Food Chemistry

Food Chemistry 104 (2007) 12501257 www.elsevier.com/locate/foodchem

Changes in organic acid contents during mead wort fermentation

Abstract Organic acids contained in honey inuence the fermentation rate and frequently cause mead fermentation to halt. The purpose of this study was to determine the changes that occur in a group of selected organic acids during the fermentation of mead worts and their impact on the process. Mead worts (1:2 and 1:3 (honey:water)) were fermented using Saccharomyces cerevisiae yeast of the Johannisberg-Riesling breed (OCK 105). The worts were supplemented with diammonium hydrogen phosphate (0.4 g/l) and citric acid (0.25 g/l). During fermentation, the contents of selected carboxylic acids were determined using gas chromatography. Mead worts contain relatively high amounts of medium-chain fatty acids, which are believed to inhibit fermentation. The dominant compounds of this group are decanoic (42 mg/l), dodecanoic (31 mg/l) and octanoic (26 mg/l) acids. The experiments demonstrated that during the early days of fermentation, the main acids to form are the acetic and succinic acids, which reduce the wort pH, while the content of fatty acids drops by 7080%. During fermentation, the amounts of the formic, hexadecanoic and octadecanoic acids also fall. 2007 Elsevier Ltd. All rights reserved.
Keywords: Derivatisation; Fatty acid; Fermentation; GC; Honey; Mead; Organic acid; SPE; SPME; TMS

1. Introduction Mead is an alcoholic beverage obtained by fermenting mead wort, with the possible addition of various spices and hops, which contains between 9% and 18% of alcohol by volume. Mead wort is produced by diluting bee honey with the appropriate amount of water or fruit juice. Depending on the proportion to which honey is diluted, dierent types of mead are obtained; the nest at 1:0.5 (honey: water), or at 1:1, 1:2 and 1:3. Worts that contain the most sugar (the 1:0.5 and 1:1 types) are prepared by successively adding the appropriate portions of honey so as not to stop the fermentation, due to an excessive osmotic pressure. Bee honey is a raw material characterised by low acidity. To ensure the optimum pH of mead wort, sometimes citric, tartaric or lactic acid is added (Gogol & Tus ski, 1996). Organic acids also form as the by-products zyn

Corresponding author. Tel.: +48 12 662 4979; fax: +48 12 662 4798. E-mail address: [email protected] (P. Sroka).

of ethanol fermentation. A quick increase in acidity is observed in the rst hours of the process and sometimes combines with the low buer capacity of mead wort to cause a rapid fall of pH causing the fermentation to stop. This phenomenon, caused mainly by the formation of succinic acid, is strongly dependent on the strain of yeast and the presence of nitrogen compounds (Fleet, 1994). The production of mead, as an alcoholic beverage, has been known since ancient times. Poland has many centuries of tradition in mead making, but the production of mead has suered in recent years, partially due to the lack of scientic progress in this eld. The relatively long time needed for wort fermentation and mead maturing, ranging from several months (meads made from wort 1:3) to several years (meads made from worts 1:0.5 and 1:1), requires producers to use large capacity vessels and also to spend significant sums on raw material purchases (Aleksandrowicz, 1988). Certain process diculties also result from the high acidity and the shortage of substances necessary for yeast development. For these reasons, research is conducted to optimise the production process of these beverages.

