographical and botanical origins which make it even more difficult to set boundaries. therefore, the analytical methods applied for adulteration detection must be independent from these varia-bility factors as far as possible. From the historical aspect, the introduction of stable isotope analysis for honey testing was a milestone in this respect. 13C/12C carbon stable isotope ratio mass spectrome-tric analysis (δ13C-iRMS) can be used to differenti-ate between different sugar sources. as both the honey sugars (derived from nectar and honeydew collected by the bees) and the sugar syrups which may be used as adulterant originate from plants, the isotope ratios of the sugars are influenced by the photosynthetic pathway of the plant species. thus, it is possible to distinguish different natural or synthetic sugar sources according to their δ13C isotopic values. the first iRMS method for honey was established in the late 1980s and published as aOaC method [11]. the purpose of this method was to detect sugars from C4 plants (sugar cane and corn) in honey. the δ13C values of the isolated honey protein and the bulk honey are determined

by combusting the samples in an elemental analy-zer (ea) to carbon dioxide, nitrogen and water, and measuring the 13C/12C isotope ratio in the formed carbon dioxide with an isotope ratio mass spectro-meter (ea-iRMS). in case of authentic honey, the isotope values of the honey protein and the bulk honey are very similar showing only a slight natural variability of approx. ± 1‰. the isotopic value of the honey protein serves as an internal standard for the pure honey, because it is not altered when sugar syrup is added to honey (syrup does not contain any measurable protein). if C4 sugars (δ13C value approx. -10 ‰) are admixed to honey, the isotopic value of the bulk honey (δ13C value approx. -23 to -29 ‰, depending on the honey type) will shift towards more positive values while the corre-sponding δ13C protein value will remain unaltered. the more C4 sugars are added to honey the larger the difference between δ13C protein and δ13C ho-ney. the honey must be considered as adulterated, if the difference exceeds the natural variation bet-ween both values. However, as the calculation of the C4 sugar percentage is dependent on the ab-solute δ13C values of the protein and the honey, the limit value was not set for the maximum allowable natural variation between both values but for the resulting C4 sugar percentage calculated thereof. thus, honey is considered to be adulterated, if the C4 sugar percentage is ≥ 7%. this refers to a diffe-rence of δ13C protein minus δ13C honey showing values of approx. 0.9 to 1.4 ‰.

the aOaC method can only prove adultera-tion in case of negative differences, i.e. the δ13C value of honey is more positive than that of the

protein (adulteration with C4 sugars). However, a definitive result interpretation cannot be given in the reverse case of positive differences (δ13C value of protein more positive than that of ho-ney). One possible reason for such a deviation can be the adulteration with so-called C3 sugars derived from plants like wheat, sugar beet, rice or tapioca. But also other reasons can apply. except for South and Central america, adulteration of honey with C4 sugars plays only a minor role in other regions worldwide, while adulteration with C3 sugars is prevailing in europe and asia. as the aOaC method cannot detect C3 sugar adultera-tion, other and more sophisticated techniques are required which are able to prove this type of adulteration. the absolute δ13C isotopic values cannot be used for differentiation of honeys and C3 sugars in this case, because the isotopic values of nectar and honeydew from which the honey is produced is also derived from C3 plants. Here, a specific feature of honey can be utilized: the δ13C values of honey protein and the individual sugars of honey are almost identical in authen-tic honeys [12,13]. By comparing the individual deviations between the δ13C values of the diffe-rent honey fractions it can be evaluated whether the honey is authentic or has been manipulated with foreign sugars (C4/C3). the proper technical solution for this analytical problem is the online hyphenation of liquid chromatography (lC) with iRMS (lC-iRMS), a technique which is still not very common in food analysis. the sugar fractions of honey (fructose, glucose, di- and trisaccharides) are separated by lC and subsequently chemically

Fig. 1: δ13C EA/LC-IRMS for detection of honey adulteration. The protein isolated by pre-cipitation reaction from the honey is combusted in the elemental analyzer (EA) and the 13C/12C isotope ratio of the formed CO2 gas is measured by isotope ratio mass spectrome-try (IRMS). The individual sugar fractions (fructose, glucose, disaccharides and trisaccha-rides) are separated from each other by liquid chromatography (LC), chemically oxidized to CO2 in the interface, separated from the liquid eluent and the 13C/12C isotope ratios determined by IRMS.

