Generally beer contain numerous aromatic compounds, some naturally present in the raw materials and some formed during processing. In this work were analysed beer wort flavor compounds, during primary fermentation, taking into study two beer wort types: one obtained from Pilsner malt (100%), other from a mixture of Pilsner malt (80%) and Caramel Malt (20%). An Shimadzu GC/MS-QP2010 equipment was used for samples analysis. The major compounds founded in beer wort samples, was derived from a vast array of compounds that arise from a number of sources. In this way, during fermentation and conditioning we founded different categories of secondary products: higher alcohols, esters, vicinal diketones (VDK), organic acids and aldehydes. The concentration of this aroma compounds depends on malt type and the moment of wort examination. Was noticed that some compounds disappeared and others appeared, from the day to day of primary fermentation

MICHIU, Delia

MUDURA, Elena

MUNTEAN, Florina

TOFANA, Maria

English

Publicatii USAMV Cluj-Napoca (University of Agricultural Sciences and Veterinary Medicine / Universitatea de Ştiinţe Agricole şi Medicină Veterinară din Cluj-Napoca)

309 Bulletin UASVM Agriculture, 67(2)/2010 Print ISSN 1843-5246; Electronic ISSN 1843-5386  Preliminary Research Concerning the Determination of Beer Wort Flavor Compounds During Primary Fermentation  Delia MICHIU, Maria TOFANĂ, Elena MUDURA, Florina MUNTEAN  University of Agricultural Sciences and Veterinary Medicine, Faculty of Agriculture, 3-5, Mănăştur Street, 400372, Cluj-Napoca, Romania, e-mail: deliamichiu@yahoo.com  Abstract. Generally beer contain numerous aromatic compounds, some naturally present in the raw materials and some formed during processing. In this work were analysed beer wort flavor compounds, during primary fermentation, taking into study two beer wort types: one obtained from Pilsner malt (100%), other from a mixture of Pilsner malt (80%) and Caramel Malt (20%). An Shimadzu GC/MS-QP2010 equipment was used for samples analysis. The major compounds founded in beer wort samples, was derived from a vast array of compounds that arise from a number of sources. In this way, during fermentation and conditioning we founded different categories of secondary products: higher alcohols, esters, vicinal diketones (VDK), organic acids and aldehydes. The concentration of this aroma compounds depends on malt type and the moment of wort examination. Was noticed that some compounds disappeared and others appeared, from the day to day of primary fermentation.  Keywords: beer wort; primary fermentation; flavor compounds; headspace; gas chromatography; mass spectrometry.  INTRODUCTION  The most common definition of flavor includes the four basic tastes - sourness, bitterness, sweetness, and saltiness - and the aroma perceived through tasting. A second, more precise definition that has found favor in the food science industry states that flavor is the total impression of taste, odor, tactile, kinesthetic, temperature, and pain sensations perceived through tasting. Flavor perceptions are a complicated mix of all of these factors (Bickham, 1997). Beer flavor is a very complex subject. More than 1000 different flavor elements have been identified in beer, and more are being found as increasingly sensitive analytical methods are developed. Some of these flavors are derived from raw materials (malt, adjuncts, hops, and water), but the vast majority are formed by yeast during fermentation (Bickham, 1997). Consequently, the selection of raw materials and/or yeast strain has great impact on the flavor. However, the flavor is also influenced by technological factors which affect the composition of the wort, and the conditions during fermentation, maturation, filtration and bottling. The organoleptic characteristics of beer depend mainly on the aroma-active substances produced by yeast during the fermentation of wort.   