Honeydew Honeys: A Review on the Characterization and

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Honeydew honeys: a review on characterization and authentication of the botanical and geographical origin Consuelo Pita-Calvo, and Manuel Vazquez J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05807 • Publication Date (Web): 20 Feb 2018 Downloaded from http://pubs.acs.org on February 26, 2018

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Honeydew honeys: a review on characterization and authentication of the

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botanical and geographical origin

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Consuelo Pita-Calvo and Manuel Vázquez*

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Department of Analytical Chemistry, Faculty of Veterinary Science, University of

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Santiago de Compostela, 27002-Lugo, Spain

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* Corresponding author. Email: [email protected]

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ABSTRACT

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The commercial interest in honeydew honeys (from secretions of plants or

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excretions of plant-sucking insects on plants) is increasing due to the higher therapeutic

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properties than those of most blossom honeys (from nectar). However, honeydew

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honeys have been less studied than blossom honeys. In this work, studies carried out to

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characterize and authenticate honeydew honeys by botanical and geographic origins

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have been reviewed. The identification of the honey origin has been approached by two

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ways: chemical markers and development of analytical methodology combined with

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multivariate analysis. Some compounds have been suggested as specific botanical

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markers of several honeydew honeys: quercitol and trans-oak lactone for oak honey; 2-

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aminoacetophenone and propylanisol for holm oak; and 1-chloro-octane and tridecane

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for pine honey. 3-Carene and an unidentified compound in samples were proposed to

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discriminate between Greek and Turkish pine honeys. Chemometric analysis has been

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applied on chemical composition and physicochemical, microscopic, or spectra

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parameters and proved to be a valuable way for authenticating honeydew honeys. The

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analytical methods based on spectra information are suitable for the routine control of

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the honeydew honeys origin because they are fast and require an easy sample

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preparation.

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Keywords: honey; honeydew; chemical characterization; botanical authentication;

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geographical authentication; botanical chemical markers; geographical chemical

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markers; classification; multivariate analysis

33 34

1. INTRODUCTION

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Honey is the natural sweet substance produced by bees (Apis mellifera). It is a

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compound blend primarily constituted by carbohydrates (70-80% w/w) and water (10-

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20% w/w). The most abundant carbohydrates are glucose (≈ 31% w/w) and fructose (≈

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38% w/w) and disaccharides of them 1. Honey also contains a high number of minor

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components. It possess health-promoting properties, such as antioxidant, antibacterial,

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free radical-scavenging, and anti-inflammatory activity as well as anti-tumoral efficacy

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which depend on the flora from which honey was produced

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reported to have an inhibitory effect on monoamine oxidase 5 and carbonic anhydrase 6,7

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which is considered to be derived mostly from honey polyphenols.

2–4

. Honey has been

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Honey is rich in both enzymatic (catalase, glucose oxidase, and peroxidase) and

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non-enzymatic antioxidant substances (phenolic compounds, ascorbic acid, α-

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tocopherol, carotenoids, organic acids, amino acids, proteins, and Maillard reaction

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products)

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infections. Antimicrobial activity of honeys has been attributed to the high osmolarity

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(high sugar concentration), low pH, hydrogen peroxide, bee defensin-1, and

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methylglyoxal

8,9

. Honey can be used as a topical antibacterial agent for surface wound

10

. The strong antibacterial activity of Leptospermum honeys which

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include New Zealand manuka honey (Leptospermum scoparium) and Australian jelly

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Bush honey (Leptospermum polygalifolium) is attributed to their high concentrations of

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methylglyoxal

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Revamil®, medical grade honey, and it substantially contributes to its antibacterial

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properties

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considered partially responsible for honey antibacterial activity. Honey phenolic

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composition and consequent antibacterial and antioxidant properties depend on the

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botanical origin of honey 2.

12

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. Bee defensine-1 has been identified in honey used as a source for

. Along with methylglyoxal and defensine-1, phenolic compounds are

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Honey can be classified according to the original raw plant in: i) blossom (floral)

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or ii) honeydew honey. Bees make blossom honey from the nectar of the flowers of

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blossoming plants. Otherwise, honeydew honey is produced from secretions of plants

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(genera Pinus, Abies, Castanea, and Quercus, among others) or excretions of plant-

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sucking insects, mostly from the family Aphididae, on plants (EU Directive 110/2001,

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http://eur-

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lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2002:010:0047:0052:EN:PDF).

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The honeydew secreted by insects cannot be considered as excrement because plant sap

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is not digested in the insect stomach 13.

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The commercial interest in honeydew honeys is increasing due to higher

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therapeutic properties than those of blossom honeys. Higher antibacterial and

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antioxidant properties have been found in honeydew honeys than in blossom honeys

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8,14,15

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honeys 16. Therefore, discrimination between honeydew and blossom honeys is of great

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interest to avoid adulterations and frauds.

. Furthermore, the food industry appreciates the strong flavour of this kind of

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Honeydew honeys and blossom honeys show different chemical composition

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and physicochemical and biochemical properties as concluded from studies carried out

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that were reviewed recently

. Honeydew honeys are darker than the most blossom

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honeys. They are also usually described by higher values of several physicochemical

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variables used in routine quality control of honeys such as electric conductivity (EC),

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acidity, pH, or ash. EC is used in quality control to discriminate between both types of

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honeys. The European legislation (EU Directive 110/2001) states that blossom honey

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and blends of these honeys must have EC values ≤0.8 mS/cm. This rule does not apply

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to certain honeys such as bell heather (Erica), strawberry tree (Arbutus unedo), manuka

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or jelly Bush (Leptospermum), ling heather (Calluna vulgaris), tea tree (Melaleuca sp.),

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lime (Tilia sp.), and eucalyptus honeys. Honeydew and chestnut honeys or

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combinations of them with blossom honeys (except those cited) must have EC ≥ 0.8

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mS/cm. An extremely high EC value, 3.07 mS/cm, was found in Salix sp. honeydew

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honey 18.

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Another important physical property which can be used to differentiate blossom

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honeys from honeydew honeys is optical rotation. Honey exhibits the property of

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rotating the polarization plane of polarized light. Its specific optical rotation depends to

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a large extent on the types and relative amounts of carbohydrates present in honey.

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Honeydew or adulterated honeys are generally dextrorotatory whereas blossom honeys

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are levorotatory 19.