0308-8146/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2007.01.046

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Frequently, during fermentation, the appropriate alcohol content is not reached within the appropriate time. The few studies published on this subject dealt with the characteristics of mead (Kime, McLellan, & Lee, 1991), the breeding of yeast adapted to high sugar concentrations (Sawicka, 1970), thermal processing (Wintersteen, Andrae, & Engeseth, 2005), and methods of preparing worts as well as the processes of their fermentation and maturing ska, (Wzorek & Lisak, 1980; Wzorek, Konieczna, Szyman & Bugajewska, 1983). Experience with grape wine production indicates that changes in the concentration of organic acids can cause the fermentation to halt. Non-dissociated particles of the octanoic, decanoic and acetic acids are antiseptic, and in greater concentrations can completely stop yeast develop -Correia, ment (Brul & Coote, 1999; Cabral, Viegas, & Sa 2001). Fatty acids can also cause an increased activity of certain enzymes, like ATP-ase. Higher concentrations of octanoic acid activate that enzyme and at the same time stop cell growth (Arneborg, Jespersen, & Jakobsen, 2000). The antiseptic eect of medium-chain fatty acids usually prolongs the lag phase and reduces the growth and the cell weight output, in some cases also contributing to cell death. Octanoic acid retards the biosynthesis of ace -Correia, tic and succinic acids by yeast cells (Viegas & Sa 1995). Decanoic acid intensies the outow of amino acids from the cell, improves the passive ow of protons through the cell membrane, and can even cause it to be broken leading to outow of cytoplasm (Stadford & Anslow, 1996). It should be mentioned that the reduction of the yeast biomass growth by acetic acid also has a partially benecial impact on the capacity of ethanol production (Taherzadeh, Niklasson, & Liden, 1997). The biochemical transformations of organic acids have been relatively well researched and described for the fermentation of grape must (Fleet, 1994). However, mead worts are characterised by a lower pH and a dierent combination of acids originating from bee honey. So far, no research has been done on the transformations of organic acids during mead wort fermentation. Due to the high sugar content, the fermentation process progresses very slowly, requires the right pH, temperature, and specic yeast breeds and biocatalysts growth. The identication and elimination of factors which restrict cell activity in these conditions could shorten the process and thus, cut costs. The purpose of this study was to identify the quantity of carboxylic acids in mead worts and the changes that occur in a group of these compounds during the fermentation. 2. Materials and methods 2.1. Biological material Free cells of Saccharomyces cerevisiae yeast, JohannisbergRiesling (JR) breed, obtained from the Collection of Pure Industrial Microorganism Cultures of the Institute

of Fermentation Technology and Microbiology, Technical University of Lodz (collection no. OCK 105) were used. 2.2. Mead wort preparation and fermentation Buckwheat honey (Bartnik Sadecki, Poland) was mixed with potable water (66.1 mg/l Ca2+, 9.0 mg/l Mg2+) in the following proportions: 1:2 (v/v) and 1:3 (v/v), heated and gently boiled for 10 min, then topped up with diammonium hydrogen phosphate(V) (0.4 g/l) and citric acid (0.25 g/l); the extract was checked after mixing. The hot wort was poured into conical asks (1.5 l of wort into 3 l asks) stopped with sterile fermentation trap tubes. After the wort was cooled down to approximately 30 C, a precisely dened amount of starter yeast was added (0.5 g/l calculated for the dry substance); this yeast was prepared in a three-stage culture (agar medium, 9% brewers wort, still culture (10 ml) and shaken culture (100 ml)). The fermentation progressed at room temperature (20 C to 22 C) until two subsequent weights of samples measured at an interval of 4 days did not dier by more than 1 g. All fermentation experiments were conducted twice. 2.3. Analytical methods 2.3.1. General The ethanol content, pH, the total and volatile acidity were determined using ocial methods (O.I.V., 1990). 2.3.2. Acetic and formic acid determination 2.3.2.1. General. The analysis was performed by gas chromatography after rst esterifying the analysed acids with benzyl bromide and extracting them using headspace solid-phase microextraction (HS-SPME) (Wittman, Van Langenhove, & Dewulf, 2000). 2.3.2.2. Sample preparation. A 15 ml vial was lled with 1.5 ml of ClarkLabes buer (pH 7), 0.2 ml of the sample to be analysed and 0.02 ml of a 50% benzyl bromide solution in benzyl alcohol. The vial was sealed with a screw cap with a Teon liner and heated at 50 C for 4 h, during which time it was mixed using a magnetic stirrer. Then, the sample was cooled in an ice bath, 0.99 g of NaCl was added, and the sample was placed in an incubator (30 C, magnetic stirrer). After 30 min, an SPME (PDMS, 100 lm, Supelco) bre was placed in the headspace for another 30 min. The analytes absorbed were desorbed for a time of 3 min in the sample injector of a gas chromatograph. 2.3.2.3. Chromatographic separation conditions. The chromatographic analysis was performed using an HP 5890, series II gas chromatograph, with a ame ionization detector (FID) and an HP5 capillary column (dimensions: 30 m 0.53 mm 2.65 lm). The temperature of the injector and detector was set to 250 C. A programmed increase

