Volatile compounds of unifloral honey and floral nectar from Quillaja saponaria

1 Department of Plant Science, School of Agronomy and Forestry.Pontificia Universidad Católica of Chile, Casilla 306-22, Santiago, Chile. 2 DICTUC Taste and Smell Center and ASIS‐UC Interdisciplinary Research Program on Tasty and Healthy Foods, Pontificia Universidad Católica of Chile, Casilla 306-22, Santiago, Chile. Address Correspondence to: Francisca Santander, e-mail: f.santander.cadegan@gmail.com Recibido / Received 20.XI.2012. Aceptado / Accepted 15.VII.2013. Resumen. Actualmente, la búsqueda de marcadores químicos relacionados con el origen botánico de las mieles es de gran interés debido a su uso potencial como una herramienta complementaria al análisis melisopalinológico. El objetivo de este estudio fue comparar (1) los contenidos de compuestos volátiles del néctar de Quillaja saponaria Mol. (Fam. Quillajaceae) con mieles monoflorales de esta misma especie, y (2) los compuestos volátiles de mieles de Q. saponaria recolectadas provenientes del mismo origen geográfico. Para la identificación y semicuantificación de compuestos volátiles se utilizó Cromatografía de Gases con Espectrometría de Masa (GC-MS). El néctar de Q. saponaria presentó compuestos volátiles diferentes a los identificados en las mieles, los que podrían ser precursores de los compuestos presentes en dichas mieles. Diez compuestos volátiles fueron encontrados en las cinco mieles de Q. saponaria analizadas: ácido 2-metil butírico (2 – 21,6 μg/L), benzil alcohol (1 – 6 μg/L), 2-feniletanol (16 – 125,3 μg/L), ketoisoforona (2,6 – 15,9 μg/L), linalool (2,4 – 13,8 μg/L ) y sus óxidos 1 y 2 (6 – 13,3 μg/L y 3 – 7 μg/L respectivamente), βdamascenona (4 -12 μg/L), pantolactona (2 – 7,5 μg/L) y furfural (7 – 44,2 μg/L), siendo estos compuestos comunes en mieles monoflorales de distintas fuentes florales descritas en mieles de otros países. Estos resultados indicarían que las mieles de Q. saponaria no presentan un tipo particular de compuestos volátiles que permitan diferenciarlas claramente respecto de otras mieles monoflorales.


INTRODUCTION
Honey aroma has been studied for several years due to its relation to organoleptic quality and authenticity (Pérez et al., 2002;Montenegro et al., 2008).Each unifloral honey has distinct aroma due to specific volatile compounds that may be derived from floral nectar; therefore, its organoleptic quality depends mainly on the origin floral source (Kaskoniene & Venskutonis, 2010).The differences in sensory properties of unifloral honey make possible to establish a relation between the main species present in honeys, and one or more compounds responsible for honey aroma and other volatile compounds identified by chemical analysis (Piasenzotto et al., 2003;Bogdanov et al., 2004;Manyi-Loh et al., 2011).
The volatile compounds could be used to determine the main floral source in unifloral honey, providing a tool to detect fraud by addition of pollen and misdescription of pollen grains (Radovic et al., 2001;Bogdanov & Martin, 2002;Arvanitoyannis et al., 2005).Furthermore, it has been reported that pollen grains of kanuka (Kunzea ericoides) and manuka (Leptospermum scoparium) are indistinguishable by melisopalinological analysis (Stephens et al., 2010); therefore, alternative tools have to be developed to identify the real botanical origin of this kind of unifloral honeys to guarantee honey authenticity.
In previous research, volatile composition analysis and sensory evaluation were performed on three types of native unifloral honey from Chile.This allowed the researchers to relate distinctive aroma descriptors in unifloral honey to the presence of terpenes, norisoprenoids, and phenolic derivatives.Nevertheless, researchers had difficulty recognizing aroma in Quillaja saponaria Mol.(Fam.Quillajaceae) honey, possibly due to the use of different types of unifloral Q. saponaria honey.However, it may also be due to the fact that the honey contains other floral influences (Montenegro et al., 2009).
Plant species have a particular chemical composition.Because honey comes from the flower nectar of certain species, finding a correlation between the chemical composition and the species that originated each particular honey would be expected.
Chile has a great diversity of native flora that can be used by bees (Apis mellifera L.) for honey production (Montenegro, 2002;Montenegro et al., 2010).Nevertheless, little information exists about the volatile compounds present in Chilean native honey, and information about the chemical composition of native floral nectar has not yet been described.
The objective of this research was to compare the (1) volatile compounds of Quillaja saponaria Mol.(Fam.Quillajaceae) floral nectar with those of unifloral honey of this same species, and (2) the volatile compounds of Q. saponaria honeys from the same geographical origin to establish possible volatile compounds as chemical markers for Q. saponaria honey.