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Honeydew honeys also present a higher oligosaccharides content, mainly

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melezitose and raffinose, and a lower content of monosaccharides than blossom honeys

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17

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honeys

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they increase the population of bifidobacteria and lactobacilli in human gut 21.

. Melezitose (a trisaccharide) is considered as a distinguishing feature of honeydew 20

. Oligosaccharides from honey showed probable prebiotic activity because

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Honeydew honeys have been less studied than blossom honeys. Given the

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growing interest in honeydew honeys, especially for their superior therapeutic

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properties, it is interesting to summarize the studies that have been carried out to

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characterize and authenticate the botanical and geographic origins of this kind of

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honeys. To our knowledge, no review articles on honeydew honeys exist about this

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subject.

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Studies of authentication of honeydew honeys have been approached in two

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ways: On one way, the isolation and identification of specific chemical markers for their

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botanical or geographical origin; on the other way, the development of analytic methods

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joined to the chemometric processing of data for the authentication of honeydew honey

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origin. In the next sections, these two approaches will be discussed.

110 111

2. CHARACTERIZATION OF HONEYDEW HONEYS BY BOTANICAL AND

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GEOGRAPHICAL ORIGIN: CHEMICAL MARKERS

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Honeydew honeys of several botanical and geographical origins have been

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characterized based on physicochemical parameters and chemical composition. In the

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last years, there has been great interest in characterizing honeydew honey using specific

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markers to authenticate its origin. Markers compounds have been identified amongst

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phenolic and volatile constituents of these honeys.

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Finding reliable chemical markers for the botanical origin is rather difficult

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because honey chemical composition depends not only on the species of plant that

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produces honeydew or nectar but also on other factors such as the geographic area, bee

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species, collection season, weather conditions, time of honey ripening, nature of bees

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pastures, storage mode, and harvest conditions and technology, among others

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Moreover, honeys of the same botanical origin may result in a different chemical

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composition depending on the analytical technique and sample preparation procedure

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employed.

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The following subsections are dedicated to different categories of compounds:

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carbohydrates, volatile constituents, and phenolic compounds. Compounds suggested as

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botanical chemical markers of several honeydew honeys are listed in Table 1. Several

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compounds were proposed to discriminate several honeydew honeys from each other.

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They are shown in Table 2. Compounds suggested as geographical chemical markers of

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the same botanical origin are shown in Table 3.

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2.1. Carbohydrates. Honey carbohydrates are the result of the action of several

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bee saliva enzymes on sugars of nectar or honeydew. Honeydew honeys of different

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countries and botanical origins were characterized on the basis of their carbohydrate

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composition. However, no chemical marker has been found within this category of

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compounds. Results about carbohydrate composition is shown in Table 4. Cyclitols

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(carbohydrate-related compounds) were studied to investigate their possible use as

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markers of Quercus sp. honeydew honeys.

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Chromatographic techniques are widely used to isolate, identify, and quantify

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carbohydrates in honeys. They include high-performance liquid chromatography

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(HPLC) with several detectors such as refractive index detector (RID), diode array

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detector (DAD), pulsed amperometric detection (PAD) or evaporative light scattering

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detector (ELSD). Gas chromatography (GC) with flame ionization detector (GC-FID) or

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mass spectrometry (GC-MS) are also commonly used. The GC methods require a

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previous derivatization in order to obtain volatile carbohydrate compounds 11.

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GC-MS was used to determine the carbohydrate composition in Spanish honeys

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from Abies sp. (fir) and Quercus ilex (holm oak). In Abies sp. honeys, the amounts of

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fructose and glucose were 33.1% (w/w) and 24.2% (w/w), respectively. In the sample of

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Quercus ilex honey analyzed, the amounts of fructose and glucose were 38.3% (w/w)

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and 26.3% (w/w), respectively. The main disaccharides found in Abies sp. samples

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were: turanose (4.56), maltulose (3.48), isomaltose (3.1), kojibiose (2.95), and

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trehaluose (2.15). In Quercus ilex honeys were: maltose (2.3), turanose (2.1), maltulose

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(1.9), and kojibiose (1.9). In relation to the trisaccharides, Quercus ilex honeys

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presented higher amounts of raffinose, 0.1% (w/w); kestose, 0.36% (w/w); and

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melezitose, 0.3% (w/w) than Abies sp. honeys. Furthermore, raffinose was not detected

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in Abies sp. samples

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composition between the two botanical origins, results should be taken with caution due

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to the small number of samples analyzed (holm oak: n=2; fir: n=1).

24

. Although differences were found in the carbohydrate

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HPLC-RID was applied to the determination of carbohydrates in Turkish

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honeys. In this case, samples belonged to Quercus robur L. (oak) (n=3) and Pinus L.

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(pine) (n=4) honeys. The amounts of fructose and glucose found in pine honey were

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39.80% (w/w) and 23.67% (w/w), respectively; in oak honey were 43.28% (w/w) and

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21.73% (w/w), for fructose and glucose, respectively. In pine honeys the amounts of

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trehalose, melezitose, maltose, and melibiose found were (% w/w): 0.23, 0.62, 0.54, and

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0.35, respectively. In oak honey the amounts of trehalose, melezitose, and maltose

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found were (% w/w): 0.42, 0.94, and 0.19, respectively; melibiose was not detected.

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Sucrose was not detected in any of the two types of honey studied

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sugar was detected in Spanish honeys from Quercus ilex (0.79% w/w) and Abies sp.

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(0.13% w/w) by GC-MS

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2.59% w/w)

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performance anion exchange chromatography (HPAEC-PAD). This could be explained

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by the higher sensitivity of both techniques.

26

24

25

. However, this

, in fifty-seven samples of French Abies sp. honeys (0.45-

and in New Zealand Nothofagus sp. (beech) honey (n=1)

27

by high-

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The average amounts of fructose, glucose, erlose, raffinose, and melezitose

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found in Abies sp. honeys (n=57) from French origin were 33.37, 25.63, 0.82, 1.56, and

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2.22% (w/w), respectively, with wide concentration ranges for erlose (0.320-1.11),

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raffinose (0.920-2.860), and melezitose (0.960-3.120) 26. In Quercus sp. honeys (n=19)

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of Spanish origin, the amounts of fructose and glucose found were 32.80% (w/w) and

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27.22% (w/w), respectively 28.

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Isomaltose was the most abundant disaccharide found in Nothofagus sp. honey

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(n=1) from New Zealand, while melezitose, panose, and maltotriose were significant

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trisaccharides 27. Metcalfa honey is a type of honeydew honey produced mainly in Italy.

