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Food Chemistry 126 (2011) 574582

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Inuence of pollen addition on mead elaboration: Physicochemical and sensory characteristics

A. Roldn a,, G.C.J. van Muiswinkel b, C. Lasanta a, V. Palacios a, I. Caro a
a b

Department of Chemical Engineering and Food Technology, Cdiz University, Aptdo 40, Polgono Ro San Pedro, 11510 Puerto Real, Cdiz, Spain Department of Agrotechnology and Food Sciences, Wageningen University and Research Center, Bomenweg 2, 6703 HD Wageningen, The Netherlands

a r t i c l e

i n f o

a b s t r a c t
Mead (honey-wine) results from the alcoholic fermentation of diluted honey using a wine yeast strain. However, mead elaboration can be hampered by several problems, including delayed or arrested fermentation, production of an unpleasant aroma, poor quality and inconsistency of the nal product. These difculties are due to honeys low nutrient content, its natural antifungal components, and the inability of the yeast strain to adapt to these unfavourable growth conditions. In this study, we evaluated the results of adding pollen at concentrations ranging from 10 to 50 g/l as a fermentation activator to improve the fermentation kinetic and the quality of meads. The effect of pollen addition on the honey must, fermentation kinetics, physicochemical characteristics, aroma proles and sensorial aspects of the obtained meads were evaluated. The results showed that pollen addition improved fermentation rates, alcohol yields, and the nal characteristics of meads. An increase in the volatile contents of the meads and an improved sensory prole was observed with pollen addition; however, this improvement was not correlated with the concentration of pollen. The adequate dose of pollen was determined by the nal characteristics and sensory prole of the meads. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 3 May 2010 Received in revised form 25 September 2010 Accepted 10 November 2010

Keywords: Mead Honey Honey wine Fermentation Pollen Aroma prole

1. Introduction Honey, the sweet substance produced by honey bees, has been used for centuries to prepare traditional, homemade drinks. Honey can be fermented to produce different types of mead (honey wine), sherry type wine, sparkling wine and fruit-honey wine, which may have different avours depending the oral source of the honey and the additives and yeast used in fermentation (Gupta & Sharma, 2009). Honey production has a signicant economic importance in several countries, and many scientic works about it have been published (Baroni et al., 2009; Estevinho, Pereira, Moreira, Dias, & Pereira, 2008;), and about the health benets of honey (Bogdanov, Jurendic, Sieber, & Gallmann, 2008; Sato & Miyata, 2000). However, there are few scientic studies about mead and other honey products (Pereira, Dias, Andrad, Ramalhosa, & Estvinho, 2009; Sroka & nski, 2007). Tuszy All honeys share certain general characteristics, including a moisture content below 20%, a sugar content of 7080%, an ash content ranging from 0.1% to 0.2%, and a pH between 3.8 and 4.7 (Nagai, Inoue, Kanamori, Suzuki, & Nagashima, 2006; Ouchemoukh, Louaileche, & Schweitzer, 2007; Silva, Videira, Monteiro,

Corresponding author.
E-mail address: (A. Roldn). 0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.11.045

Valento, & Andrade, 2009). Proteins, free amino acids (principally proline), organic acids, aromatics, and vitamins and minerals are minor components (Hernndez, Fraga, Jimnez, & Arias, 2005; Pisani, Protano, & Riccobono, 2008). Honey also contains antimicrobial and antioxidant properties (Gomes, Leandro, Moreira, Rodrigues, & Estevinho, 2010; Nagai et al., 2006). Mead is an alcoholic beverage containing 818% (v/v) ethanol, obtained by the alcoholic fermentation of diluted bee honey with an appropriate amount of water. Fruits, juices and spices may also be added (Gupta & Sharma, 2009). The time needed for fermentation and maturation ranges from several months to several years. During that time, several problems can occur: fermentation may be delayed or arrested; the yeast may referment; volatile acidity can increase; bacteria may contaminate the mead, changing its organoleptic quality; and the nal product may lack uniformity nski, 2007). (Pereira et al., 2009; Sroka & Tuszy Experience with grape wine production indicates that these problems are usually associated with the yeast strains inability to adapt to unfavourable growth conditions, such as limitations in nutrients, osmotic stress, ethanol toxicity and temperature shock stresses (Atteld, 1997; Bauer & Pretorius, 2000; Bisson, 1999). Yeast from the Saccharomyces cerevisiae strain, used in wine and beer production, has also been used as a starter in mead production. Recently, the capacity of S. cerevisiae strains, isolated from Portuguese honeys, to produce mead has been evaluated by Pereira

A. Roldn et al. / Food Chemistry 126 (2011) 574582


et al. (2009). Under the stress conditions (ethanol, sulphur dioxide and osmotic stresses) studied by these authors, the yeast strains isolated from honey behaved similarly to the commercial wine strains used in enology. That study also showed that the mead production outcomes depend on the composition of the honey used nski and the supplements added to it. Moreover, Sroka and Tuszy (2007) showed that the acetic and succinic acids formed during diluted honey fermentation reduce the meads pH and lead to a increased non-dissociated fatty acid content, which, in addition to the presence of relatively large amounts of medium-chain fatty acids, may cause the fermentation to slow down or stop. The nutritional requirements of yeast for fermenting grape must have been relatively well researched and described (Alexandre & Charpentier, 1998; Bell & Henschke, 2008; Bisson & Butzke, 2000). These nutrients, particularly yeast-assimilable nitrogen (YAN), are essential for the optimal growth and development of the yeast (Barre, Blondin, Fuillat, Sablayrolles, & Salmon, 1998; Riberau-Gayn, Dubordieu, Doneche, & Louvand, 2000; Bell & Henschke, 2008). However, the nitrogen level of the fermentation media modies the sensory character of mead (Vidrih & Hribar, 2007) because the amino acid composition affects the yeasts metabolism and thereby the production of the volatile aromatic compounds. The amino acid compounds are precursors to fermentation (Bouseta, Scheirman, & Collin, 1996; Vilanova, Ugliano, Siebert, Pretorius, & Henschke, 2007). The lower pH and the low mineral content of light honey versus dark honey can decrease the growth of yeast (Pereira et al., 2009). Fruit juices, salts and acids have been used as additives to stimulate the fermentation and improve the mead production process (Gupta & Sharma, 2009). Formulations with different amounts of ammonium phosphate, potassium sodium tartrate, magnesium sulfate, calcium sulfate, citric acid, tartaric acid and vitamins (biotin, pyridoxine, thiamin) have been used as supplements in many

