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Acta Alimentaria, Vol. 36 (2), pp. 151162 (2007) DOI: 10.1556/AAlim.36.2007.2.

STUDIES ON THE SENSORY PROPERTIES OF MEAD AND THE FORMATION OF AROMA COMPOUNDS RELATED TO THE TYPE OF HONEY
R. VIDRIH* and J. HRIBAR
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1111 Ljubljana. Slovenia (Received: 03 November 2004; revision received: 22 August 2006; accepted: 17 January 2007)

Three different types of mead were prepared from chestnut, lime and honeydew varieties of honey. All three types of honey were diluted with water until the solution reached 25 Brix. The solution was inoculated with a selected yeast strain and allowed to ferment at 15 C. Fermentation of chestnut mead was completed first (in 24 days), while the fermentation of lime and honeydew types took 39 days to complete. At the end of the process, all three meads contained 14.2% vol. of ethanol. During fermentation, more higher alcohols (n-propanol, iso-butanol, iso-amyl alcohol) and ethyl acetate were produced in chestnut mead compared to lime or honeydew type mead. A panel test ranked chestnut and lime mead as equal, followed by the honeydew types. The panellists also preferred mead with a higher amount of reducing sugar (80 g l1) over mead with a lower amount of reducing sugar. Keywords: fermentation, higher alcohols, honey, mead, sensory evaluation

Honey is a sweet substance produced by honey bees from the nectar of blossom or from secretions of or on living parts of plants, which they collect, transform and combine with specific substances, and store in honeycombs. In prehistoric times, honey represented the only concentrated sweet substance (MORSE & STEINKRAUS, 1975). Honey was used to prepare possibly the first alcoholic drink known to Indians thousands of years ago (MORSE, 1980; JOSHI et al., 1990). Today, the technical term mead is used for honey wine that is made by the fermentation of diluted honey. Prior to or after fermentation, various additives such as fruit pulps or juices, citric acid, etc. may be added (JOSHI et al., 1990).

* To whom correspondence should be addressed. Phone: +386 1 4231161; fax: +386 1 2566296; e-mail: rajko.vidrih@bf.uni-lj.si 0139-3006/$ 20.00 2007 Akadmiai Kiad, Budapest

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Additives are also used to speed up the fermentation. The most common additives are the following: ammonium sulphate, potassium phosphate, magnesium chloride, citric acid, sodium citrate, biotin, pyridoxine, meso-inositol, calcium panthotenate, thiamine, peptone and ammonium sulphate (MORSE & STEINKRAUS, 1975). Various yeast nutrients are found in honey, depending on the honey type. Buckwheat honey was found to ferment much more rapidly than clover honey. The authors also suggested that dark honeys contain more nutrients than white honeys (MORSE & STEINKRAUS, 1975). Fermentation of honey solution usually starts with delay and proceeds slowly. MORSE and STEINKRAUS (1975) found fermentation of clover honey solution did not start after 18 days and contained only 6% alcohol after 54 days of fermentation. Fermentation of honey solutions is known to be difficult due to their high sugar content (WZOREK et al., 1993) or presence of some inhibitory agents (PONS & SHUTZE, 1994). Yeasts used in wine technology usually show weaker fermentation abilities that result in slower fermentation and a lower alcohol content. Strains of Sacharomyces bayanus usually perform fermentation well and may produce up to 14.8% alcohol (WZOREK et al., 1993). Higher alcohols are synthesised from oxo-acids during fermentation as a result of yeast activity. Oxo-acids have their origin in amino acids or in sugar metabolism (MANGAS et al., 1994). Saccharomyces cerevisae forms only amyl alcohol from the amino acid isoleucine, but it produces amyl, iso-amyl and n-propanol from threonine (REAZIN et al., 1973). In cider processing, the synthesis of higher alcohols corresponds to the quantity of fermented sugars; major sugar conversion yields more higher alcohols (MANGAS et al., 1994). Ten % of higher alcohols are synthesised from the corresponding amino acids, 65% from other amino acids and 25% from sugars (RIBEREAU-GAYON & SUDRAUD, 1991). Applying the appropriate technology is a tool for regulating the synthesis of higher alcohols. Removal of insoluble particles before must fermentation yields wines with less higher alcohols (RIBEREAU-GAYON & SUDRAUD, 1991), which are more fruity, fresh and delicate in taste (KLINGSHIRN et al., 1987). Temperature during fermentation is known to influence the synthesis of higher alcohols (KOSMERL & KORDIS-KRAPES, 1997). An increase of iso-butanol, amyl alcohol, iso-amyl alcohol and 2-phenyl ethanol results from an increased temperature during must fermentation (OUGH et al., 1966). The total nitrogen content in fermentation broth also plays a role (OUGH & BELL, 1980); iso-butyl and iso-amyl alcohol correlate negatively with total nitrogen content, while npropanol correlates positively. From sensory point of view, higher alcohols are aggressive in taste and flavour (ROUS et al., 1983) and resemble fresh cut grass; KLINGSHIRN and co-workers (1987) found wines with a greater higher alcohol content harsh in taste. RAPP and MANDERY (1987) suggested a total higher alcohol content in wine of 300 mg l1 as the sensory threshold. A concentration up to 300 mg l1 provokes a pleasant flavour and taste, but above 400 mg l1 causes an unpleasant flavour and taste.