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It is rich in maltotriose and contains particularly high amounts of dextrins 29,30.

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As far as we know, very few studies on the carbohydrate composition of

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different honeydew honey types have been carried out. Moreover, they have been only

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focused on a few botanical origins and countries. In most of these studies, the number of

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samples analyzed is insufficient to characterize honeydew honeys and, additionally,

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only a few carbohydrates have been identified and quantified. Obviously, a lot of

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studies are needed to characterize honeydew honeys of different botanical and

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geographical origins on the basis of their carbohydrate composition and draw clear

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conclusions. It would also be interesting to know to what extent the composition of

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carbohydrates and, in general, the chemical composition of honeydew honeys from a

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certain botanical origin can be affected by geographical location within the same

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country as well as the other factors mentioned at the beginning of this section.

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Quercitol (acorn sugar or 1,3,4/2,5-cyclohexane-pentol) is a minor constituent in 31

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Quercus acorns, leaves, and bark

. Quercitol and other cyclitols possess antiradical

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activity. Quercitol also shows inhibitor activity of glucosidase which blocks the

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absorption and metabolism of carbohydrates

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healthy properties of oak honey

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carbohydrates by GC-FID and proposed as a specific marker for Quercus sp. honeys. In

33

32

. Therefore, it can also contribute to the

. Quercitol has been determined along with

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the seventeen samples of honeydew honey analyzed, its content ranged from 0.013 to

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1.5% (w/w) and in the thirteen blossom honeys analyzed between 0 (most samples) and

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0.010% (w/w)

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blossom and honeydew honeys

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the two samples of Quercus ilex honey analyzed (0.23 and 0.49% w/w), which would be

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in agreement with the proposal of considering quercitol as a marker for Quercus sp.

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honeys

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resonance (NMR) spectroscopy 34. Using this technique, quercitol was identified in the

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fifteen samples of oak honey analyzed whereas the samples of blossom honey (n=6) and

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other honeydew honeys (from fir and spruce) studied (n=2) did not contain it.

21

21

. Other researchers also analyzed quercitol and other polyalcohols in 24

. Noticeable amounts of quercitol were only found in

. Quercitol was also identified in oak honeydew honeys by nuclear magnetic

211 212

2.2. Volatile compounds. Aroma is a very sought-after property in honey. The

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honey aroma is formed by volatile substances which may come from honeydew or

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nectar used by the bees to make honey. Therefore, aroma depends significantly on the

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source plant and maturity of the honey. Additionally, honeybees can produce flavor

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components or these originate during the thermal processing and storage of the honey 22.

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Volatile substances in honey belong to a great variety of chemical compounds.

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Results about the characterization of honeydew honeys on the basis of their volatile

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composition are summarized in Table 5. The study of botanical chemical markers

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should be dedicated to find constituents derived from plants such as benzene derivatives,

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norisoprenoids, terpenes, and terpene derivatives

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derivatives cannot be considered as potential biomarkers because they are present at

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high concentrations in all types of honey. They include benzaldehyde, benzyl alcohol,

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2-phenylethanol, and phenylacetaldehyde, among others

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to measure volatile compounds in honey is GC-MS with different extraction methods:

16

. However, several benzene

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headspace solid-phase micro-extraction (HS-SPME/GC-MS), solid-phase micro-

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extraction (SPME/GC-MS), purge and trap (purge and trap/GC-MS), ultrasonic solvent

228

extraction (USE/GC-MS), and simultaneous distillation-extraction (SDE/GC-MS).

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Different volatile composition has been reported depending on the extraction procedure

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employed and, if applicable, polarity of the solvent used. This fact makes it difficult to

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characterize honeys according to their origin and the search for chemical markers. As an

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example, methyl salicylate, a compound suggested as chemical marker for Salix sp.

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honeys, was identified in headspace volatiles by HS-SPME/GC-MS whereas it was not

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detected when USE was use as a extraction method 18.

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Simultaneous distillation–extraction, ultrasonic extraction, and other traditional

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extraction techniques such as supercritical fluid extraction, liquid–liquid extraction, or

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solid-phase extraction are laborious and time-consuming. Moreover, they require the

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use of large quantities of solvents, which are generally toxic and costly with the

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consequent danger to human health and the environment. On the other hand, it has been

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reported that the recovery of terpenic and norisoprenoid compounds in rosemary

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blossom honey was better using SDE than other methods, such as liquid-liquid

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extraction or solid-phase extraction 37. HS-SPME is the most commonly technique used

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for extraction of volatile fraction from honeys. When compared with traditional

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techniques, it shows important advantages; it is a solvent-free simple technique which

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integrates sampling, isolation and enrichment of analytes into one step. However, it

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requires the optimization of the conditions of its use in terms of SPME fiber type,

247

temperature and sorption time, as well as desorption temperature with consequent time

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consumption 38.

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In some cases, HS-SPME/GC-MS and USE/GC-MS using separately different

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extraction solvents were applied simultaneously. Both techniques provided different

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chromatographic profiles for the samples analyzed, which were complemented allowing

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a better characterization of honeys aroma 18,39–41.

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Some studies have been carried out to characterize honeydew honeys from

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different botanical origins and countries, mainly from Spain, Greece, Turkey, and

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Croatia, on the basis of their composition in volatile compounds. A micro-scale SDE/GC-MS method was used to study the volatile profile of

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16

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Spanish holm oak, oak, and forest (blend of holm oak and oak) honeys

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abundant of the sixty-six volatile compounds found in samples (n=9) analyzed was

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phenylacetaldehyde. Certain norisoprenoids, such as isophorone and ketoisophorone,

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were also plentiful in all samples. The terpene fraction included: two isomers of

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epoxylinalool, linalool oxide, α-terpineol, eugenol, car-2-en-4-one, hotrienol, p-cymen-

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8-ol, and 2-hydroxycineol. Hydroxyketones (principally 3-hydroxy-2-butanone) were

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also found in the volatile fraction. Some compounds were suggested as chemical

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markers: 2-aminoacetophenone (no detected in oak honey) and propylanisol for holm

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oak honey, and trans-β-methyl-γ-octalactone (no detected in holm oak honey) for oak

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honey. However, 2-aminoacetophenone has also been suggested as marker for chestnut

267

honey

268

botanical of holm oak honey. Trans-β-methyl-γ-octalactone is also known as trans-oak-

269

lactone because it has been found only in oak wood. This compound contributes

270

significantly to the aroma of oak honey and can be found in two isomeric forms that

271

have very low odour thresholds: 1 µg/kg for the cis isomer and 2 µg/kg for the trans

272

isomer 44.