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of the column temperature to 40 C (5 min), and to 250 C (7 C/minute) and 1 min of nal heating were applied. The carrier gas was helium at 20 ml/min. Signals were identied by comparing their retention times with formic and acetic acid esters. The recovery of formic and acetic acids was relatively high, at 96.4% and 95.0%, respectively. 2.3.3. Fatty acid determination 2.3.3.1. General. Fatty acids were analysed as their volatile methyl esters (Gallart, Francioli, Viu-Marco, Lopez-Tamames, & Buxaderas, 1997). 2.3.3.2. Determination of non-volatile carboxylic acids. Non-volatile organic acids (hydroxy- and multi-carboxylic derivatives) were determined by gas chromatography in the form of trimethylsilyl derivatives (TMS) after preliminary purication on ion-exchanger resin. The samples for determination were prepared using a modied J.T. Baker procedure (Phillipsburg, NJ) (Application Notes). 2.3.3.3. Sample preparation. A BAKER SPE column processor and SPE-SAX 3 ml columns (Supelco) lled with 500 mg of ion-exchange resin (quaternary amines) were used for the preparation. Prior to the analysis, the columns were conditioned by rinsing them with the following solutions: methanolwater 1:1, 2 ml, 0.3 ml/min; NaOH 1 M, 2 ml, 0.3 ml/ min; 30 ml of redistilled water, 3.0 ml/min; CH3COOH solution (pH 2.05), 5 ml, 0.5 ml/min; CH3COOH solution (pH 4.5), 45 ml, 3.0 ml/min. The sample (0.5 ml) supplemented with 0.2 ml of internal standard (malonic acid 4576 mg/l) was fed into the prepared column at 0.3 ml/min, the column was then rinsed with 3 ml of distilled water, dried for 5 min and eluted with 5 M formic acid. The eluant was gathered into 4 ml glass vials, water was added to 4 ml and the contents were mixed. One millilitre of the resulting solution

was measured, transferred to a dry 4 ml vial, dried at 45 C, dissolved in 0.5 ml of freshly-distilled pyridine (dried over KOH), after which 0.1 ml of BSTFA (N,O-bis-(trimethylsilyl) triuoroacetamide) was added. After 10 min, the sample was separated on a gas chromatograph. 2.3.3.4. Chromatographic separation conditions. The chromatographic analysis was conducted using the apparatus described above. This time the column was heated from 55 C (6 min) to 260 C (at 8 C/min), with 5 min of nal heating. Signals were identied by comparing the retention time of trimethylsilyl-derivatives (TMS) of succinic, tartaric, lactic and citric acids. 2.4. Statistics The calculated average deviations were plotted on graphs. For selected series, a one-factor analysis of variance (ANOVA) was calculated using StatSoft Statistica 6.1 software (signicance level a = 0.05). The dierences between the averages were veried using the Duncan test (p < 0.05). 3. Results and discussion 3.1. General The process of mead fermentation is particularly dicult because of the high sugar concentration of the wort and the resultant high osmotic pressure. In the rst week, the 1:3 mead wort fermented almost twice as fast as the 1:2 mead wort (Fig. 1). The rate of the process probably depended on the osmotic pressure (1:2 worts with approx-

20

15

Ethanol (% v/v)

10

0 0 5 10 15 20 25 30

Time (days) Wort 1:3 Wort 1:2

Fig. 1. Changes in the contents of ethanol during the fermentation of mead worts.