studies (Gupta & Sharma, 2009; Pereira et al., 2009). In a previous study by our research group (Roldn, van Muiswinkel, Lasanta, & Caro, 2008), different grape juice fermentation activators were studied to improve diluted honey fermentation, including thiamine chlorhydrate, yeast extract, pollen and royal jelly (the last two of which are derived from beehives). The results showed that pollen improved the fermentation kinetics the most and was the best fermentation activator. Pereira et al. (2009) showed that the greatest difculty in mead production came from attempting to ferment honey with a low pollen content. Pollen is collected from owers and is the most important source of proteins, lipids, minerals and vitamins for bee survival. Recently, increasing evidence suggests its potential therapeutic benets, including antioxidant properties (Leja, Mareczek, Wyzgolik, Klepacz-Baniak & Czekonska, 2007) and bioactive properties as functional dietary food supplement (Kroyer & Hegedus, 2001). In addition to sugars, pollen contains 7.4% moisture, proteins (at least 20%), 6% lipids and 2.2% ash, plus minerals, vitamins and carotenoids (Almeida-Muradian, Pamplona, Coimbra, & Monika Barth, 2005; Human & Nicolson, 2006). Proline, aspartic acid, phenylalanine and glutamic acid are the primary amino acids in pollen (Gonzlez, Gmez, Cordn, Garca-Villanova & Snchez, 2006). The aim of this work was to evaluate the inuence of pollen addition on mead elaboration. Different concentrations of pollen were used as an activator to improve fermentation and the nal characteristics of meads. The fermentation kinetics, physicochemical characteristics, aroma proles, and sensory aspects of the nal products were determined.

2. Materials and methods 2.1. Mead elaboration Others authors instructions were followed to produce mead (Gupta & Sharma, 2009; Pereira et al., 2009; Vidrih & Hribar, 2007). Commercial honey (Valencia, Spain) (75 Brix) of multioral origin ($3 kg for each test) and always from the same marketing rm was diluted with water until a diluted honey of approximately 12 Be (2022 Brix) was obtained. The acidity was corrected to an approximate pH of 3.6 with tartaric acid, because according to Gupta and Sharma (2009), tartaric acid is less easily metabolised by undesirable lactic acid bacteria. Potassium metabisulphite was added (up to 50 mg/l) to prevent lactic acid bacteria growth. The diluted honey was divided into 5 l fermentation tanks, and commercially produced pollen (Valencia, Spain) was added in concentrations of 10, 20, 30, 40 and 50 g/l (P10 to P50). A pollen-free sample (sample B) was also included. Once enriched diluted honey, the turbidity and yeast-assimilable nitrogen (YAN) contents of the honey must were determined (Table 2). The honey must was inoculated with a commercial wine yeast strain of S. cerevisiae, ENSIS-LE5 (Tensum Tecnologicas, Barcelona, Spain), at a dose of 15 g/hl and incubated at 25 C. Throughout the fermentation process, the density and biomass concentrations of the must were routinely measured. Before each determination, the tanks were homogenised with a charge/discharge of honey must. The sample was centrifuged (10000 rpm, 10 min), and the density was determined using an electronic DMA 48 densimeter (Anton-Paar, Net IterLab. Salt, Madrid, Spain). The yeast cell biomass was determined by a staining method; the sample was stained with methylene blue to distinguish between live and dead cells, and examined under a light microscope in a hemocytometer (Neubauer counting chamber) (Paiting & Kirsop, 1990). Once the fermentations had nished (the density measure was unchanged and the residual sugar <5 g/l), the meads were cold

Table 1 Volatile compounds identied, odour descriptor, odour series and literature odour threshold. Compound Isovaleric acid Octanoic acid Ethyl 3-hidroxy butanoate 3-etoxy-1-propanol Isoamyl acetate Ethyl hexanoate Ethyl octanoate Diethyl succinate 2-phenylethanol Hexanoic acid 1-hexanol Phenylacetic acid Phenylacetaldehyde Phenylethyl acetate Benzaldehyde Buthyl lactate Ethyl lactate Ethyl acetate Isoamyl alcohols Acetaldehyde
a b c d e f

Odour descriptor Acid, sour, cheese Oily, rancid, butter, cheese Racid Fruity Banana Green apple, fruity Pear, pineapple Fruity Roses, lilac Plant, herbs Herbs, cut herbs Honey, pollen Honey, beeswax Honey, sweet Bitter almond Creamy, milky, sweet Fruity, buttery Solvent Solvent, alcohol Bitter almond, pineapple

Odour Series (OS) Oxidation (DO) Oxidation (DO) Oxidation (DO) Fruity (F) Fruity (F) Fruity (F) Fruity (F) Fuity (F) Floral (FL) Green (G) Green (G) Honey (H) Honey (H) Honey (H) Sweet (S) Sweet (S) Other Other Other Other (O) (O) (O) (O)

Threshold 33 lg/la 500 lg/lb 67000 lg/lc 50000 lg/lc 30 lg/la 5 lg/lc 5 lg/la 1200 lg/lb 10000 lg/lc 420 lg/la 1100 lg/lb 10000 lg/le 5 lg/lf 250 lg/ld 2000 lg/lb 1000 lg/lb 15000 lg/lb 12 lg/le 30 lg/lf 10 g/lc

Escudero, Hernndez-Orte, Cacho, and Ferreira (2000). Peinado et al. (2004). Mayn et al. (2005). Selli, Cabaroglu, Canbas, Erten, and Nurgel (2003). Gmez-Mguez, Gmez-Mguez, Vicario, and Heredia (2007). Ferreira, Ortn, Escudero, Lpez, and Cacho (2002).