273

42

and rhododendron honey

43

. The most

, leaving in question this compound as marker

Spanish honeydew honeys from Abies sp. (n=1) and Quercus ilex (n=2) were 24

274

analyzed using HS-SPME/GC-MS

. Comprehensive information on the volatile

275

composition of the three samples analyzed are not indicated, only the main volatile

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compounds found. In any case, studies with a greater number of samples should be

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carried out in order to characterize these honeydew honeys. In the Abies sp. honey

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sample, the main compounds found were: butanoic acid, methyl butyrate, α-pinene, α-

279

phellandrene, and 1,8-cineole. In Quercus ilex honeys were: dimethyl sulfide, 3-methyl-

280

1-butanol, 3-methyl-3-buten-1-ol, cis-linalool oxide, and isophorone. High amounts of

281

acetic acid were also found; these compounds except dimethyl sulfide, were also

282

identified in oak, holm oak, and forest honeys of Spanish origin. High concentrations of

283

acetic acid seem to be characteristic of honeydew honeys 45,46.

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Volatile composition of Croatian Quercus frainetto honeys (n=2) was studied

285

using HS-SPME/GC-MS and USE/GC-MS. A more complete and representative

286

information of the volatile composition of this kind of honeys was obtained using

287

simultaneously both techniques

288

volatile organic headspace compounds and obtain a reliable composition of the most

289

volatile organic constituents of honey samples. The most abundant organic compounds

290

in the headspace, of the thirty found, were terpenes (cis- and trans-linalool oxides,

291

hotrienol, and epoxylinalool), mainly the isomers of linalool oxide. These compounds

292

(linalool oxide as the isomer Z) were also found in Spanish oak, holm oak, and forest

293

honeys. Another important group of organic compounds found in samples were

294

aliphatic acids: octanoic, nonanoic, decanoic, and hexadecanoic acids. USE

295

methodology allowed to know volatile and less-volatile organic composition. This

296

extraction procedure does not require heat and thus a thermal generation of artifacts is

297

avoided. Two solvents, dichloromethane and a mixture (1:2 v/v) of pentane-diethyl

298

ether, were studied separately which led to different analytical results. Phenylacetic acid

299

was the most abundant of shikimic pathway derivatives. This compound was not found

300

in the samples of Spanish honeys which may be due to the use of a different extraction

39

. HS-SPME/GC-MS allowed to identify the most

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method. Other abundant benzene derivatives were also not identified in the Spanish

302

honeys:

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hydroxycinnamic acid, and methyl syringate. Although Quercus frainetto belongs to the

304

oak species, trans-oak-lactone, proposed as a chemical marker for oak honey, was not

305

detected in the two analyzed samples.

benzoic

acid,

4-hydroxybenzoic

acid,

4-hydroxybenzyl

alcohol,

4-

Greek Pinus sp. honey (n=39) has been characterized according to its volatile

306

47

307

composition (HS-SPME/GC-MS) and physicochemical parameters

308

compound classes were identified: esters, alcohols, carboxylic acids, aldehydes, ketones,

309

hydrocarbons, among others. Most of the identified esters may originate from resinous

310

components of pine tree. Numerous volatile compounds found have been previously

311

identified in Greek and Turkish pine honeys

312

worth noting the presence of 2,3-butanediol in the samples. This compound has been

313

considered by several researchers as characteristic of the honeydew honeys

314

contrast to 47, other researchers have identified 1-chloro-octane and tridecane in Greek

315

and Turkish pine honeys using purge and trap/GC-MS and have proposed them as

316

botanical chemical markers for pine honey 13.

13

. Various

as well as some blossom honeys. It is

45,46

. In

317

Other researchers have suggested several volatile compounds, identified by HS-

318

SPME/GC-MS, as chemical markers to discriminate between Greek Pinus sp. (n=39)

319

and Abies sp. (n=31) honeys. On the one hand, several compounds were identified only

320

in

321

ylbicyclo[3.1.0]hexan-3-one (β-thujone) in pine honey; and 6-methyl-5-hepten-2-one,

322

2-hydroxy-3,5,5-trimethylcyclohex-2-en-one, 3,4,5-trimethylphenol, and 6,10-dimethyl-

323

5,9-undecadien-2-one (geranyl acetone) in fir honey. On the other hand, higher amounts

324

of certain compounds were found in honey samples of a particular botanical origin:

325

ethyl esters of the hexanoic, heptanoic, octanoic, nonanoic, decanoic, dodecanoic, and

one

kind

of

honey:

1-nonanol

and

(1S,4S,5R)-4-methyl-1-propan-2-

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326

tetradecanoic acids, nonanal, nonane, 1-(2-furanyl)-ethanone, and (Z)-5-methyl-4-

327

nonene in fir honey; and octane in pine honey 48. Other researchers did not identify β-

328

thujone in pine honeys of Greek and Turkish origin using purge and trap/GC-MS which

329

may be due to the different extraction technique used 13.

330

Polish fir (Abies alba Mill.) honeydew honey was characterized using three

331

analytical techniques (HPLC-DAD, USE/GC-MS, and HS-SPME/GC-MS) which

332

allowed to identify a wide range of compounds. Typical physicochemical parameters

333

were also determined. The chemical profiles were dominated by source plant-originated

334

benzene derivatives: 3,4-dihydroxybenzoic acid (protocatechuic acid) determined by

335

HPLC-DAD, methyl syringate by USE/GC-MS or benzaldehyde by HS-SPME/GC-MS.

336

Other important compounds identified were terpenes including norisoprenoids, mainly

337

4-hydroxy-3,5,6-trimethyl-4-(3-oxobut-1-enyl)cyclohex-2-en-1-one (USE/GC-MS), and

338

monoterpenes, mainly linalool derivatives as well as borneol (HS-SPME/GC-MS)

339

The results obtained in this study differed a lot from those obtained by other researchers

340

in fir honeys from Greece

341

enyl) cyclohex-2-en-1-one, coniferyl alcohol isomers, borneol, and benzaldehyde were

342

proposed for fir honeydew honey screening. According to the authors of this study,

343

there was no report on occurrence of coniferyl alcohol isomers in any honey variety

344

type including honeydew honeys. On the other hand, protocatechuic acid was suggested

345

as a potential marker of fir honeydew honey regardless of the geographical origin

346

because this compound was also previously found in fir honeys by several researchers

347

41

48

and Spain

24

41

.