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oxidized to carbon dioxide using an interface spe-cifically designed for this purpose. the carbon dio-xide is separated from the eluent with a selective gas-permeable membrane and transferred into the iRMS for δ13C measurement using helium as a purge and carrier gas. However, the δ13C value of the isolated honey protein cannot be measured in this way and must be determined by conventi-onal ea-iRMS instead (Fig. 1).

a reference database of authentic honeys has been built up in order to set purity crite-ria and limit values for authentic honeys. this reference database has been continuously ex-panded over the years and consists nowadays of more than 20’000 samples worldwide. the following limit values were defined: the δ13C values of fructose and glucose shall not differ more than ± 1‰; the δ13C values of protein, bulk honey, fructose, glucose, di- and trisaccha-rides shall not differ more than ± 2.1‰ [12-14]. if the honey in question exceeds these limit va-lues, it can be assumed that the honey is adul-terated with foreign sugars (Fig. 2).

the δ13C ea/lC-iRMS technique has become established over the last years and proved suc-cessful in the general routine screening for honey adulteration. thus, it is nowadays an inherent part of the current authenticity assessment of honey. it provides significant advantages com-pared to the aOaC method, as detection is in-dependent of the sugar type (C4/C3). However, like any other screening technique, there are also some drawbacks that have to be mentioned. in practice, often neither the honey type or its ori-

gin, nor the possible types of adulterant (syrup) are known. this makes it difficult to perform a more sophisticated evaluation and define stricter limit values depending on the honey variety and the type of sugar syrup. therefore, the defined limit values are a reasonable compromise regar-ding detection sensitivity and prevention of false

positive findings due to the natural variability of honey and considering commercial honey blends. Furthermore, this screening technique is primarily a qualitative method, because calculation of the percentage of foreign sugar is difficult and can lead to misinterpretations when the exact com-position of the pure honey and the syrup, respec-tively, are unknown. the general detection limits were empirically evaluated by spiking experiments and are typically ≥ 1% for C4 sugars and ≥ 10% for C3 sugars [12, 13]. nevertheless, sometimes the detection limits can be higher, particularly when honeys and syrups are mixed together which show very similar isotopic patterns (e.g. rape honey and rice syrup). in these cases, where the detection limit of the screening method is not suf-ficient, isotopic screening has to be complemen-ted with alternative analytical methods which are more specific and more sensitive for these types of adulteration. examples for such complemen-tary methods are the foreign enzymes methods detecting enzymes like beta-fructofuranosidase and beta-/gamma-amylases which are used in the production of invert sugar syrups from sucrose or starch [15,16]. additionally, there are methods detecting specific marker substances indicating the presence of sugar syrups in honey [17-20] by gC-MS, lC-MS or lC-elSD, for example the honey-foreign oligosaccharides (oligosaccharide ≥ DP 4) which are a remainder of the enzymatic starch degradation and do not occur naturally in flower or honeydew honey (Fig. 3).

Fig. 3: LC-ELSD chromatogram of authentic and adulterated honey. The adulterated sam-ple shows honey-foreign oligosaccharides (DP 5 to DP 10) which serve as marker sub-stances to detect adulteration.

Fig. 2: Excerpt from reference [13]. Example of a flower honey adulterated with approx. 11% rice syrup (C3 sugar). δ13C values: fructose -27,4 ‰, glucose -27,0 ‰, disaccharides –26,4 ‰, trisaccharides -24,3 ‰, honey-foreign oligosaccharides (DP >4, originating from rice syrup adulterant) -26,7 ‰. Difference of isotopic values: 3,1 ‰, failing the limit value of ± 2.1‰

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Of course, the disadvantage of these specific marker methods is that they can only detect and prove one certain type of adulteration.