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imately 35% of extract) and the longer time over which yeast cells could adapt to the specic environmental conditions. In the rst week of fermentation, total acidity increased markedly, up to 4.5 g/l (Fig. 2). This increase in acidity was caused mainly by the yeast cells synthesising acetic acid (0.71.0 g/l) and the succinic acid (0.10.3 g/l) (Figs. 3 and 4). This changed concentration of organic acids reduced pH to 3.03.2, which remained practically unchanged until the fermentation end. The quick pH drop in the fermenting worts is benecial, as it stops the development of foreign microora. In the second week of fermentation, acidity dropped by between 8% and 22%, and in the following weeks changed

only insignicantly. The total acidity of 1:3 samples was usually 0.10.7 g/l lower (Fig. 2). 3.2. Acetic acid Volatile acidity increased during fermentation, mainly as a result of acetic acid synthesis, whose concentration in the 1:2 samples reached a maximum (1.3 g/l) on the 24th day of fermentation (Fig. 2). The acetic acid content of 1:3 worts (less concentrated) was 60% (0.44 g/l) lower in the second week. The acetate quantity depends mainly on the concentration of carbohydrates and the source of nitrogen as well as the pH (Fleet, 1994). Acetic acid content increases signif-

Total acidity (g/l), Volatile acidity (g/l)

0 0 4 7 11 14 17 21 24 28

Time (days)

Total acidity (1:3 wort) Volatile acidity (1:3 wort)

Total acidity (1:2 wort) Volatile acidity (1:2 wort)

Fig. 2. Changes in total and volatile acidity during the fermentation of mead worts.

1.6

1.4

Concentration of acids (g/l)

1.2

1.0

0.8

0.6

0.4

0.2

0.0 0 4 7 11 14 17 21 24 28

Formic acid

Time (days) Acetic acid

Succinic acid

Fig. 3. Changes in the contents of formic, acetic and succinic acids during the fermentation of 1:3 mead worts.

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1.6

ski / Food Chemistry 104 (2007) 12501257 P. Sroka, T. Tuszyn

1.4

Concentration of acids (g/l)

1.2

1.0

0.8

0.6

0.4

0.2

0.0 0 4 7 11 14 17 21 24 28

Formic acid

Time (days) Acetic acid

Succinic acid

Fig. 4. Changes in the contents of formic, acetic and succinic acids during the fermentation of 1:2 mead worts.

icantly during alcoholic fermentation if the sugar concentration is high. If yeast is subjected to osmotic stress, the reduction of acetaldehyde to ethanol is frequently observed to slow down, as a result of which more acetic acid is synthesised (Erasmus, Merwe, & Vuuren, 2003). A similar eect was noted if the wort had a low pH (Shimazu & Watanabe, 1981; Monk & Cowley, 1984). The acetic acid in the fermenting worts changes yeast metabolism, reducing the glycerol synthesis and the biomass output. Yeast cells use more ATP to transform acetic ions into acetylCoA and remove the protons coming from the acid dissociation, which leads to a slight increase in ethanol production (Taherzadeh et al., 1997). 3.3. Formic acid Another component which had a degree of impact on the volatile acidity was formic acid. The formate contained in the wort, originating from honey, was partially metabolised by yeast, so its content in the 1:2 mead wort dropped from 0.28 to 0.20 g/l (Fig. 4). However, in 1:3 mead wort, the concentration dropped only by some 0.03 g/l, reaching 0.16 g/l in the fourth week of fermentation (Fig. 3). Like acetic acid, formic acid also signicantly increases volatile acidity, which in the case of mead should not exceed 1.4 g/l recalculated for acetic acid (Dziennik Ustaw, 2004). These compounds also slow down fermentation rate (Arneborg et al., 2000). 3.4. Medium-chain fatty acids Of the medium-molecule fatty acids determined in mead worts, comparatively high concentrations were detected for decanoic (1342 mg/l), dodecanoic (731 mg/l) and octanoic acids (526 mg/l) (Figs. 5 and 6). Tetradecanoic acid was present in lower quantities (815 mg/l). During the rst two weeks of 1:2 mead wort fermentation, the concentra-