. 4-Hydroxy-3,5,6-trimethyl-4-(3-oxobut-1-

. However, protocatechuic acid was also found in Turkish oak and pine honeys 25.

348

SPME/GC-MS has been applied to the analysis of phenolic and other aromatic

349

compounds in different types of European honeys. Honeydew honeys included: Abies

350

alba Miller (France and Italy), Quercus sp. (France), Pyrenees (France), and forest

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Journal of Agricultural and Food Chemistry

351

honey (Italy); the last two being a mixture of honeydew honeys. French Pyrenees honey

352

could be differentiated from the others honeys by the absence of cinnamic acid. Only a

353

small number of samples were analyzed (one or two of each kind). Additional studies

354

with a larger number of samples are necessary to confirm this finding 49. Cinnamic acid

355

was also identified in Croatian Quercus Frainetto honey by other researchers 39.

356

Salix sp. (willow) honey can be produced from honeydew or nectar resulting in

357

two completely different honeys. Croatian willow honeydew honey was analyzed using

358

USE/GC-MS and HS-SPME/GC-MS with two different chromatographic columns

359

Volatile compounds with different functionality, molecular weight, and polarity were

360

extracted and identified, including benzoic acid and its derivatives, fatty acids and

361

alcohols, hydrocarbons, and others. Among the identified compounds, thirty-three were

362

only identified in headspace volatiles. USE was applied using four solvents in order to

363

obtain a more complete volatile profile: pentane, diethyl ether, diethyl ether (after

364

pentane extraction), and a mixture of pentane:diethyl ether (1:2 v/v). In total, one

365

hundred and one volatile compounds were identified. It has been pointed out as

366

differentiator of this botanic origin high concentrations of benzoic acid, phenylacetic

367

acid, 2-hydroxybenzoic acid (salicylic acid), and 4-hydroxyphenylacetic acid with

368

minor amounts of 4-hydroxy-2-phenylethanol, 4-methoxybenzoic acid, and 4-

369

hydroxybenzoic. All these compounds probably came from Salix plant which is a source

370

of salicylic acid and its derivatives.

18

.

371

The main volatile compounds identified by HS-SPME/GC-MS were nonan-1-ol,

372

hotrienol, dimethyl sulfide, benzaldehyde, 2-phenylethyl acetate, 3-methylbutanoic acid,

373

isophorone, hexanoic acid, benzyl alcohol, and 2-phenylethanol. A small amount of

374

methyl salicylate was detected in samples, in comparison with the highest concentration

375

of this compound found in Spanish willow blossom honeys 24. Other researchers did not

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376

find methyl salicylate in Croatian willow blossom honey neither by HS-SPME/GC-MS

377

nor by USE/GC-MS, although it should be noted that its shikimate pathway precursors

378

(salicylic and benzoic acids) were detected

379

detected in Croatian willow honeys made from nectar or honeydew using reverse phase

380

(RP)-HPLC-DAD

381

salicylate as a specific marker for willow honeys due to the fact that it has not been

382

detected in other honeydew honeys or blossom honeys

383

with more samples are necessary to confirm this compound as a specific marker of Salix

384

sp. honeys.

50

40

. In addition, methyl salicylate was not

. Therefore, there is controversy about the possible use of methyl

18

. Further studies performed

385

Using RP-HPLC-DAD, several compounds were identified which can serve as

386

markers for differentiating willow blossom honey from willow honeydew honey.

387

Abscisic acids (ABA) (not detected in willow honeydew honey) could be considered

388

characteristic of willow blossom honey, predominantly the isomer (Z,E)-ABA more

389

than the (E,E)-ABA. On the other hand, kynurenic acid and salicylic acid could be

390

characteristic of willow honeydew honey. Salicylic acid was only detected in willow

391

honeydew honey and a greater amount of kynurenic acid was found in the samples of

392

willow honeydew honey than in those of willow blossom honey 50.

393

Other researchers detected salicylic acid in willow honey (from nectar or

394

honeydew) by USE/GC-MS but much higher concentrations were found in the samples

395

of honeydew honey 18 than in those of blossom honey 40. Salicylic acid was also found

396

in Quercus frainetto honey using this same technique

397

agreement with those previously reported by several researchers which found higher

398

amounts of salicylic acid in honeydew honeys (0.71-7.06 µg/g) than in blossom honeys

399

group (0-0.48 µg/g). They suggested the salicylic acid as a marker to discriminate

400

between blossom honeys and honeydew honeys 49.

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. These findings are in

16

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Journal of Agricultural and Food Chemistry

401

NMR spectroscopy is also a valuable technique for the characterization of

402

honeys. It allows to research specific chemical markers for particular kinds of honeys

403

regarding their geographical and botanical origin. Moreover, NMR spectroscopy is a

404

powerful technique used to obtain structural information and on a wide range of

405

chemical species in a single experiment 51,52. The use of this technique does not require

406

complex pre-treatment of the honey samples. However, the high spectral complexity

407

requires the use of multivariate techniques to extract the useful information 51,53.

408

Italian commercial honeys of twenty botanical origins, including blossom

409

honeys and honeydew honeys, were analyzed by 1H NMR spectroscopy to seek

410

botanical chemical markers. A previous solid-phase extraction was used before analysis

411

by NMR. Three samples of honeydew honeys were studied, two labeled as “metcalfa”

412

and one generically as “wood honeydew”. They gave almost identical NMR profiles,

413

containing the typical resonances of an aliphatic component, very likely deriving from

414

honeydew. The authors of the study suggested these aliphatic resonances as a marker

415

for honeydew honeys. In addition, these resonances can be used to detect honeydew in

416

blossom honeys because they were observed at lower intensities in almost all the spectra

417

of blossom honeys studied. Markers for blossom honeys of several floral origins were

418

also proposed 54. NMR spectroscopy has also been used by other researchers to detect

419

possible chemical markers for several kinds of Italian honeys: acacia, linden, orange,

420

eucalyptus, chestnut, and honeydew. The NMR spectra of chloroform extracts of the

421

honey samples (n=353) were analyzed and specific markers were identified for each

422

botanical origin. A diacylglycerilether was identified as a specific marker for honeydew

423

honeys which can be useful to discriminate blossom honeys from honeydew honeys

424

52,55

.