An important consideration in beer fermentation and flavour maturation is the fine-tuning of the concentration of all volatile compounds.The main flavor characteristics are the bitter taste derived primarily from the hops, an alcoholic note from ethanol and a carbonation mouthfeel from CO2. Secondary flavor notes include fruity estery flavors, alcoholic notes from higher alcohols and various sulphur components. However, most of the flavor compounds are present in very small quantities (below their individual taste threshold) and act synergistically to provide the balanced and refreshing taste of good beer. Occasionally 310 undesirable flavour components may appear, giving the beer various off-flavors. Of all secondary metabolites, higher alcohols are generally produced by yeast in the highest absolute concentrations. Those of importance for beer aroma are: propanol (solvent-like), isobutanol (alcoholic), isoamyl alcohol (fruity, sweet) and 2-phenyl ethanol (rose, floral). The brewing industry is the largest user of malt, and malt is arguably the most important ingredient in beer. Malt is the major supplier of the fermentable sugars that are converted to alcohol by the yeast. Pilsner malts are the most common type of malt. They are the major ingredient in the light lager beers that are so popular around the world. These beers require a malt with maximum extract and good enzyme potential but with limited development of color and flavor. Pale lager malts are generally made from two-rowed barley with average levels of protein. Caramel malt is unlike other colored malt in that fully modified green malt are roasted in a sealed roaster (65–70) prior to drying. As a result, the starches of caramel malts are degraded to simple sugars during this early stewing stage of kilning, in contrast to standard malts where the majority of starch conversion occurs later in the brewhouse. Caramel malt give a sweet, caramel flavor to beer (Edney, 2003). In the brewing process, the eﬃciency of fermentation and the character and quality of the final product are intimately linked with the amount and health of the yeast being pitched. The fermentation performance of yeast is also influenced by external factors, like wort clarity, lipid composition, wort oxygenation, pitching-rate and temperature, among others. Higher temperatures accelerate the yeast's metabolism and the yeast will be able to consume the sugars faster and generally more complete. But the production of unwanted flavor compounds at higher temperatures limits the fermentation temperature (Guido et al., 2003).  MATERIALS AND METHODS  Two types of beer wort were taken in study: one obtained from Pilsner malt (100%), other from a mixture of Pilsner malt (80%) and Caramel malt (20%). For the experiment, was used an industrial lager brewing yeast strain in a pilot-scale brewery. Pilsner malt and Caramel malt were grinded by a laboratory mill in grist, with fine granulation for a maximum effective power. The grist obtained, was mixed with water at 40°C in 1:4 ratio, followed the brewing process. After filtration, the sugar solution, in brewers’ jargon called ‘wort’, was transferred to the brewing kettle, where it is boiled 60 minutes with the addition of hops (Hallertau Magnum). Was used a few grams of hops (8 g/hl wort), a major ingredient with crucial impact on well-defined beer features. After cooling and removal of spent hops, the liquid, known as ‘hopped wort’ is pumped into fermentation vessels and yeast is added under aeration for growth. During the anaerobic phase, yeast cells convert sugars to ethanol and carbon dioxide. ‘Bottom fermentation’ was conducted: the temperature during primary fermentation process was 10°C, for 7 days.  The flavor profile was followed in the 5th and 6th days of primary fermentation when the characteristic flavor is stabilized. In beer wort samples, flavor compounds were quantified by HS/GC/MS. For this were used an Shimadzu GCMS-QP2010 and an AOC-5000 (CombiPAL) autosampler. 5 mL of sample was heated for 15 min. at 80/85°C in the HS. Separation was achieved on a capillary column: 50mx0.32mmx0.25µm, Phenomenex, USA. The following conditions were applied: oven temperature 40°C for 5 min., increase at 8°C/min. to 250°C, hold for 10 min. Helium was used as carrier gas.  The obtained chromatograms were analysed using Spectres library.  