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Table 2 Pollen inuence on physicochemical characteristics of honey musts (mean SD, n = 3). B B TA (g TH2/l) pH Abs 280 nm Abs 420 nm Turbidity (NTU) YAN (mg/l) 11.1 0.1 1.01 0.09 3.60 0.01 2.57 0.32 0.17 0.09 137 10 53.2 5.0 P10 11.9 0.2 1.20 0.10 3.62 0.02 3.88 0.57 0.29 0.10 2377 150 70.0 8.5 P20 12.1 0.2 1.46 0.19 3.64 0.02 5.18 0.71 0.35 0.05 3740 200 92.4 6.3 P30 12.5 0.1 1.78 0.11 3.55 0.01 6.78 0.43 0.42 0.04 5367 180 120.4 6.1 P40 12.7 0.1 2.00 0.11 3.60 0.02 7.65 0.52 0.51 0.07 8688 175 140.0 7.2 P50 12.9 0.1 2.23 0.12 3.66 0.01 8.72 0.39 0.61 0.11 >10000 168.0 5.7

B, control must honey; P10P50, must honey with pollen from 10 g/l until 50 g/l; TA, total acidity in g of tartaric acid/l; abs, absorbance at 280 and 420 nm; YAN: yeastassimilable nitrogen.

(6 C) for one week, and subsequently treated with gelatine (4 g/hl) and bentonite (40 g/hl). Finally, the meads were ltered and bottled. All trials were triplicated to ensure statistical signicance. 2.2. Nitrogen, protein and amino acid analysis of honey and pollen The total nitrogen was determined with the Kjeldahl method adapted for the Velp Scientica digestion (model DK6) and distillation (model UDK127) units (Velp Scientica s.r.l., Milano, Italy). The total protein was calculated by multiplying the honey and pollen nitrogen content by 6.25. Amino acids were isolated from commercial honey and pollen according to the method described by Gonzlez et al. (2006). The derivatization mixture was prepared by dissolving 50 mg of reagent OPA (phthaldialdehyde) in 2.5 ml of ethanol, 25 ll 2mercaptoethanol (both reagents from SigmaAldrich Qumica S.A., Madrid, Spain), and 4.45 ml of borate buffer (0.4 M, pH 9.5). The derivatization procedure was as follows: 50 ll borate buffer 0.4 M (pH 9.5), 60 ll OPA reactive and 1 ml of isolated amino acids. The borate buffer and OPA reagent were added by the autosampler/dilutor automatic injector at 1 min before the injection. Direct injection of derivatised samples was made on a Gilson, Inc., liquid chromatograph (322 Pump equipment and UV/VIS detector model 151) tted with a SUPELCOSIL LC-18 column (3 lm, 150 4.6 mm) set at room temperature. The chromatographic conditions were as follows: 0.8 ml/min; volume injection 20 ll; and solvents A (sodium acetate buffer (50 mM, pH 6.8): methanol (9:1) with 2% of tetrahydrofurane) and B (methanol with 0.5% of tetrahydrofurane). The gradient consisted of 85% A, 72% A (3 min), 56% A (25 min), 44% A (35 min) and 20% A (45 min). Fluorimetric detection was carried out using excitation and emission wavelengths of 324 and 420 nm, respectively. Identication and quantication was achieved through retention times obtained from pure compounds and the calibration curves of a kit of high purity L-amino acids (SigmaAldrich), respectively. 2.3. Analysis of honey must and meads Several measurements of the honey musts were taken: Beaum degree, pH, total acidity, sulphurous anhydride and yeast-assimilable nitrogen contents (YAN). Alcohol content, total acidity (TA), volatile acidity (VA), pH, residual sugars (RS), absorbance at 280 nm and 420 nm and major and minor aromatic compounds were determined in the meads. The Beaum degree was determined by areometry, using Dujardin-Salleron areometers (Laboratoires Dujardin-Salleron, France). The pH was determined by measuring the samples directly, using a digital micro-pHmeter CRISON-2001 (Crison Instruments Corp., Barcelona, Spain) with automatic temperature compensation. The turbidity was determined by nephelometry (NTU) with a 2100AN equipment (Hach Company, Loveland, USA). The quantity

of yeast-assimilable nitrogen (YAN) was determined according to the abbreviated formol index method proposed by Aerny (1997). The alcohol content, total acidity and volatile acidity were determined according to the ofcial methods of wine analysis (OIV (Ofce International de la Vigne et du Vin), 1990). Residual sugars were measured using an enzymatic test with ViniTest equipment (VinoBios, Denmark). Absorbance was measured with a Genesys 10UV spectrophotometer (Thermo, USA). Absorbance at 280 nm indicated the total phenol index (TPI). 2.4. Analysis of volatile compounds Major volatile compounds (acetaldehyde, ethyl acetate, methanol, 2-methylbutanol and 3-methylbutanol) were analysed using a GC equipped with an FID detector (HP 5890 Series II) on a Carbowax 20 M column (50 m, 0.25 mm ID, 0.25 lm). The injector and detector temperatures were 175 and 225 C, respectively. The carrier gas was hydrogen (1 ml/min). The oven temperature was 35 C for the rst 5 min, with a ramp of 5 C/min until the temperature reached 100 C. A direct injection of 20 ll of distilled sample was made. For identication and quantication of major volatile compounds, 4-methyl-2-pentenol was added as internal standard and pure standards compounds (SigmaAldrich Qumica, S.A., Madrid, Spain) were used to determine the retention times and calibration. Minor volatile compounds were determined by GCMS, after a solid phase extraction (SPE) following the method described by Di Stefano (1991). A GCMS model Voyager (Termoquest, Milan, Italy) was used with a Supelcowax-10 column (L 60 m, ID 0.32 mm, PD 0.5 lm). The operation conditions of the GC were the following: injector and detector temperature, 250 C; oven temperature of 40 C for 5 min, followed by a ramp of 2 C/min, and 200 C for 5 min); 2 ll of sample volume in splitless mode (40 s); and carrier gas, He (1 ml/min). The MS conditions were as follows: electronic impact mode (EI+) at 70 eV; origin temperature, 220 C; interface temperature, 320 C; scan index at 1 scan/s; and mass acquisition range of 45400 amu. Identication of the minor volatile compounds was performed by GCMS retention indices (authentic chemicals) and mass spectra comparisons (authentic chemicals and Xcalibur spectral library collection). All the volatile compounds standards used for the identication and quantication were obtained from SigmaAldrich (SigmaAldrich Qumica, S.A. (Madrid, Spain). Semiquantitative analyses were carried out, assuming a response factor equal to one. 2.5. Analysis of odour activity values (OAVS) To simplify the aromatic prole, odour activity values (OAVs) were calculated as the ratio between the concentration of each compound and its perception threshold (Table 1). Only compounds with OAVs greater than 1 were considered contributors to the meadsaromatic proles. The volatile compounds were grouped in odour series according to similar aroma descriptors in order to