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Journal of Agricultural and Food Chemistry

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425

The properties and composition of the honeys not only depend on the botanical

426

origin but also on other factors as already indicated in previous paragraphs. Great

427

interest has also been put in the authentication of the geographic origin of the honeydew

428

honeys because the most important characteristics of honey also depend on the

429

geographic area in which it was produced. Pine honey is very appreciated in Greece and

430

Turkey and it is produced from honeydew secreted by the insect Marchalina hellenica

431

(Gennadius) 56. Differentiation between Greek and Turkish pine honeys on the basis of

432

their physicochemical parameters was not possible due to the broad variation of the

433

variables values within of each type of honey. However, pine honeys of both countries

434

could

435

trimethylbicyclo[4.1.0]hept-3-ene) and one unidentified compound (m/z: 55, 79, 91,

436

107, 123, 165). These compounds were found in 100% of Turkish honey samples and

437

were not detected in any sample of Greek honey 13.

be

differentiated

by

two

volatile

compounds:

3-carene

(3,7,7-

438 439

2.3. Phenolic compounds. In recent years, there has been an increasing interest

440

in the determination of the antioxidant activity of honeys. It has been reported that

441

botanical origin has a greatest influence on antioxidant activity of honey whereas other

442

factors such as processing, handling, and storage affect only to a minor degree.

443

Moreover, antioxidant activity widely varied with the botanical source

444

method for honey antioxidant activity determination does not exist. Several assays can

445

be used: FRAP (ferric reducing antioxidant power), DPPH (1,1-diphenyl-2-

446

picrylhydrazyl) free radical scavenging activity, ORAC (oxygen radical absorbance

447

capacity), TEAC (Trolox equivalent antioxidant capacity), and ABTS (2,20-azinobis (3-

448

ethylbenzothiazolin)-6-sulfphonate), among others. It has been suggested to use a

449

combination of antioxidant tests and compare results in order to have a more reliable

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57,58

. An official

18

Page 19 of 60

Journal of Agricultural and Food Chemistry

450

criterion of honey antioxidant behaviour because several phenolic compounds can react

451

differently depending on the test used

452

researchers use the same test, different modifications are often included making it

453

difficult to compare the results of the different studies.

59–62

. Another problem is that when different

454

Many studies have suggested that phenolic compounds are mostly responsible

455

for antioxidant activity of honey because a high correlation between the two parameters

456

is found. In addition, high correlations between color of honey and their TPC and

457

antioxidant activity were found. Therefore, darker honeys such as honeydew honeys and

458

some blossom honeys (chestnut, heather, buckwheat, anzer, and manuka, among others)

459

have higher antioxidant activity due to their higher TPC 4,8,57,59,63–66. Several researchers

460

found a high correlation between antioxidant capacity and TPC but also with other

461

parameters such as pH, EC, acidity, and net absorbance 67.

462

In some studies, a correlation between TPC and antioxidant activity was not

463

found. The Folin-Ciocalteu spectrophotometric method is usually used to determine

464

TPC in honeys and it is based on chemical reduction of the reagent which can also be

465

easily reduced by other compounds in honey. Therefore, this assay should be seen as a

466

measure of total antioxidant capacity rather than TPC; many non-phenolic substances

467

show significant reactivity to Folin-Ciocalteu reagent such as many vitamins, amino

468

acids, reducing sugars, and even some inorganic ions such as Fe(II) and Mn(II) 68. This

469

could be the reason why TPC of Anatolian heather honeys was higher than those of oak

470

and chestnut honeys, although the antioxidant capacity of these was higher than that of

471

heather honeys. Heather honey samples distinguished from the other honeys by high Fe

472

amounts, and Fe shows considerable reactivity towards the Folin-Ciocalteu reagent.

473

Similarly, the high Mn contents of oak honeys are probably responsible for their TPC

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Journal of Agricultural and Food Chemistry

Page 20 of 60

474

besides phenolic substances 69. Interferences of hydroxymethylfurfural were also noted

475

in the determination of total flavonones with the reagent 2,4-dinitrophenylhiydrazine 64.

476

TPC of honeydew honeys belonging to several botanical and geographical

477

origins has been reported. Data are shown in Table 6. Turkish Quercus Robur L. honey

478

had a higher TPC and antioxidant capacity than those of Pinus brutia L. honey and it is

479

also darker. The TPC found in samples of oak and pine honey were 120.04 and 61.42

480

mg of gallic acid equivalent (GAE) per 100 g of honey, respectively

481

honeys had similar amounts (Quercus Robur L.: 94.5-129.8 mg GAE/100 g; Pinus L.:

482

58.6-74.6 mg GAE/100 g)

483

L. (78-91 mg GAE/100 g) and Pinusburia L. (36.7-48.3 mg GAE/100 g) honeys and

484

they found lower values for each type of honey 4.

69

25

. Anatolian

. Other researchers analyzed the TPC in Turkish Quercus

485

TPC was evaluated in Spanish honeys from different botanical origins. A TPC

486

of 101 mg GAE/100 g was reported in Quercus sp. honey 28. Values between 100-154.4

487

mg GAE/100 g were found in Quercus pyrenaica (Pyrenean oak) honey

488

Portuguese and Spanish Quercus rotundifolia Lam. (holm oak) honeys, a TPC of 81.03

489

mg GAE/100 g was found 71.

70

. In

490

Several studies showed a TPC in Slovenian Abies alba Mill. (fir), Picea abies

491

(L.) Karst (spruce), and forest honeys of 24.14, 21.75, and 23.39 mg GAE/100 g,

492

respectively

493

Italian forest (insect: metcalfa pruinosa) and fir (Abies alba Mill. and Picea abies L.)

494

honeys contained a TPC of 80.1 and 59.6 mg GAE/100 g, respectively 62. On the other

495

hand, much higher TPC and antioxidant activity have been reported in willow

496

honeydew honey samples (119.79 mg GAE/100 g) than in those of willow blossom

497

honey (50.86 mg GAE/100 g). In relation to this, willow honeydew honey is almost

57

. A moderate-high TPC was found in Polish Abies alba Mill. honey

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.

20

Page 21 of 60

Journal of Agricultural and Food Chemistry

498

black with the lightness indicator, L, very low (L*=7.0), whereas willow blossom is

499

much clearer (L*=64.3) 50.