Conclusively, several complementary methods have to be applied in order to perform a com-prehensive and reliable assessment of the honey authenticity. to date, there is no universal me-thod available for routine control being able to determine all the different types of honey adul-terants with sufficient detection sensitivity and robustness at the same time. the availability of such a universal method has been a long time requirement for a faster and more cost effective control of honey authenticity, but will still be part of current and future research.

as a lookout to future developments the current investigation of using nMR profiling technology for honey adulteration detection can be mentioned. nMR profiling has already been applied successfully for authenticity screening of fruit juices and wines. While nMR has been used in the context of honey so far mainly as an alternative or complementa-ry methodology to iRMS in order to determine δ13C/2H isotope ratio values of honey (so-called SniF-nMR) or for the botanical differentiation of different honey types [21-24], the 1H-nMR profi-ling approach for honey adulteration detection is rather new [25,26]. Here, an untargeted profiling of all substances contained in the honey and their concentration levels is performed and compared with the respective compound spectra of authen-tic honeys using chemometrics. Honeys showing an ‘untypical’ 1H-nMR profile (transgression of typical concentration ranges found for authentic honeys, detection of syrup marker compounds) are automatically considered as non-authentic or adulterated, respectively. However, there are still major challenges in this context: (i) to build up very comprehensive and representative reference data-bases which fully reflect the natural variability of the honey composition depending on the botani-cal and geographical origin, (ii) the consideration of possible compositional variations due to seasonal, productional or climatic factors and (iii) feasibility to detect the relevant adulteration markers despite the fact that nMR is not the method of choice for trace substances (i.e. the marker substances) occur-ring only at very low concentration levels in adulte-rated honeys. at present, nMR profiling has to be complemented with other analytical methods like stable isotope analysis and mass spectrometric tar-get screening of adulteration markers in order to achieve reasonable detection levels (5-10% foreign sugars) at all times. Furthermore, there is still the difficulty in proving the causal relationship bet-ween an untypical 1H-nMR profile and the admix-ture of foreign sugars in case no signals of known adulteration markers (e.g. honey foreign oligosac-charides) are visible. Further experience will tell

whether this new analytical approach will become a regular feature in the authenticity assessment of honey in future.

References:[1] FAOSTAT (http://faostat.fao.org)[2] EU Honigrichtlinie 2001/110/EG in der Fassung

vom 23.06.2014 (http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:02001L0110-20140623&from=EN)

[3] Codex Alimentarius Standard of Honey, 12-1981, Rev. 2-2001 (http://www.codexa-limentarius.org/download/standards/310/cxs_012e.pdf)

[4] Economically Motivated Adulteration of Honey: Quality Control Vulnerabilities in the International Honey Market (http://www.foodprotection.org/files/food-protection-trends/Jan-Feb-14-everstine.pdf)

[5] Food Fraud and “Economically Motivated Adulteration” of Food and Food Ingredients, CRS Report Januar 2014 (http://fas.org/sgp/crs/misc/R43358.pdf)

[6] European Commission, REPORT on the food crisis, fraud in the food chain and the control thereof (2013/2091(INI)),

[7] Food Fraud Database (www.foodfraud.org)[8] EU RASFF Portal (https://webgate.ec.europa.

eu/rasff-window/portal/)[9] DIN 10760, Honig - Untersuchung der rela-

tiven Pollenhäufigkeit[10] International Honey Commission, Har-

monized methods of Melissopalynology, Apidologie 35 (2004) S18–S25

[11] AOAC official methods of analysis, Kapitel 44.4.18A, Methode 998.12 (1998, rev. 2013): C-4 plant sugars in honey, internal standard stable carbon isotope ratio method

[12] Cabañero A.I., Recio J.L., Ruperez M. (2006), Liquid chromatography coupled to isotope ratio mass spectrometry: a new perspective on honey adulteration detection, J. Agric. Food Chem. 54, 9719–9727.

[13] Elflein L., Raezke K.-P. (2008), Improved detection of honey adulteration by measu-ring differences between 13C/12C stable carbon isotope ratios of protein and sugar compounds with a combination of elemental analyzer - isotope ratio mass spectrometry and liquid chromatography - isotope ratio mass spectrometry (δ13C-EA/LC-IRMS), Api-dologie 39, 574-587

[14] Fei X. et al., Honey adulteration detection using liquid chromatography / elemental analysis – isotope ratio mass spectrometry (2011), Chin. J. Chromatogr. 29, 15-19

[15] Bednar M. and Titera D. (2010), Non-authen-tic enzymes in honey, Journal of ApiProduct and ApiMedical Science 2: 102 - 128

[16] Elflein L. et al. (2012), Honey Authenticity: Overview of state-of-the-art methodology and new analytical developments for the detection of honey adulteration with su-gar syrups, II International Symposium on Bee Products, Braganca, Portugal (http://www.ipb.pt/ihc2012/imagens/itf147.pdf)