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relate the quantitative information of the chemical analysis with sensorial perceptions, resulting in a more objective approach (Peinado, Moreno, Bueno, Moreno, & Mauricio, 2004). An odour series, depending of their main odour descriptor, was assigned to each component. The following aromatic series were chosen as descriptors for meads: green (G), fruity (F), oral (FL), oxidation (DO), sweet (S), honey (H) and others (O). The sum of the OAVs obtained for each aroma descriptor was calculated. 2.6. Sensory evaluation The sensory evaluation of meads was performed by a panel of ten panellists, ages 3050 ages (four female and six male) with wine tasting experience, and trained in the evaluation of meads. Sensory analysis was performed in individual booths with controlled illumination located in the tasting room of the Andaluz Center of Wine Research (CAIV, Puerto Real, Cdiz). The meads were presented in standard tasting glasses 3591 (ISO 3591, 1997) and covered with watch glass to minimise the evaporation of volatile compounds. The tastings were conducted between 11:30 am and 14:00 pm and the meads were at a temperature of 1012 C. Each taster was given specic tasting notes to evaluate the visual (turbidity and colour), aroma (quality and intensity) and taste (quality and intensity) characters, as well as the general acceptability of the product. Each character was scored from 0 to 5 according to the increasing intensity. Some aspects of interest such as characteristic odour (honey, fruity, ower) and defects odour (rancid, oxidised, musty) were also considered. 2.7. Statistical analysis Means and standard deviations were calculated and signicant differences were evaluated using Students t-test. Statistical processing was carried out using the STATISTICA Release 7 (Statsoft, Inc.-USA) statistical package.

3. Results and discussion 3.1. Effects of pollen addition on honey must composition The diluted honey had a density of 11.1 Be (188.7 g/l of sugars), a turbidity of 137 NTU, a pH of 4.42 and total acidity of 0.7 g tartaric acid/l. After pollen was added in different concentrations (050 g/l), the Baum degree, total acidity, pH, total polyphenol index (TPI, absorbance 280 nm), turbidity and yeast-assimilable nitrogen (YAN) of each sample were measured (Table 2). This table shows the change in the Baum degree with pollen addition (R2 equal to 0.9847), which corresponded to approximately 0.4 g of sugar per gram of pollen added. Therefore, pollen addition contributed to the increased probable alcohol content of the mead. Total acidity was also enhanced by pollen addition (R2 = 0.9909), with a 0.02 g increase in tartaric acid per gram of pollen. However, the pH values remained constant (mean pH 4.4) indicating that, given the low buffering capacity of the honey must, the increase in the total acidity after pollen addition was due to the weak acids. Adding pollen increased the total polyphenol index by approximately 0.130 absorbance units per gram of pollen, indicating that certain compounds of pollen (polyphenol substances, mainly avonoids) were solubilised in honey must. The honey must colour was also modied by the addition of pollen, as can be observed in the absorbance, at 420 nm, values (0.09 absorbance units per gram of pollen). This increase in absorbance values resulted in more yellow and brown honey musts. The turbidity and YAN levels also increased with the addition of pollen, and both measures correlated

closely with the pollen concentration (YAN: R2 = 0.9951; turbidity: R2 = 0.9847 for YAN). YAN constitutes of ammoniacal nitrogen, amino acids, small peptides and nitrogen that can be easily assimilated and is essential for yeast growth (Alexandre & Charpentier, 1998; Bell & Henschke, 2008; Bisson & Butzke, 2000) and for the complete development of fermentation (Bisson, 1999; Bisson & Butzke, 2000). Pollen is a major source of YAN and could be used to enrich nitrogen-poor media. The nitrogen and protein contents of pollen were 2.52% (0.60) and 15.74% (0.60), respectively, while the nitrogen and protein contents of honey were 0.10% (0.07) and 0.63% (0.07), respectively. As expected, the honey that was used for our mead exhibited a low YAN concentration, and activators were necessary to optimally ferment the honey must. The total amino acid contents of the pollen and honey were 15.1% and 0.70%, respectively (Table 3). The proportions of free amino acids were approximately the same in both samples: 14.2% of the total amino acid content for pollen and 14.3% for honey. Honey and pollen amino acids were mainly proteins and peptides. Pollen had a higher amino acid content than honey; however, the pollen/honey ratio (P/H) was very similar for the different amino acids measured, which indicates that both substrates are closely correlated and both have the same origin. The pollen content of honey was indicated by its amino acid proportions. Consequently, pollen addition provides a good supply of YAN, and the overall percentage of each amino acid remains constant when pollen is added. Moreover, the amino acid content of honey must is enriched in a balanced way by pollen addition. The main amino acids in both pollen and honey are proline ($52%), glutamic acid ($15%) and phenylalanine ($14%), followed by aspartic acid ($3%) and threonine, tryptophan and valine ($2%). These results are in accordance with previous reports that pollen contains 16% amino acids, with major contributions of proline, glutamic acid, aspartic acid, lysine and leucine (Human & Nicolson, 2006). Honey only contains 1% (w/w) amino acids, primarily proline, phenylalanine, tyrosine and lysine (Bouseta et al., 1996; Hermosn, Chicn, & Cabezudo, 2003). Each 10 g/l of pollen added to a fermentation medium provides on average 70% of the amino acids that were already present in 1 l of honey-must. The YAN levels in the P40 and P50 meads were above 140 mg/l, the minimum amount necessary to complete the