MATERIALS AND METHODS
Nectar and honey samples.Floral nectar and honey samples were collected from four sampling sites (Petorca, Limache, Huayacán, and Alicahue) in the Valparaiso Region (32° 02' -33° 57', V Region) in Central Chile (Fig. 1).Native vegetation was predominant in all of the sampling sites.
During Q. saponaria flowering (November and December), a representative number of trees were identified and labeled.Plants with leaf damage were discarded.Floral nectar from Q. saponaria flowers (250 flowers) was taken randomly from 10 trees.The floral nectar was collected with glass capillary tubes as described by Southwick (1983) and Vidal et al. (2006).Honey and nectar from Quillaja saponaria During the corresponding harvest season, fifty-seven honey samples from the same geographical area were collected and studied using melisopalinological analysis to certify the Q. saponaria botanical origin as described in norm NCH 2981.OF2005 (Montenegro et al., 2008).The pollen grains were compared with a Pollen Grain Library for their identification.The data analysis was conducted using Statistical Analysis of Proportions, using maximum likelihood estimation to construct confidence intervals (Mead et al., 1993).Species that presented proportions lower than 0.03 (considering confidence interval) were discarded (Loveaux et al., 1978;Moar, 1985).Results and corresponding statistics are shown in Appendix 1 (Tables 6 to 62) (www.revistaphyton.fundromuloraggio.org.ar/vol83.html).
According to this analysis, five unifloral Q. saponaria honey samples were identified and selected for the chemical research.The botanical origin (percentage of Q. saponaria), and companion species of these honey samples are described in Table 1.Chemical analysis.One sample of floral nectar and five certified Q. saponaria honey samples were kept in a cold chamber before the chemical analysis.A 2 cm 50/30 μm DVB/Carboxen/PDMS StableFlex fiber (Supelco, Inc., Bellefonte, PA) was used for aroma extraction.
The honey solution plus sodium chloride (0.2 g) and internal standard (1 μL of 4-nonalol solution 3.460 mg/mL) were placed in a 20 mL vial tightly capped with a Teflon/ silicone septum (catalog number S126-0020, I-CHEM).The sample was equilibrated at 35 °C in a water bath for 5 min and extracted after stirring for 1 hour at the same temperature.After extraction, SPME fiber was inserted into the injection port of the GC/MS to desorb the analytes.GC/MS analyses were carried out on a HP 6890 Gas Chromatograph, coupled to a 5972A MSD Hewlett Packard mass spectrometer, and equipped with a 60 m x 0.25 mm x 0.25 μm DB-WAXETR capillary column ( J&W Scientific).The injector was set at 260 °C and column oven temperature was held for 5 min at 40 °C, then raised 3 °C/min until it reached 240 °C, at which point it was held for 25 min.Mass spectra was obtained by electron impact ionization (70 eV) scanning a mass range of 35-350 m/z.The MS quadrupole and MS source temperatures were 150 °C and 220 °C, respectively.The spectrometric data was compared with those from NIST-EPA-NIH libraries (http:// www.nist.gov/srd/nist1a.htm)that have more than 130000 entries.Semiquantitative analysis was carried for the volatile compounds identified.
The repeatability of the extraction procedure was evaluated using nonalol as internal standard.The relative standard deviations (RSD) of the 10 common honey sample compounds are shown in Table 2.
Table 2. Relative standard deviation (% RSD) of 10 compounds obtained from a honey sample, estimated from 3 extractions.