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The literature regarding mead processing and related aroma compounds is scarce. The aim of our study was to clarify the influence of honey type on the synthesis of aroma volatiles during mead processing, and to reveal their effect on the sensory properties of mead. 1. Material and methods Three different honeys were used to prepare mead. The chestnut and lime honeys are nectar type honeys, while the third honey was dew type. In our case, honeydew was produced by the insect Metcalfa pruinosa on different plants and were collected by bees. All three types were classified as pure honeys matching the corresponding source of origin. According to BERTONCELJ and co-workers (2002), these three types of honey differ in electrical conductivity and ash content. Honeydew honey has the highest electrical conductivity and ash content, followed by chestnut and lime (BERTONCELJ et al., 2002) Chestnut honey has the highest water insoluble solids content, followed by lime (GOLOB & PLESTENJAK, 1999). Honeydew honey is known to have the lowest content of water insoluble solids (GOLOB, personal communication). The aroma of both nectar honeys resembled the corresponding flowers of lime and chestnut. The aroma of honeydew honey resembled the smell of caramel, which is typical of honey produced by the insect Metcalfa pruinosa. 1.1. Mead preparation Each type of honey was diluted with water (1:4) until the solution contained 25% of soluble solids. Fifty ml of each honey solution were put in a glass vessel and inoculated with 20 g of lyophilised yeast of Sacharomyces bayanus strain R2 (Lalvin). After inoculation, each glass vessel was sealed with a bubbler. The mead was allowed to ferment at 15 C until the equilibration point in soluble solids was reached. During fermentation, samples of approximately 200 ml were taken twice a week and frozen at 25 C for further analyses. At completion of fermentation, the mead was decanted and SO2 was added to reach 25 mg l1 (determined as free SO2). Addition of SO2 is necessary to prevent oxidation and better preserve the original aroma of the mead. 1.2. Soluble solids assay Soluble solids were measured by means of a hand refractometer (Atago) twice a week during fermentation. 1.3. GC assay conditions Volatiles (higher alcohols, acetaldehyde, ethyl acetate, methanol) were analysed by GC (Agilent technologies 6890 N) equipped with an HP-FFAP (50 m0.2 mm0.3 m) column and FID detector. Temperature programming was as follows: 6 min isothermal at 40 C, then a linear temperature rise of 25 C min1 to 220 C. Higher alcohol

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analyses were performed as follows; 100 ml of mead was provided with an internal standard (n-pentanol) and distilled on Gibertini automatic distillation equipment. One hundred ml of distillate was collected in a volumetric flask at 0 C to prevent losses due to evaporation; 1 l of distillate was injected into the column and peaks were recorded. 1.4. Determination of total nitrogen Content of total nitrogen was determined according to the method described by HACH and co-workers (1987). 1.5. Sensory evaluation A ranking test according to total impressions was performed by 11 trained panellists on meads and honey solutions. Honey solutions were prepared by dissolving honey in water containing 80 g l1 of reducing sugars. Ranking test of honey solutions was performed to determine which honey type would be a better primary material for mead processing. After fermentation and maturation, meads contained less than 4 g l1 reducing sugars. Honey was added to meads to reach 40 or 80 g l1 reducing sugars. A ranking test of total impressions was carried out in order to determine how the reducing sugars influence sensory properties. 2. Results and discussion 2.1. Total nitrogen content Chestnut honey contained 0.061% total nitrogen, lime honey 0.041% and honeydew type 0.10% total nitrogen. 2.1. Soluble solids assay According to the assay of soluble solids and volatiles, the whole process of fermentation can be divided in into two parts: main fermentation that took c.a. 20 days and maturation that took c.a. 25 days. The change of soluble solid content could sufficiently describe the fermentation process. The fermentation endpoint coincided with the equilibrium of soluble solids. Chestnut type mead fermented first and reached an equilibrium in soluble solids (% Brix) after 20 days (Fig. 1). The reason for the faster fermentation of chestnut mead is the higher content of nutrients in chestnut honey. Chestnut honey contains more pollen than other types of honey. Pollen is a good source of proteins used by yeasts. It took 40 days for lime and honeydew type mead to reach the equilibrium point.