tions of octanoic (42%) and decanoic (51%) acids dipped signicantly, but went up again in the third and fourth weeks (Fig. 6). The contents of dodecanoic and tetradecanoic acids also fell in the rst week of the process (by 28% and 60%, respectively), but they did not change much at later fermentation stages. In 1:3 mead worts, only the dodecanoic acid concentration shrunk markedly (Fig. 5). The decreasing concentrations of fatty acids during the rst two weeks of fermentation results from their metabolisation by yeast cells (Fleet, 1994). It is much more dicult to explain the increase of their concentrations in the third week of the process, the probable cause being the solution of the fatty acids in the ethanol created. The determined concentrations of medium-chain fatty acids were almost ten times greater than those so far determined in fermenting grape musts (Ancin, Ayestaran, & Garcia, 1998). These compounds probably slow mead wort fermentation down. The octanoic and decanoic acids are present in the fermenting wort mainly in their non-dissociated form. Their molecules, which have an amphipathic structure, are easily adsorbed on yeast surfaces and penetrate into the cells. Non-dissociated fatty acids can also adsorb on the insides of fermentation vessels and on matter suspended in mead worts. The increasing concentration of medium-chain fatty acids in cell membranes reduces their hydrophobic characteristics, impacts the operation of enzymes and reduces the cells resistance to ethanol. Octanoic acid is very toxic to yeast cells at concentrations as low as 16 mg/l (Viegas & -Correia, 1995). The octanoic as well as other lipophilic Sa weak acids, particularly at low pH, kill microorganisms by making their cytoplasm acidic and accumulating toxic anions inside the cells. Fat-soluble acids have a signicant impact on the spatial organisation of cell membranes, causing the energy necessary to transport protons to be exhausted and as a result bring about a drop of the pH inside the cell (Cabral et al., 2001). Other acids, like dodecanoic (lauric) and tetradecanoic acids, which were

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350

Concentration of acids (g/l)

300

250

200

150

100

50

0 0 4 7 11 14 17 21 24

Time (days) C8 C10 C12 C14 C16 C18 C18:2

Fig. 5. Changes in the contents of octanoic (C8), decanoic (C10), dodecanoic (C12), tetradecanoic (C14), hexadecanoic (C16), octadecanoic (C18) and octadecadienoic acids (C18:2) during the fermentation of 1:3 mead worts.
400

350

Concentration of acids (g/l)

300

250

200

150

100

50

0 0 4 7 11 14 17 21 24

Time (days) C8 C10 C12 C14 C16 C18 C18:2

Fig. 6. Changes in the contents of octanoic (C8), decanoic (C10), dodecanoic (C12), tetradecanoic (C14), hexadecanoic (C16), octadecanoic (C18) and octadecadienoic acids (C18:2) during the fermentation of 1:2 mead worts.

present in relatively high concentrations (Figs. 5 and 6), probably have a similar inuence. The high osmotic pressure of mead worts may increase the toxicity of these acids to yeast cells and thus, slow the fermentation down. It should be emphasised that short-chain fatty acids improve the taste of mead and also form esters, which give mead its bouquet. If these components are removed using sorbents, the sensory characteristics of the product may change (Bugajewska et al., 1998). Processing wine with ion-exchange resins does allow all anions of the undesirable acids to be removed, but the process is illegal in Poland (Dziennik Ustaw, 2004).