Table 3 Distribution of total and free aminoacids (as mg/g sample) for commercial honey and pollen, and pollen/honey (P/H) ratio (mean SD, n = 4). Total aminoacids Honey Ala Arg Asp Cys Glu Gly His Ileu Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Total 0.07 0.01 0.12 0.02 0.23 0.05 0.03 0.00 1.07 0.08 0.04 0.01 0.02 0.00 0.04 0.01 0.08 0.02 0.05 0.03 0.02 0.00 1.00 0.15 3.74 0.78 0.07 0.02 0.15 0.01 0.16 0.00 0.04 0.00 0.14 0.02 7.07 0.83 Pollen 1.49 0.49 3.46 0.76 4.78 1.33 0.50 0.05 22.81 1.02 0.82 0.31 0.37 0.04 0.80 0.34 1.63 0.57 1.18 0.53 0.38 0.07 21.80 1.09 78.91 1.63 1.41 0.32 3.90 0.31 3.09 0.10 0.75 0.17 2.93 0.86 151.0 2.43 P/H 21.6 30.1 21.0 20.0 21.3 20.5 21.8 22.2 21.2 22.7 21.1 21.8 21.1 20.4 25.8 19.3 21.4 20.9 21.4 Free aminoacids Honey nq 0.02 0.01 0.03 0.01 nq 0.16 0.01 0.01 0.00 nq nq 0.01 0.00 0.01 0.01 nq 0.15 0.02 0.56 0.02 0.01 0.01 0.02 0.01 0.02 0.01 nq 0.02 0.01 1.02 0.03 Pollen 0.02 0.00 0.31 0.07 0.69 0.23 0.07 0.01 2.92 0.10 0.11 0.01 0.05 0.00 0.11 0.02 0.24 0.02 0.17 0.03 0.06 0.01 3.10 0.14 11.59 0.62 0.27 0.10 0.45 0.03 0.42 0.15 0.18 0.05 0.44 0.11 21.40 0.09 P/H 18.2 20.3 18.3 18.3 21.8 21.3 21.1 20.7 24.5 20.5 20.0 21.0 20.9

Nq, no quantied (<0.01 mg/g sample).


A. Roldn et al. / Food Chemistry 126 (2011) 574582

18 16 14 9 8 7 6
Vm ax t m ax

fermentation in grape must (Barre, Blondin, Feuillat, Sablayrolle, & Salmon, 1998; Riberau-Gayn, Dubordieu, Doneche, & Louvand, 2000). Based on this data, pollen provides a good complement of amino acids for the honey-must fermentation process. At industrial level, turbidity is usually related to the quantity of nutrients in the raw material; it is the rst checkpoint in proper fermentation development. Hidalgo (2003) set the optimal turbidity values from 50 to 200 NTU for grape must. However, the addition of pollen, even at lower doses, signicantly increases the turbidity values, exceeding 2000 NTU in all cases.

mg/l days

12 10 8 6 4

4 3 2 1 0 0 10 20 30 40 50 60

3.2. Pollens inuence on fermentation kinetics The evolution curves of the relative density during the fermentation of the different honey musts are shown in Fig. 1. The fermentation kinetics were signicantly different between the control must (B) and the honey must with added pollen. In all cases, fermentation was complete, and all of the produced meads reached a residual sugar content of less than 0.6 g/l. Without a fermentation activator like pollen (B), the fermentation lasted approximately 6 weeks, conrming that honey is low in nutrients for yeast fermentation (Bahiru, Mehari, & Ashena, 2001; Gupta & Sharma, 2009) and that, as Barre et al. (1998) showed, this lack prolongs the fermentation process. Comparing between the different concentrations of pollen, the fermentation rate (Fig. 1) increased with pollen addition. Fig. 2 shows the maximum fermentation rate (Vmax) and the time required to reach the maximum rate (tmax) for the different concentrations of added pollen. The control mead (0 g/l added) showed a signicantly lower fermentation rate and a higher time to reach the maximum rate than the honey musts with added pollen. An increase in fermentation rate (y = 0.0074x2 + 0.6017x + 3.9225, R2 = 0.9916) and reduction of the time to maximum with increasing concentrations of pollen were observed. The honey musts containing more than 30 g/l of added pollen showed the highest fermentation rate and the shortest maximum time. The increase in the fermentation rate was correlated with increase in YAN and turbidity values, and resulted in improving the fermentation kinetics (Alexandre & Charpentier, 1998; Bisson & Butzke, 2000). These ndings correspond with the results of Vilanova et al. (2007), which showed that the fermentation rates increased in response to increased nitrogen concentrations in chemically dened fermentation media. Moreover, the high levels of polyunsaturated fatty acids found in pollen, such as linoleic and linolenic acids (Xu, Sun, Dong, & Zhang, 2009), must be taken into account because these are metabolised by the

2 0

Pollen concentration (g/l)

Fig. 2. Maximum fermentation rate (Vmax in mg/lday) and time to reach the maximum rate (tmax in days) reaching with pollen addition.