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Fig. 1. Decrease of soluble solids during fermentation of chestnut, lime and honeydew type mead. : Chestnut; - - - -: lime; - - - -: honeydew

2.2. Volatiles assay Nearly all volatiles assayed reached their maximum concentration in the first 20 days and remained more or less stable during the maturation period. The final concentrations of all volatiles assayed are presented in Table 3. Acetaldehyde was synthesised predominantly during the initial phase of fermentation (Fig. 2). After 15 days, its concentration tended to decrease slightly or remained constant. Throughout the whole fermentation process, the lime mead showed a lower concentration of acetaldehyde, while the honeydew type reached the highest concentration. Ethyl acetate is an ester synthesised during fermentation as a result of yeast activity (Fig. 3). Chestnut mead showed a higher accumulation of ethyl acetate at the beginning of fermentation, while later it tended to decrease to be equal to the other types of mead. Lime mead possessed the highest and honeydew type the lowest concentration (Table 1).
Table 1. Concentration of volatiles and SD in meads made from chestnut, lime and honeydew type honey Volatile/Type of honey Acetaldehyde (mg l1) Ethyl acetate (mg l1) n-Propanol (mg l1) Iso-butanol (mg l1) Iso-amyl aclohol (mg l1) 2-Phenyl ethanol (mg l1) Chestnut 1370.10 8 16.40 0.04 39.10 0.02 30.46 0.01 112.95 0.2 6.26 0.1 Lime 608.98 11 23.21 0.4 20.79 0.1 24.92 0.3 72.81 0.1 4.39 0.1 Honeydew 1619.62 3 14.62 0.4 22.60 0.2 25.22 0.2 59.45 0.1 4.20 0.19

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Fig. 2. Production of acetaldehyde during mead fermentation from chestnut, lime or honeydew type honey. Data represent means of three replicates SD. : Chestnut; - - - -: lime; - - - -: honeydew

Fig. 3. Production of ethyl acetate during mead fermentation from chestnut, lime or honeydew type honey. Data represent means of three replicates SD. : Chestnut; - - - -: lime; - - - -: honeydew

Normal-propanol started to be accumulated at the very beginning of fermentation and almost reached its maximal concentration ten days after the start of the process (Fig. 4). Afterwards, its concentration remained stable during maturation (Table 1).

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Chestnut mead produced double the amount of n-propanol compared to the other two types. OUGH and BELL (1980) found a positive correlation of n-propanol synthesis with total N content in grape must. Another reason for an increased synthesis of n-propanol is a larger O2 entrapment before the start of fermentation (MAURICIO et al., 1997).

Fig. 4. Production of n-propanol during mead fermentation from chestnut, lime or honeydew type honey. Data represent means of three replicates SD. : Chestnut; - - - -: lime; - - - -: honeydew

Chestnut honey has more pollen particles compared to lime or honeydew. Pollen particles are good substrates for entrapment of O2 and other gases before the start of fermentation. Iso-butanol is a product of yeast metabolism and is produced mainly in the first 10 days of fermentation and does not change noticeably afterwards (Fig. 5). Its concentration is similar to that of n-propanol, but nearly three times lower than that of iso-amyl alcohol (Table 1). It is harsh in taste and constitutes an unpleasant flavour, if present in higher concentrations. Iso-amyl alcohol proved to be the most abundant of all the higher alcohols. As happened in the case of n-propanol, chestnut type honey produced double the amount of iso-amyl alcohol compared to lime or honeydew type honeys (Table 1, Fig. 6). Maceration of grapes provoked an increase of iso-amyl alcohol, probably because of higher oxygen entrapment (GOMIS et al., 1991; BOSSO, 1993; MAURICIO et al., 1997).