500

The main sources of phenolic compounds in honeys are plants. Therefore,

501

phenolic compounds could be an interesting tool to know the botanical origin of

502

honeydew honeys. Several phenolic compounds have already been suggested as

503

botanical markers of blossom honeys

504

characterize honeydew honeys of different botanical origins on the basis of their

505

phenolic composition.

22

. Few studies have been carried out in order to

506

Analysis of phenolic compounds in honey is usually carried out by HPLC.

507

Honey is a complex matrix with a high concentration of sugars being phenolic

508

compounds present in very low concentrations. Therefore, analysis of phenolic

509

compounds usually requires a previous clean-up step to remove sugars and other

510

interfering compounds and pre-concentration of analytes. The most used detectors are

511

DAD and UV/Vis detector. Other analytical techniques with higher levels of sensitivity

512

and selectivity have also been used. HPLC-DAD coupled to mass spectrometry with an

513

electrospray ionization interface (HPLC-DAD-ESI-MS) has been used to identify and

514

quantify flavonoid aglycons and ABA isomers in Slovenian honeys from both

515

honeydew or nectar. A previous solid-phase extraction using Strata-X SPE cartridges

516

was used 72. UHPLC-ESI-MS/MS was applied to the analysis of phenolic compounds

517

in blossom and honeydew honeys using the same solid-phase extraction procedure

518

Several researchers identified and quantified phenolic compounds in blossom and

519

honeydew honeys by HPLC-ESI-MS/MS without a previous cleaning or pre-

520

concentration procedure 73. SPME/GC-MS has also been applied to the determination of

521

phenolic and other aromatic compounds in honeydew and blossom honeys 49.

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.

21

Journal of Agricultural and Food Chemistry

Page 22 of 60

522

HPLC-ESI-MS/MS has also used for the determination of thirty phenolic

523

compounds and other honey constituents in Brazilian Mimosa scabrella Bentham

524

(bracatinga) honeydew honeys 74. Benzoic acid (1.2-11.05 µg/g), 3,4-dihydroxybenzoic

525

acid (1.28-1.78 µg/g), and salicylic acid (0.82-2.02 µg/g) were found to be most

526

abundant in the nine samples analyzed. According to the authors of this study, several

527

phenolic compounds were identified for the first time in honeydew honeys: luteolin

528

(6.54-9.74 µg/100 g), hesperidin (0-18.06 µg/100 g), isorhamnetin (8.73-10.64 µg/100

529

g), pinobanksin (2.86-6.64 µg/100 g), and coniferaldehyde (0-9.04 µg/100 g). p-

530

Aminobenzoic acid was also identified for the first time in honeydew honeys, but it

531

could not be quantified. Except pinobanksin, which was identified in Slovenia

532

honeydew and blossom honeys 72, the other phenolic compounds could be considered as

533

characteristic of Brazilian bracatinga honeydew honeys.

534

In a study of several Turkish honeys (from nectar or honeydew), Quercus robur

535

L. and Pinus brutia L. differed considerably from one another in their phenolic

536

composition

537

detector. The major compounds found in samples of oak honey (n=3) were rutin,

538

protocatechuic acid, gallic acid, and p-OH benzoic acid. In pine honey (n=4), the main

539

compounds were catechin, protocatechuic acid, p-OH benzoic acid, and vanillic acid.

540

Gallic acid was proposed as a chemical marker to differentiate oak from pine honeys

541

because it was only detected in oak honey and in a relatively high amount (82.49 µg/g).

542

Moreover, the absence of gallic acid in pine honey was also reported by other

543

researchers

544

considered as markers because much higher amounts of them were found in oak honeys

545

(protocatechuic acid: 744.60 µg/g; rutin 538.68 µg/g) than in pine honeys

546

(protocatechuic acid: 81.19 µg/g; rutin: 11.64 µg/g).

25

75

. Phenolic compounds were analyzed by RP-HPLC with UV-Vis

. Other compounds, such as protocatechuic acid and rutin can also be

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Page 23 of 60

Journal of Agricultural and Food Chemistry

547 548

3. ANALYTICAL METHODS AND MULTIVARIATE ANALYSIS

549

Another valuable tool to authenticate the botanical or geographical origin of

550

honey, especially when no specific markers are detected, is the use of analytical

551

methodology combined with a chemometric approach. In the classification techniques,

552

mathematical models are constructed capable of predicting the belonging of a given

553

sample to a class or category, honey type, on the basis of the characteristics of the

554

sample. In order to construct the model, it is necessary to have a set of samples,

555

"training set", in such a way that the class to which they belong is beforehand known as

556

well as the values of the prediction variables. Predictor variables must contain

557

discriminant information, i.e., they must be able to distinguish between classes. Once

558

constructed, the model is used to predict the category of new samples from the

559

measurement of their predictor variables. These chemometric techniques are framed

560

within what is known as supervised analysis where to build the model is previously

561

known the class to which each sample belongs. Various types of classifying techniques

562

are used such as lineal (LDA) or quadratic (QDA) discriminant analysis, artificial neural

563

network (ANN), soft independent modelling of class analogy (SIMCA), k-nearest

564

neighbors (KNN), among others. Conversely, the unsupervised analysis, such as cluster

565

analysis (CA) and principal component analysis (PCA), help to know if the samples

566

form groups or classes. In this case the existence of categories is not known or

567

deliberately ignored. The use of analytical methods with data multivariate analysis has

568

the drawback that a large amount of reference samples is required to build a robust

569

database or model to discriminate or classify samples.

570

In this review, only those chemometric/analytical methods that involve

571

honeydew honeys have been included and they are briefly described in Table 3

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Journal of Agricultural and Food Chemistry

Page 24 of 60

572

(geographical origin) and Table 8 (botanical origin). They were classified in three types

573

according to the kind of parameter used: i) analytical methods on the basis of

574

physicochemical parameters and/or chemical composition, ii) analytical methods on the

575

basis of microscopic parameters, and iii) analytical methods on the basis of spectra

576

information. In any case, the analytical results were chemometrically processed,

577

allowing the discrimination or classification of the honey samples according to their

578

botanical or geographical origin. As it can be observed in Tables 3 and 8, the most used

579

classification technique is LDA.

580 581

3.1. Analytical methods based on physicochemical parameters and chemical

582

composition. Several analytical methods have been developed which allowed to

583

classify honeys only with physicochemical parameters. For instance, Turkish pine

584

honeys and several types of unifloral honeys could be successfully classified using pH

585

values and LDA; a correct classification rate of 100% was obtained for all honey types

586

studied

587

and a polyfloral group could be classified by ANN using values of EC and chromatic

588

characters (a*, b*). About 95% of honeys were appropriately categorized 71.