nski, 2007) and yeast cells during fermentation (Sroka & Tuszy may improve the fermentation kinetics. To study the effect of pollen addition on the alcoholic fermentation yield and efciency, the yield [produced alcohol (% w/v)/consumed sugars (% w/v) 100] and efciency [(produced alcohol/ theoretical alcohol from consumed sugars) 100] were calculated. The results (Table 4) show that the diluted honeys alcoholic fermentation yield (B) was about 37 g ethanol/100 g fermentable sugars, representing an alcoholic fermentation efciency of about 81%. The addition of pollen improved the fermentation yield and efciency by more than 7% and 10%, respectively. 3.3. Pollens inuence on the physicochemical characteristics of meads The physicochemical analyses of the nal meads are shown in Table 5. The nal characteristics depended on the alcoholic fermentation and the amount of pollen added to the honey must. The total acidity increased and the pH decreased during alcoholic fermentation. These results conrmed those of other authors nski, 2007), who afrm that as honey must fer(Sroka & Tuszy ments, succinic and acetic acids are formed. These acids increase the meads total acidity and reduce their pH. As can be observed in Table 5, the meads total acidity decreased with pollen addition, which could be primarily due to a small deviation for the yeasts metabolism toward organic acid production, principally acetic acid. In addition, pollen provides potassium and calcium salts (Ouchemoukh et al., 2007; Silva et al., 2009) that may lead to salinization, resulting in decreased acidity. As a result of this decrease in acidity, the nal mead pH increased to more than 4.0 with pollen addition. These high pH values, especially for additions of 40

1,005 0,995 0,985 0,975

B P10 P20 P30 P40 P50

Density (g/ml)

0,965 0,955 0,945 0,935 0,925 0,915 0,905 0 10 20 30 40

Table 4 Alcoholic fermentation yield and efciency in meads (mean SD, n = 3). Yield (%) B P10 P20 P30 P40 P50 36.7 0.5 44.5 0.2 44.0 0.3 43.4 0.5 43.0 0.4 43.3 0.8 Efciency (%) 81.4 0.8 98.7 0.5 97.5 0.3 96.2 0.4 95.2 0.2 90.7 0.5


time (days)
Fig. 1. Relative density evolution of the honey-musts during fermentation. (B) Control honey must.; P10P50: honey musts with pollen from 10 g/l to 50 g/l.

B, control must honey; P10P50, must honey with pollen from 10 g/l until to 50 g/l.


A. Roldn et al. / Food Chemistry 126 (2011) 574582 Table 5 Pollen inuence on physicochemical characteristics of meads. (mean SD, n = 3). B EtOH (% v/v) pH TA (g TH2/l) VA (g AcH/l) Abs 280 nm Abs 420 nm RS (g/l) 9.04 0.02 2.95 0.15 4.87 0.21 1.370 0.179 4.22 0.20 0.080 0.002 <0.6 P10 11.74 0.06 3.59 0.09 4.05 0.17 0.477 0.029 5.54 0.23 0.152 0.011 <0.6 P20 11.80 0.10 3.78 0.11 3.40 0.08 0.515 0.065 7.71 0.54 0.205 0.005 <0.6 P30 12.03 0.08 3.94 0.07 3.29 0.07 0.481 0.014 10.62 0.31 0.284 0.024 <0.6 P40 12.09 0.05 4.04 0.06 3.15 0.10 0.433 0.077 13.29 0.13 0.327 0.008 <0.6 P50


12.39 0.12 4.05 0.05 3.37 0.09 0.710 0.104 19.02 0.27 0.572 0.083 <0.6

B, control must honey; P10P50, must honey with pollen from 10 g/l until to 50 g/l; TA, total acidity in g of tartaric acid/l; VA, volatile acidity in g of acetic acid/l; abs, absorbance at 280 and 420 nm; RS, residual sugars.

and 50 g/l of pollen (P40 and P50), might reduce the microbial stability of meads and could justify correcting their pH after fermentation. The volatile acidity was elevated in all cases, reaching levels of almost 0.5 g/l. Moreover, the control mead showed acetic acid levels above the sensory threshold for table wines (0.7 g AcH/L). Low amounts of YAN favour the production of acetic acid (Vilanova et al., 2007), and yeast metabolism may benet by the accumulation of acetic acid as an intermediate secondary product (Barre et al., 1998) during a slow fermentation. Mead colour, generally measured at 420 nm (a yellow hue), increased with the increasing content of pollen, as did its absorbance at 280 nm, indicating its total phenol index (TPI). Phenolic compounds are naturally present in honey and pollen but vary widely depending on the oral origin (Nagai et al., 2006; Ouchemoukh, Louaileche, & Schweitzer, 2007). Polyphenols are of great importance to the colour, astringency and antioxidant properties, which are highly correlated (Paixo, Perestrelo, Marques, & Cmara, 2007). A signicant increase in the meads absorbance at 280 nm values (PTI) was observed after alcoholic fermentation. In the honey musts, this increase was linearly correlated with the amount of pollen added; however, in the meads, this increase was exponential (y = 4.1933e0.0299x, R2 = 0.9976). This nonlinear behaviour could be due to the proportion of alcohol present in the mead. As can be seen in Table 5, the TPI value increased with the alcoholic level (y = 0.049x + 11.46, R2 = 0.9835). Therefore, alcohol favours the extractability of compounds that absorb at 280 nm, such as polyphenols, avonoids, phospholipids, and proteins. Ethanol could expand the outer layer of the pollen wall (exine) and improve the solubilisation of its bioactive compounds. The residual sugars were below the detection limit of the ViniTest equipment (<0.6 g/l) for all meads. This result indicates that all honey-musts sugars were converted to ethanol and/or other secondary metabolites of fermentation. It appears that using pollen as a fermentation activator not only improves the fermentation kinetics, but also contributes to improve the meads nal characteristics.
Table 6 Major aromatic compounds of the obtained meads (mg/l). (mean SD, n = 3). Compound Isoamyl alcohols 2-methyl butanol 3-methyl butanol Average total isoamyl alcohols Others Methanol Acetaldehyde Ethyl acetate Average total others Total major aromas B 24.11 0.16 91.04 0.92 115.15 1.06 5.66 0.64 8.01 0.37 21.80 12.98 35.47 5.02 150.63 2.41 P10 61.55 0.41 187.22 1.88 248.78 2.29 11.80 2.00 19.08 0.91 13.15 7.83 43.03 10.91 291.81 5.22 P20