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Fig. 5. Production of iso-butanol during mead fermentation from chestnut, lime or honeydew type honey. Data represent means of three replicates SD. : Chestnut; - - - -: lime; - - - -: honeydew

Fig. 6. Production of iso-amyl-alcohol during mead fermentation from chestnut, lime or honeydew type honey. Data represent means of three replicates SD. : Chestnut; - - - -: lime; - - - -: honeydew

Chestnut type mead contains more insoluble solids due to its higher pollen content. As demonstrated by KLINGSHIRN and co-workers (1987), a positive correlation exists between insoluble solids and synthesis of iso-amyl alcohol. The other factors that

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influence the synthesis of iso-amyl alcohol are the content of total N prior to fermentation and the temperature during fermentation. Total N is negatively correlated with the synthesis of iso-amyl alcohol, while the temperature correlates positively. The lowest accumulation of iso-amyl alcohol in honeydew type mead can be explained by the highest content of total N in honeydew type honey. It is a general practice to add nitrogen, usually as ammonium salts, before fermentation in order to stimulate yeast growth, as well as to reduce the synthesis of higher alcohols. Fermentation temperature is maintained between 15 and 20 C, because higher temperatures stimulate the synthesis of iso-amyl alcohol as well as of isobutanol. Cider produced at fermentation temperatures maintained at a lower level were classified as more delicate and fresh in odour (GOMIS et al., 1991). Chestnut type mead also produced three times as much 2-phenyl ethanol compared to the other two types (Table 2, Fig. 7). It was synthesised predominantly at the beginning of fermentation, and afterwards its concentration rose only slightly. A low concentration of nutrients is a cause of increased synthesis of 2-phenyl ethanol (LEA, 1995). Chestnut and lime type honeys contain less total N than honeydew type honey, which can explain the lowest concentration of 2-phenyl ethanol in honeydew type mead.
Table 2. Ranking test for total impression of three honey water solutions, each containing 80 g l1 reducing sugars from honey Total number of panellists 11 Chestnut 6 Number of panellists Lime 3 Honeydew 2

Fig. 7. Production of 2-phenyl ethanol during mead fermentation from chestnut, lime or honeydew type honey. Data represent means of three replicates SD. : Chestnut; - - - -: lime; - - - -: honeydew

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2. 3. Sensory evaluation test The smell and taste of mead resemble the smell and taste of honey from which it is processed. The ranking test on samples according to total impressions classified chestnut honey solution as the best. Six out of 11 panellists choose the chestnut honey solution as superior, followed by lime (3 out of 11) and honeydew type honey (2 out of 11) solutions as presented in Table 2. Chestnut honey is rather bitter in taste, but it is probably the bitterness that contributes to the body and quite pronounced and lasting aftertaste. Panellists preferred chestnut honey solution predominantly because of its superior body and aftertaste compared to the other two honey solutions. According to ranking test, chestnut honey seems to be the most suitable for mead processing. All three types of mead benefited from the addition of reducing sugars. Panellists preferred meads with 80 g l1 sugar over meads with 40 g l1 reducing sugar (Table 3). The difference was the highest in case of chestnut honey (10:1), followed by honeydew type (8:3) and lime type (7:4). In the case of chestnut mead, reducing sugars mask the bitter taste that originates from chestnut honey. A higher content of reducing sugars (80 g l1) contributed to the body and harmony of taste. Dry mead with no reducing sugar is rather flat in taste and poor in body.
Table 3. Results of ranking test for total impression of three types of mead, each with 40 or 80 g l1 reducing sugars from honey (ciphres represent number of panellists) Chestnut 40 g Chestnut 80 g Honeydew 40 g Honeydew 80 g Lime 40 g Lime 80 g Better 1 10 3 8 7 4 Worse 10 1 8 3 4 7 Total number of panellists 11 11 11 11 11 11

Contrary to the honey solutions, panellists gave the same score to chestnut and lime type mead (4 out of 11), while three panellists preferred honeydew type of mead, as presented in Table 4. The fermentation process made lime and chestnut type meads equally acceptable to the panellists. Compared to honey solutions, the fermentation process improves the bouquet of lime mead, while the chestnut type retained its stronger body.
Table 4. Results of ranking test for total impression of three types of mead, each with 80 g l1 reducing sugars from honey Total number of panellists 11 Chestnut 4 Number of panellists Lime 4 Honeydew 3

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3. Conclusion Although mead production dates back in prehistoric times, we did not find much literature regarding mead processing. Mead retains the taste and smell of the honey from which it is made, although the fermentation process modulates the sensory properties of the primordial honey taste and flavour. Chestnut honey is known to have a strong body due to its higher pollen content compared to other types of honey. On the other hand, lime mead benefited from fermentation as regards its acceptable boquet. However, insoluble solids in chestnut honey are the main cause of an increased production of higher alcohols. An increased production of higher alcohols worsens the sensorial properties of fermented beverages in general.

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