RESULTS AND DISCUSSION
The main factors that affect the composition of volatile compounds in honeys are: floral source, geographical origin, honey maturity, and harvest season (Gheldof & Engeseth, 2002;Kaskoniene & Venskutonis, 2010).Botanical origin seems to be the main factor (Kaskoniene & Venskutonis, 2010).The five Q. saponaria honey samples were selected from the same geographical area (V Region of Central Chile) during the same season (2011)(2012).For these reasons, differences in the content of volatile compounds in Q. saponaria honey might be attributed mainly to the honey samples companion species and postharvest conditions.Santander F et al., FYTON 83 (2014) Different methods of extraction for the determination of volatile compounds in honey by GC-MS have been described (Alissandrakis et al., 2003;Bianchi et al., 2005;Castro-Vázques et al., 2010).The use of CAR/PDMS columns has been described as an effective fiber for the isolation of aromatic compounds in honey (Plutowska et al., 2011), and was selected for this research.The RSD of the 10 common compounds in the five honey samples were found in a range of 0.2 -32.7%.Similar values have been described by Alissandrakis et al. (2003) and Piasenzotto et al. (2003).Nevertheless, our results showed higher RSD for 2-methyl-butyric acid and pantolactone.
Volatile compounds in Q. saponaria nectar and comparison to its unifloral honey.An example of a Chromatogram obtained by GC-MS of floral nectar and the corresponding volatile compounds is shown in Figure 2.
The volatile compounds identified in the Q. saponaria nectar differ both in the type and relative concentration when compared to the five unifloral honey samples (Table 3, 4 and  5).Nevertheless, there were two compounds (nonanal and benzaldehyde) found in both nectar and honey of Q. saponaria.Nonanal was found in higher mean concentrations in honey (9.8 μg/L) than nectar (3.8 μg/L), whereas bezaldehyde was found in higher concentration in nectar (9.6 μg/L) than honey (1.3 μg/L) (Table 5).These differences may be explained by the presence of compounds in nectar that could be precursors of other compounds present in honey, and agree with results obtained by other researchers (Rowland et al., 1995;Alissandrakis et al., 2003, Alissandrakis et al., 2007a, Alissandrakis et al., 2007b) where floral nectar and honey do not always share identical volatile compounds.
Comparison of volatile compounds in Q. saponaria honey.An example of Chromatograms obtained by GC-MS of each Q. saponaria honey, and the corresponding volatile compounds, are shown in Figure 3.
The volatile compounds identified in the five Q. saponaria honey samples differ both in type and relative concentration.In the five honey samples, 2-methyl butyric acid was found as the only type of acid in concentrations between 2.0 -21.6 μg/L (Table 3).Among alcohols, benzyl alcohol and 2-phenylethanol were identified in concentrations between 1.0 -6.0 μg/L and 16.0 -125.5 μg/L, respectively (Table 3).Ketoisophorone was the only ketone identified in the five honey samples in concentrations between 2.6 -15.9 μg/L (Table 4).Furans were identified in the form of furfural with concentrations that varied between 7.0 -44.5 μg/L (Table 5).Linalool and oxide 1 and 2 were the only terpenes found in all five samples with concentrations that oscillated between 6.0 -13.3 μg/L, 3.0 -7.0 μg/L and 3.0 -13.8 μg/L, respectively (Table 4).One norisoprenoid was identified in the form of β-damascenone in concentrations between 3.4 -12.0 μg/L and one lactone was identified in the form of pantolactone in concentrations of 2.0 -7.7 μg/L (Table 5).
These results could be explained by the different companion species in each honey.To be classified as a unifloral honey by melissopalinoligical analysis, at least 45% of the honey sample's pollen grain must come from a single species (Montenegro et al., 2008).The botanical origin results in this research indicate that the five unifloral honey samples had a Q. saponaria percentage of between 55.3 -87.2%, and the companion species oscillated between 22.8 -44.7% (Table 1).An important variety of the companion species was identified in the five honey   The nature of the honey used in most of this type of research makes it possible to easily correlate results of chemical composition among the different honey samples.This is because mostly cultivated species are used to obtain honey (citrus, lavender, eucalyptus, salvia, among others), which results in a very high percentage of the predominant species in each honey and a lower variability between honeys of the same botanical origin.This research, on the other hand, was focused on sampling sites where non-cultivated species (mostly native plants) predominated.This situation meant that there were a wider variety of species where honeybees collected nectar, resulting in a lower percentage of the predominant species.
Honey samples N° 1, 2, 3 and 4 had similar concentrations of pantolactone (Table 5), linalool and its oxides (Table 4) and benzil alcohol (Table 3).Linalool and its oxides have been described in citrus, thyme, quillay, Christ thorn, cotton, chestnut, heather, rape, sunflower, lavender, false acacia and acacia honey (Radovic et al., 2001;Alissandrakis et al., 2005b;Castro-Vásquez et al., 2007;Jerkovic et al., 2007;Montenegro et al., 2009;Jerkovic et al., 2009).Linalool has cytotoxic activity over fungus and several bacteria, which could be related to the Santander F et al., FYTON 83 (2014) Table 4. Ketone, terpene, aldehyde and ester composition (µg/L) of flower nectar and five Chilean unifloral honey samples of the same botanical origin (Quillaja saponaria) obtained via SPME-GC/MS.Tabla 4. Composición de cetonas, terpenos, aldehídos y ésteres obtenidos mediante SPME-GC/MS de néctar de flores y cinco mieles monoflorales chilenas del mismo origen botánico (Quillaja saponaria).antibacterial activity of this honey (Bakkali et al., 2008;Montenegro et al., 2009).Benzyl alcohol has been described in citrus, cotton, Christ thorn and false acacia honey (Alissandrakis et al., 2005b;Castro-Vásquez et al., 2007;Jerkovic et al., 2007;Jerkovic et al., 2009).Pantolactone has been found in chestnut honey and eucalyptus extracts, and honey from Spain (Castro-Vázquez et al., 2003;Castro-Vázquez et al., 2010).In the work of Castro-Vázquez et al. (2010), this compound was described as having a woody and toasty caramel taste.Honey sample N°5 had higher contents of furfural and 2-phenylethanol (Tables 3 and 5) than the rest of the samples.Furans were detected in a significantly higher concentration in two out of the five honey samples analyzed (Table 5).Furan derivatives have been described as indicators of heat treatments during extraction and storage conditions.Since all honey used was harvested in the same season, these results might indicate that honey samples N°2 and N°5 were exposed to high temperatures during the extraction process and/or storage, which is congruent with the HMF content in honey N°5 (Alissandrakis et al., 2003;Piasenzotto et al., 2003).Moreover, honey N°5 presented high concentrations of 2-phenylethanol.This compound in high concentrations has been described in citrus, false acacia, Christ thorn and cambará honey (Piasenzotto et al., 2003;Alissandrakis et al., 2005a;Moreira & De Maria, 2005;Castro-Vázquez et al., 2007;Jerkovic et al., 2007;Jerkovic et al., 2009).Wani et al. (2010) reported that phenylethanol has anti-  septic activity and this activity may also be associated with the antibacterial activity of Q. saponaria honey as reported by Montenegro et al. (2009).Phenylethanol has also been described as a characteristic compound of the first stage of the fermentation process in honey.The presence of 2-phenylethanol has been associated with exposure to high temperatures during the process of extraction and/ or storage (Vidrih & Hribar, 2007).Because of this, the high concentration of these compounds in honey samples N°4 and N°5 may be attributed to the inadequate storage after their harvest.