3.5. Long-chain fatty acids The concentration of octadecanoic (stearic) acid fell during the rst week of fermentation, to rise again in the following two (in a 1:3 mead wort) up to 224 mg/l (Figs. 5 and 6) The hexadecanoic (palmitic) acid concentration was lower (69151 mg/l), and decreased slightly during the process. The decreasing amount of fatty acids can be explained by their being metabolised by yeast cells. The increased stearic acid content in the second week of fermentation may have been the result of the increasing content of ethanol, in which waxes originating from honey

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Table 1 Concentration of non-volatile acids in fermenting and fermented worts Compound Succinic acid (mg/dm3) Citric acid (mg/dm3) Lactic acid (mg/dm3) Tartaric acid (mg/dm3) nd, not detected. Mead worts 1:3 nd 223 34 720 20 43 12 1:2 nd 250 9 1070 100 45 12 Fermented mead worts 1:3 351 10 216 11 620 20 51 8 1:2 93 20 194 30 1130 110 40 3

mentation. The main compounds in this group were the following acids: decanoic (42 mg/l), dodecanoic (31 mg/l) and octanoic (26 mg/l).  A higher extract content and the increase in general acidity of worts cause the yeast to synthesise more acetic acid, leading to an increased content of non-dissociated fatty acids, which slow down ethanol fermentation. References
czesnos c . Aleksandrowicz, J. (1988). Miody pitne tradycja i wspo Przemys fermentacyjny i owocowo-warzywny, 7, 2224. Ancin, C., Ayestaran, B., & Garcia, A. (1998). Evolution of fatty acid contents in Garnacha and Viura musts during fermentation and the aging of wine. Zeitschrift fu r Lebensmitteluntersuchung und -Forschung A, 206, 143147. e. Application Notes, Extraction of organic acids from fruit juice and pure Baker, <www.jtbaker.com>. Arneborg, N., Jespersen, L., & Jakobsen, M. (2000). Individual cells of Saccharomyces cerevisiae and Zygosaccharomyces bailii exhibit dierent short-term intracellular pH responses to acetic acid. Archives of Microbiology, 174, 125128. Brul, S., & Coote, P. (1999). Preservative agents in food. Mode of action and microbial resistance mechanisms. International Journal of Food Microbiology, 50, 117. _ en cukro w, wybranych Bugajewska, A. (1998). Wpyw wysokich ste z w i zabiego w selekcyjnych na wyniki fermentacji nastawo w aktywatoro re cechy droz _ dz _ y. Ph.D. Dissertation, SGGW, winiarskich oraz niekto Warszawa. -Correia, I. (2001). Mechanisms Cabral, M. G., Viegas, C. A., & Sa underlying the acquisition of resistance to octanoic-acid-induced-death following exposure of Saccharomyces cerevisiae to mild stress imposed by octanoic acid or ethanol. Archives of Microbiology, 175, 301307. w winiarskich, Dziennik Ustaw, (2004). O wyrobie i rozlewie wyrobo obrocie tymi wyrobami i organizacji rynku wina, nr 34, poz. 292. Erasmus, D. J., Merwe, G. K., & Vuuren, H. J. J. (2003). Genom-wide expression analyses: Metabolic adaptation of Saccharomyces cerevisiae to high sugar stress. FEMS Yeast Research, 3, 375399. Fleet, G. H. (1994). Wine microbiology and biotechnology. Sydney: Harwood Acad. Pub. (pp. 166179). Gallart, M., Francioli, S., Viu-Marco, A., Lopez-Tamames, E., & Buxaderas, S. (1997). Determination of free fatty acids and their ethyl esters in must and wines. Journal of Chromatography A, 776, 283291. ski, T. (1996). Miody pitne tradycje, surowce Gogol, D., & Tuszyn i technologie. Przemys Fermentacyjny i Owocowo Warzywny, 8, 2527. Kime, R. W., McLellan, M. R., & Lee, C. Y. (1991). An improved method of mead production. American Bee Journal(June), 394395. Monk, P. R., & Cowley, P. J. (1984). Eect of nicotnic acid and sugar concentration of grape juice and temperature on accumulation of acetic acid during yeast fermentation. Journal of Fermentation Technology, 62, 515525. O.I.V. International Organisation of Vine And Wine (1990). Compendium of international methods of wine and must analysis, Paris. _ dz _ y do fermentacji miodo w Sawicka, R. (1970). Selekcja i adaptacja droz w i Laboratorio w Badawczych Przemysu pitnych. Prace Instytuto Spoz _ywczego, 20(3), 411424. Shimazu, Y., & Watanabe, M. (1981). Eects of yeats strains and environmental conditions on formation of organic acids in must during fermentation. Journal of Fermentation Technology, 59, 2732. Stadford, M., & Anslow, P. A. (1996). Comparison of the inhibitory action on Saccharomyces cerevisiae of weak-acid preservatives, uncouples and medium-chain fatty acids. FEMS Microbiology Letters, 142, 5358. Taherzadeh, M. J., Niklasson, C., & Liden, G. (1997). Acetic acid friend or foe in anaerobic batch conversion of glucose to ethanol by Saccharomyces cerevisiae? Chemical Engineering Science, 52, 26532659.