[17] Megherbi M. et al. (2009), Polysaccharides as a Marker for Detection of Corn Sugar Syrup Addition in Honey, J. Agric. Food Chem. 57, 2105-2111

[18] Ruiz-Matute A.I. et al. (2007), A new tech-nology based on GC-MS to detect honey adulteration with commercial syrups, J. Agric. Food Chem. 55, 7264-7269

[19] Rommerskirshen F. and Elflein L., Deve-lopment and validation of simple, cost effective and easy to use tests which can be used by the honey industry to verify honey authenticity concerning adultera-tions with sugar syrup. National Honey Board, USDA/AMS Final Project Report, September 2012

[20] Xue X. et al. (2013), 2-Acetylfuran-3-Gluco-pyranoside as a Novel Marker for the Detec-tion of Honey Adulterated with Rice Syrup, J. Agric. Food Chem. 61, 7488-7493

[21] Giraudon S. et al. (2000), Deuterium Nuclear Magnetic Resonance Spectroscopy and Stable Carbon Isotope Ratio Analysis/Mass Spectrometry of Certain Monofloral Honeys, J. AOAC Int. 83, 1401-1409

[22] Cotte J.F. et al. (2007), Study and validity of 13C stable carbon isotopic ratio analysis by mass spectrometry and 2H site-specific natural isotopic fractionation by nuclear magnetic resonance isotopic measure-ments to characterize and control the authenticity of honey, Anal. Chim. Acta 582, 125–136

[23] Consonni R. et al. (2013), Geographical discri-mination of honeys by saccharides analysis, Food Control 32, 543-548

[24] Ohmenhäuser M. et al. (2013), Qua-litative and Quantitative Control of Honeys Using NMR Spectroscopy and Chemometrics, ISRN Analytical Chemistry, Article ID 825318, 9 pages, http://dx.doi.org/10.1155/2013/825318

[25] Bertelli D. et al. (2010), Detection of Honey Adulteration by Sugar Syrups Using One-Dimen-sional and Two-Dimensional High-Resolution Nuclear Magnetic Resonance, J. Agric. Food Chem. 58, 8495-8501

[26] Spiteri M. et al., Fast and global au-thenticity screening of honey using 1H-NMR profiling, Food Chemistry, available online 25 Nov. 2014, http://dx.doi.org/10.1016/j.food-chem.2014.11.099

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Ensuring Food Safety from Farm to Fork.

Benefi t from our global network

which can provide you with a complete

range of tailor-made services along the whole

supply chain.

Intertek Food Services GmbHOlof-Palme-Straße 828719 Bremen, GermanyT.: +49 421 65 727 390food@intertek.com

www.intertek.com/food Watch the video and see how

we can support your business:

Analysis and Testing - food, food packaging and food contact materials - Sensory testing- Microbiology- Nutritionals- Residues (like pesticides, veterinary drugs) and contaminants (like heavy metals and mycotoxins)- GMO- Authenticity- Interpretation of results acc. to current food law

Auditing & Certifi cation- IFS, BRC, FSSC 22000, Halal- supplier audits

Inspection & Sampling

Labelling & Marketability

Risk analysis &

Supply chain management

Training & Workshops

Reliable services for safe food

Benefi t from our global network

which can provide you with a complete

Ensuring Food Safety from Farm to Fork.

Benefi t from our global network

which can provide you with a complete

range of tailor-made services along the whole

supply chain.

Intertek Food Services GmbHOlof-Palme-Straße 828719 Bremen, GermanyT.: +49 421 65 727 390food@intertek.com

www.intertek.com/foodWatch the video and see how

we can support your business:

Analysis and Testing - food, food packaging and food contact materials - Sensory testing- Microbiology- Nutritionals- Residues (like pesticides, veterinary drugs) and contaminants (like heavy metals and mycotoxins)- GMO- Authenticity- Interpretation of results acc. to current food law

Auditing & Certifi cation- IFS, BRC, FSSC 22000, Halal- supplier audits

Inspection & Sampling

Labelling & Marketability

Risk analysis &

Supply chain management

Training & Workshops

Reliable services for safe food

Benefi t from our global network

which can provide you with a complete

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