3.4. Pollens inuence on aromatic characteristics of meads Secondary metabolites like aromas are generally produced by yeasts (Fleet, 2003). Their production is inuenced by the yeast strain used and the compounds that provide the essential nutrients for yeast metabolism and serve as precursors for the nal aromas. The results for the major aromatic compounds are shown in Table 6. The higher alcohols contributed most to the major volatile compounds. In general, isoamyl alcohols increased with the addition of pollen, which agrees with our previous experiments related to honey fermentation (Roldn et al., 2008; Vidrih & Hribar, 2007). The sensory threshold value of isoamyl alcohols is 300 mg/l; however, they produce an unpleasant avour and taste in wines in concentrations higher than 400 mg/l (Gil, Cabellos, Arroyo & Prodanov, 2006). The concentrations in the meads did not exceed this limit. Isoamyl alcohols are derived from amino acids, and pollen components appear to contribute to their formation. The methanol content in the control samples was low, but it increased with higher pollen concentrations. This increase might be due to the large amount of pectins in pollen and the enzymatic hydrolysis during fermentation that results in methanol. The methanol levels correlated with the amount of pollen added (y = 0.4616x + 6.609, R2 = 0.9904), indicating that the methanol content was not derived from the yeasts activity during fermentation. Moreover, the acetaldehyde concentrations were the lowest in the control mead and higher for the rest of meads. The ethyl acetate values were related to the acetic acid content obtained during fermentation, and were expressed as volatile acidity. The mead with higher values of volatile acidity also had higher levels of ethyl acetate. Regarding the total minor volatile content (Table 7), pollen addition increased the control value by 266310%. P30 had the least increase in minor volatile content (166%) and P10 and P50 had the highest (210%). There was no correlation between the amount of added pollen and the total minor volatiles content. The substrates necessary for the production of these volatiles were obtained even with low concentrations of pollen (10 g/l). The main contributors to the total minor volatiles content in meads were

P30 71.55 0.48 211.08 2.12 282.62 2.61 19.91 2.25 22.15 1.04 8.75 5.21 50.81 7.19 333.43 5.34

P40 78.97 0.53 234.82 2.36 313.79 2.89 25.54 2.88 27.61 1.29 13.68 8.15 66.83 9.46 380.62 6.09

P50 83.50 0.56 234.10 2.36 317.60 2.93 29.05 3.28 30.18 0.62 19.25 11.46 78.48 8.70 396.08 6.07


73.74 0.41 218.86 1.88 292.60 2.70 17.13 1.93 18.11 0.85 15.33 9.13 50.57 7.16 343.17 5.49


B, control must honey; P10P50, must honey with pollen from 10 g/l until to 50 g/l; OS, odour series of the corresponding chemical compound. G, green odour; F, fruity odour; DO, oxidation odour; O, other odour; S, sweet odour; FL, oral odour; H, honey odour.


A. Roldn et al. / Food Chemistry 126 (2011) 574582

Table 7 Minor aromatic compounds of the obtained meads (lg/l). (mean SD, n = 3). Compound Acids Isovaleric acid Hexanoic acid Octanoic acid Phenylacetic acid Average total acids Percentage of total Esters Isoamyl acetate Ethyl hexanoate Ethyl lactate Ethyl octanoate Buthyl lactate Ethyl 3- hidroxy butanoate Diethyl succinate Phenylethyl acetate Average total esters Percentage of total B 109.14 30.05 124.66 20.77 133.50 13.06 96.79 42.47 464.09 78.10 8.2 2.12 0.90 0.74 0.64 14.01 6.23 ND ND 4.72 1.02 54.55 7.32 18.92 0.22 95.06 18.25 1.7 P10 572.09 157.53 903.12 150.46 1840.15 180.04 204.59 89.77 3519.95 592.39 20.0 61.83 26.30 22.50 19.53 54.36 24.18 80.40 27.41 ND 25.45 5.51 168.63 22.64 226.47 2.67 639.64 122.80 3.6 P20 325.69 89.68 1285.48 214.16 2137.20 209.10 268.12 117.64 4016.49 675.96 23.1 64.06 27.25 33.95 29.49 38.52 17.14 113.56 36.52 ND 26.41 5.72 218.95 29.39 205.05 2.41 700.49 134.49 4.0 P30 396.04 109.05 1067.85 177.85 1452.43 142.11 151.31 66.39 3067.62 516.22 20.3 58.46 24.87 32.23 27.99 42.85 19.06 99.00 12.03 1.22 0.54 37.26 8.07 187.85 25.22 250.09 2.94 708.96 136.11 4.7 P40 493.42 135.87 1195.46 199.16 1408.94 137.85 209.56 91.95 3307.38 556.62 21.1 73.17 31.12 33.09 28.73 15.59 6.94 81.58 23.78 1.70 0.38 42.06 9.11 220.80 29.64 306.20 3.60 774.19 148.64 4.9 P50 614.81 169.29 1337.90 222.90 1589.03 155.47 205.12 90.00 3746.86 630.58 21.3 77.62 33.01 36.03 31.29 627.34 279.11 72.40 35.41 45.71 20.68 32.27 6.99 702.39 94.29 128.64 1.51 1722.40 330.69 9.8 OS DO G DO H


B, control must honey; P10P50, must honey with pollen from 10 g/l until to 50 g/l; OS: odour series of the corresponding chemical compound. G, green odour; F, fruity odour; DO, oxidation odour; O, other odour; S, sweet odour; FL, oral odour; H, honey odour.