76

. Spanish and Portuguese honeys from eleven unifloral varieties, holm oak,

589

Blossom honeys of seven botanical origins and Quercus sp. honeys, all them of

590

Spanish origin, have been classified with metal content data and physicochemical

591

parameters. By far, the most abundant element was K. Other majority elements were Na,

592

Mg, Ca, and Al. Elements present at medium levels were Mn, Fe, Zn, and Cu. Trace

593

elements included Co, Cr, Ni, Cd, and Pb. Significant differences were found among all

594

honeys in terms of the mean concentration of all variables, except, apparent sucrose,

595

hydroxymethylfurfural, Fe, and Zn. A procedure using a sequence of seven discriminant

596

analysis (linear or quadratic) was developed because a single step did not good results.

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Page 25 of 60

Journal of Agricultural and Food Chemistry

597

A correct classification percentage close to 90% was achieved following cross-

598

validation 77.

599

Pine, acacia, and polyfloral honeys from Kashmir valley of India could be

600

differentiated applying PCA on physicochemical parameters and mineral content (Cu,

601

Mn, Fe, Zn, Pb, and Cd). The botanical origin significantly affected all variables except

602

total solids. A correct classification rate of 100% was obtained by LDA 78.

603

Stepwise LDA was applied on chromatic characters and amino acids profile in

604

order to classify Spanish honey samples into: holm oak (Quercus ilex L. subsp. ballota

605

(Desf.) Samp.), pyrenean oak (Q. pyrenaica Willd.), sweet chestnut (Castanea sativa),

606

and heather (Calluna vulgaris (L.) Hull and Erica sp.). Amino acids were analyzed by

607

RP-HPLC with (FD) fluorescence detector, previous derivatization with o-

608

phthaldialdehyde. Before chromatographic analysis, sugars were removed using ionic-

609

exchange resins. Selecting ten variables (b*, phenylalanine, valine, and seven amino

610

acids ratios), 85% of the cross-validated honeys were appropriately categorized. Within

611

the same study, a classification model was developed that allowed to discriminate holm

612

oak from pyrenean oak honeys. Selecting four variables (a*, phenylalanine, and the

613

glutamine/arginine and arginine/lysine ratios), 95% of the cross-validated honeys were

614

appropriately categorized 79.

615

Ten flavonoid aglycons and two ABA isomers were identified in Slovenian

616

honeys. Samples, five or six of each class, belonged to Abies alba Mill. (fir), Picea

617

abies (L.) Karst (spruce), forest, three varieties of unifloral honeys, and a polyfloral

618

group. Authentication of the botanical origin by specific markers was not possible

619

because profiles of these compounds were similar for all botanic sources. In addition, a

620

wide range of concentrations of these compounds was observed in each type of honey

621

and no characteristic concentration ranges could be set. Additional studies would be

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Journal of Agricultural and Food Chemistry

Page 26 of 60

622

necessary increasing the number of phenolic compounds analyzed, such as flavonoids

623

glycosides and phenolic acids, and analyzing a larger number of samples to find such

624

markers, if it were possible. Chemometric analysis proved to be more effective to find

625

the most significant compounds for botanical authentication: pinobanksin, galangin, and

626

two ABA isomers. LDA was applied on the amounts found of these compounds. The

627

overall correct classification rate was 85% (60-100%) showing than further studies are

628

required to improve these results

629

aromatic compounds in European honeys and successfully differentiated between

630

honeydew and blossom honeys using PCA 49.

72

. Other researchers analyzed phenolic and other

631

LDA was applied on physicochemical variables and volatile compounds in order

632

to classify Greek honey samples into four botanical origins: pine, fir, thyme, and orange

633

honeys. Selecting 30 volatile compounds as variables, the overall correct classification

634

rate (84.0%) was the worst. Selecting 10 physicochemical parameters or the 40

635

variables, the overall correct classification rates were 97.5% and 95.8%, respectively 48.

636

There are fewer studies on discrimination or classification methods of honeydew

637

honeys according to their geographical origin. The composition of the honey is more

638

influenced by botanical origin than by the geographical area, so it is expected a greater

639

difficulty in differentiating honeys from a given botanical source according to its

640

geographical origin.

641

A study of pine honeys (n=39) from four regions in Greece was carried out to

642

authenticate the geographical area in which honey was produced. LDA was applied on

643

physicochemical parameters values and volatile composition. The origin of honeys

644

could be determined more accurately (cross-validation) using selected volatile

645

compounds (correct classification 84.6%) than physicochemical parameter values

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(correct classification 79.5%) or a combination of both (correct classification 74.4%) 47.

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Artificial neural network using the Kohonen self-organizing map (ANN/KSOM)

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algorithm was successfully applied on volatile composition data to authenticate the

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Greek or Turkish origin of pine honey. The same discriminant effect and the same map

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were obtained if only the fifteen compounds found in 100% of samples or the most

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discriminant compounds of these were used 13.

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An analytical method based on the determination of free amino acids allowed

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geographical discrimination of Mimosa scabrella Bentham (bracatinga) honeys from

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five different areas of Santa Catarina State (Brazil). Fee amino acids were determined

655

by GC-MS after a derivatization step with an alkyl chloroformate reagent in the organic

656

phase. The techniques PCA and CA were used for the chemometric treatment of

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analytical results. According to the authors of this work, the free amino acids

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concentrations in bracatinga honeys were higher than those reported for blossom honeys

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and other honeydew honeys 80.

660 661

3.2. Analytical methods based on microscopic parameters. Studies carried

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out shown that the species composition of fungal elements in honeys is significantly

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affected by the botanical and geographical origin of the honey. Therefore, its

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identification and quantification might serve to characterize a particular kind of honey

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and to find specific microscopic indicators.

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Microscopic examination allowed to differentiate between Abies cephalonica 81

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(fir) and Pinus sp. (pine) honeys of Greek origin

. Fir honeys (n=13) had a smaller

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ratio of honeydew elements (microalgae, fungal mycelia and spores, among others) to

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pollen grains (HDE/P) and lower abundance of honeydew elements than pine honeys

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(n=60). However, they did not differ in the P value. A ratio of HDE/P >3 is generally

671

required to establish a honey sample as honeydew honey

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. In most of the honey

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samples studied, a ratio