CONCLUSION
The chemical composition of Q. saponaria nectar was different than the volatile compounds found in Q. saponaria honey, and a relationship between them could not be established.This indicates that the volatile compounds of the floral nectar should be analyzed as precursor aroma compounds present in honey, thereby establishing a relationship between both types of compounds in order to use the chemical composition of nectar to support the search for volatile compounds as chemical markers of botanical origin.
Honey and nectar from Quillaja saponaria The impact of companion species on the chemical composition of Q. saponaria honey was demonstrated by the variable volatile compositions found in the five honey samples analyzed.Even though five unifloral honey samples shared ten volatile compounds, the variable concentration of these compounds made it difficult to establish a clear relationship between the Q. saponaria honey samples.Moreover, these common chemical compounds were also present in unifloral honey of other botanical origins.For this reason, a relationship between botanical origin and volatile composition could not be established.Given this, the search for chemical compounds as indicators of botanical origin in Q. saponaria honey should be complemented with the analysis of other types of chemical compounds present in this kind of unifloral honey.

Fig. 1 .
Fig. 1.Map of Valparaiso Region, Chile.Black circles indicate the origin of the Quillaja saponaria honey samples, the white circle indicates the origin of Quillaja saponaria nectar.

Table 1 .
Percentage of Quillaja saponaria presence in honey, and companion species.