alcohols (>60%) and acids. Adding pollen increased the alcohol content, particularly 2-phenylethanol. However, their contribution to the overall volatile was 20% lower than for the control (Table 7). The contribution of the remaining aromatic families to the total aroma increased with increased pollen content. Pollen addition signicantly increased the proportion of acids in the meads aroma, particularly octanoic and hexanoic acids. Acids were increased to levels up to 500% greater than those of the control mead, but there was no correlation between the production of these acids and the pollen content of the honey must. The meads with added pollen showed an increase in the proportion of esters, especially ethyl succinate and phenylethyl acetate. In general, fruity odour was noted, and ethyl lactate and butyl lactate levels were high in P50. High butyl lactate levels in P50 could be due to the lactic acid derived from fermentation by heterolactic bacteria after alcoholic fermentation. These values could also justify the high levels of ethyl acetate and volatile acidity of this mead. The need to correct the pH value and the sulphur content in this mead once the fermentation is complete could also be justied. As can be seen in Table 7, aldehydes, particularly phenylacetaldehyde in P50, followed a trend similar to that of the esters. In general, the production of these volatile compounds in the studied meads was related to the addition of pollen. No signicant relationship with the amount of pollen added was observed. Some aromas that increased with pollen addition, such as 2-phenylethanol, benzaldehyde, phenylacetaldehyde and phenylacetic acid, are naturally present in honey, independent of its geographical or botanical origin (Bouseta et al., 1996; Fleet, 2003; Alissandrakis, Tarantilis, Harizanis & Polissiou, 2007). These compounds are usually related to the aromas of honey, pollen, and owers and to the waxy character of honeys (Castro-Vzquez, Daz-Maroto, & Prez-Coello, 2007). The increase of these compounds suggests that some pollen components, such as phenylalanine, are precursors to their formation. Attending to the odour activity values (OAVs), as can be observed in Fig. 3, in general, pollen addition was associated with increased values for all of the aromatic groups, especially for the fruity and oxidation groups, regardless of the amount of pollen added. Higher concentrations of pollen signicantly increased the aromas in the honey series, mainly associated to high levels of phenylacetaldehyde. The other aromatic series followed a different trend, reaching a maximum and a minimum for fruity and oxidation series, respectively, with the added pollen dose.


70 30




0 B P10 P20 P30 P40 P50

Pollen concentration (g/l)

Fig. 3. Inuence of pollen addition on the OAVs of meads. (B) Control must honey; P10P50: must honey with pollen from 10 g/l to 50 g/l; G: green odour; F: fruity odour; DO: oxidation odour; H: honey odour.

Therefore, the combination of increased nutrient level and a slower fermentation process might have favoured an increase in the total aroma content, as well as a different balance of aromas. P20 and P30 showed the main contribution of the fruity series and the lowest of the oxidation series. The principal contributor to the fruity series was ethyl octanoate; octanoic acid was the main contributor to the oxidation series.

3.5. Pollens inuence on sensory characteristics of meads Along with the products aromatic prole (OAVs), tasting is indispensable in evaluating the organoleptic characteristics of meads. A more complete prole is obtained by including visual, gustatory and tactile aspects along with aromas. The tasting test involved rating the following parameters along a 10-point scale: turbidity, colour, aroma quality and intensity, taste quality and intensity, and acceptability. The tasting committee members could also write down any additional aroma or

A. Roldn et al. / Food Chemistry 126 (2011) 574582

(p<0,001) Acceptability 10 8 Taste intensity (p>0,05) 4 2 0 Taste quality (p<0,001) Color (p<0,001) 6 Turbidity (p>0,05) B P10 P20 P30 P40 P50


fermentation in the production of mead. The addition of pollen improves the fermentation kinetics and physicochemical and organoleptic characteristics of the mead. In response to the sensory criteria, the dose of 30 g/hl of pollen is the most appropriate because, besides producing mead of better acceptability, it would mean less use of pollen at industrial level. References
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Aroma intensity (p<0,01)

Aroma quality (p<0,001)

Fig. 4. Effect of pollen additon on sensorial evaluation of meads. (B) Control must honey; P10P50: must honey with pollen from 10 g/l to 50 g/l.

avour characteristics they observed. The sensory analysis results are presented in Fig. 4. The p values shown in the gure indicate whether the difference presented by each parameter evaluated, at different pollen addition, compared with control mead was very signicant (p < 0.001), signicant (0.001 < p < 0.01), insignicant (0.01 < p < 0.05) or nonsignicant (p > 0.05). The most signicant differences between the added-pollen meads and the control mead were in colour, aroma quality, taste quality and acceptability. The other parameters were not signicantly different. The meads judged most acceptable (P30 and P40) had the highest aroma and avour quality ratings. Samples P10, P20 and P50 were comparably evaluated. The major difference between them was the colour value, which increased linearly (from pale yellow to amber) with pollen addition. The aroma quality appeared to be related to the sum of OAVs (Fig. 3). The controls aroma was described as oral (associated with phenylethanol) and vinegar-like acid masking other aromas, which decreased the aroma quality, as was conrmed by the presence of isovaleric and hexanoic acids and by the samples elevated total and volatile acidity (Table 5). Ethyl acetate (a solvent-like odour) was also noted in the control. P10 was described as medicinal and oral, while the P20 and P30 meads evoked almonds, dried fruits, apple, caramel and sweets. P20 was also described as smelling of pineapple associated with acetaldehyde, which was signicantly higher for this mead. P40 differed from the other samples mainly due to its profound honey aroma. P50 was characterised by toasted, bitter almond and honey scents that masked all other aromas, principally consistent with its high phenylacetaldehyde levels. The pollen content changed the mouthfeel of the meads by increasing the aromatic body of the sample, which was also stated by Vidrih and Hribar (2007). Astringency was noted for various meads, probably due to the increase in polyphenols. Flavour assessment resulted in deviations similar to those for the aroma parameters. Thus, meads P40 and P50 were highlighted for sweetness, while the control and P30 were described as sour. A slight bitterness was noted for P20. Honey avour and a oral avour were indicated for P40 and P50. The controls taste was rated signicantly lower (2.9) than the others due to its sourness and lack of desirable avours. Finally, the relative low taste value for P50 (5.1) was attributable to an excessive honey avour. 4. Conclusions According to the obtained results it can be concluded that pollen could be a good and appropriate activator of alcoholic


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