would dissolve. The octadecanoic acid represented about 50%, and the hexadecanoic acid about 30% of all mediumand long-chain fatty acids found in fermented mead worts. In fermenting worts, the level of unsaturated higher fatty acids containing two and three unsaturated bonds (Figs. 5 and 6) was also monitored. The linoleic acid concentration changed during the fermentation of both the 1:3 and 1:2 mead worts. After the rst week, the amount of this acid dropped to some 4 mg/l, but in the third and fourth weeks increased again (8.016.7 mg/l). 3.6. Non-volatile acids The acidication of mead worts is necessary for the cor ski, rect progress of their fermentation (Gogol & Tuszyn 1996). In the rst days of fermentation, the acidity of mead is insucient to ensure the optimum pH values for a microbiological purity. During the fermentation, succinic acid also formed, reaching a concentration four times greater in the 1:3 mead wort (0.100.42 g/l, Fig. 3) than in the 1:2 wort (Fig. 4). The contents of other acids, like lactic (approx. 1 g/l), citric (0.25 g/l) and tartaric acids (4050 mg/l) did not change signicantly during the fermentation (Table 1), while the dierences in their concentrations were mainly the result of dierences in the dilution of honey performed to prepare 1:2 and 1:3 worts. The acetic and succinic acids, formed during fermentation, reduce the wort pH. The increased total acidity caused the pH of the wort to drop, simultaneously reducing the dissociation of fatty acids present in the wort. The above factors, combined with the relatively high concentration of medium-chain fatty acids, may cause the fermentation to slow down, or stop. The results obtained show that during the production of mead particular attention should be paid to the contents of the above acids in the processed honey. There is a need for further research conducted in industrial-scale conditions. 4. Conclusions  Total acidity rises mainly in the rst week of mead wort fermentation. The dominant compounds determining the change in the acidity of fermenting wort are acetic and succinic acids.  Mead worts contain relatively large amounts of medium-chain fatty acids, which are believed to inhibit fer-

 ski / Food Chemistry 104 (2007) 12501257 P. Sroka, T. Tuszyn -Correia, I. (1995). Toxicity of octanoic acid in Viegas, C. A., & Sa Saccharomyces cerevisiae at temperatures between 8.5 and 30 C. Enzyme and Microbial Technology, 17, 826831. Wintersteen, C. L., Andrae, L. M., & Engeseth, N. J. (2005). Eect of heat treatment on antioxidant capacity and avor volatiles of mead. Journal of Food Science, 70(2), C119C126. Wittman, G. Y., Van Langenhove, H., & Dewulf, J. (2000). Determination of acetic in aqueous samples, by water-phase derivatisation, solid-

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phase microextraction and gas chromatography. Journal of Chromatography A, 874, 225234. rych parametro w Wzorek, W., & Lisak, M. (1980). Wpyw niekto c miodo w pitnych. Przemys Fermentacyjny i maderyzacji na jakos Owocowo-Warzywny, 2, 1316. ska, K., & Bugajewska, A. (1983). Wzorek, W., Konieczna, E., Szyman Optimization of honey wort fermentation in double mead technology. Polish Journal of Food and Nutrition Sciences, 2(1), 5765.