311 RESULTS AND DISCUSSION  The primary fermentation of beer wort supposes the metabolism of sugars, amino acids and lipids, with the formation of primary and secondary metabolic products. The type and the concentration of this compounds, is modify every day during primary cycle, leading to the formation of young beer flavour (Peacock and Deinzer, 1981). The concentrations of higher alcohols, esters and carbonyl compounds in pilsner wort and pilsner wort with caramel in the 5th and 6th day of primary concentration, shown in Tab. 1.  Tab.1 The major compounds found in beer wort samples during primary fermentation Compounds Retention time Concentration % of area Pilsner wort (100%) Pilsner wort (80%) and Caramel (20%) Pilsner wort (100%) Pilsner wort (80%) and Caramel (20%) Days 5 Days 6 Days 5 Days 6 Days 5 Days 6 Days 5 Days 6 3-metyl-butanal 4.285 4.283 4.292 4.290 8.292 4.570 4.111 1.613 2-metyl-butanal 4.448 4.446 4.459 4.450 2.802 1.027 2.516 0.464 2,3-pentandione 5.075 5.063 5.076 5.065 4.235 3.421 4.678 1.178 Vinyl acetate - 5.450 - - - 1.154 - - Acetaldehyde diethyl acetal 5.900 5.866 5.894 6.059 2.519 3.295 0.662 36.172 3-methyl-butan-1-ol 6.068 6.059 6.067 6.059 59.409 58.052 46.260 36.294 2-methyl- butan-1-ol 6.192 6.172 6.182 6.171 15.502 21.432 22.999 14.813 n-hexanal 8.017 8.004 8.017 8.031 1.196 1.442 0.793 0.619 1-butanol-3-methyl-acetate 10.415 10.399 10.401 10.392 2.380 2.697 6.827 3.303 β-mircene 13.620 13.609 13.376 10.457 1.220 0.745 0.196 0.448 Ethyl octanoate 18.412 18.398 - 13.893 0.338 1.015 - 0.080 Ethyl butanoate - 13.825 - - - 0.179 - - Ethyl decanoate - - 18.406 18.397 - - 0.989 0.733 Methyl pentanoate 22.270 - - - 0.269 - - - Ethyl propanoate - - 5.444 - - - 1.228 - Ethyl hexanoate - - 13.616 13.609 - - 7.007 2.451 Izobuthyl acetate - - - 7.216 - - - 0.218 3,7,11-trimethyl-1,3,6,10-dodecatetraene 23.040 23.036 22.250 22.246 0.566 0.327 0.427 0.377 3,7-dimethyl-1,3,7-octatriene 23.677 - 23.049 23.025 0.954 - 0.346 0.179 3-ethyl- butanoate - 22.247 - - - 0.222 - - α-cariofilene - 23.667 - - - 0.738  - Methacrylic anhydride - - 13.840 -  - 0.303 - 2-propoxy-ethanol - - - 5.523 - - - 0.618 2-nitropropan - - - 13.829 - - - 0.303  Monitoring of these compounds is important for selecting the optimum processing parameters in order to ensure the intensity of flavour in beer. The major compounds founded in beer wort samples were from different categories of secondary products: higher alcohols, esters, vicinal diketones (VDK), organic acids and aldehydes. The results for several flavor notes typically depended on malt type and on the moment of wort examination during primary fermentation. 312  In Fig. 1, are compared, the total ion chromatograms obtained from pilsner beer wort samples, in the 5th and 6th days of primary fermentation, using HS/GC/MS analysis, and in Fig. 2 from pilsner beer wort with caramel samples.                   In the Pilsner wort sample was observed an evolution of n-hexanal value from a concentration of 1.196 %  in the 5th day to a concentration of 1.442 % in the 6th day of primary fermentation, while for 3-methyl-butanal from a concentration of 8.292 % to a concentration of 4.570 % and for 2-methyl-butenal from a concentration of 2.802 % to a concentration of 1.027 %.  These modifications are due to the change of primary fermentation parameters, especially temperature changes. During the fermentation process the temperature increase 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5-1.00.01.02.03.04.05.06.07.08.09.010.011.0(x100,000)5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0(x100,000)Pale wort 100%, 6th days  Pale wort 100%, 5th days Pale wort 80% + caramel 20%, 6th days Pale wort 80% + caramel 20%, 5th days Fig.1.Comparative analysis of volatile compounds in Pilsner wort, in the 5th  and 6th days of primary fermentation Fig.2. Comparative analysis of volatile compounds in Pilsner wort 80% + caramel 20%, in the 5th  and 6th days of primary fermentation  7 10 7 9 1.  3-methyl-butanal 2.  2-methyl-butanal 3.  2,3-pentandione 4.  3- methyl – butan-1-ol 5.  2- methyl – butan-1-ol 6.  n-hexanal 7.  1-butanol-3-methyl-acetate 8.  β-mircene 9.  Ethyl hexanoate  1.  3-methyl-butanal 2.  2-methyl-butanal 3.  2,3-pentandione 4.  Acetaldehyde diethyl acetal 5.  3- methyl – butan-1-ol 6.  2- methyl – butan-1-ol 7.  n-hexanal  8.:  1-butanol-3-methyl-acetate 9.  β-mircene  10.  Ethyl octanoate  Volatile compounds in Pilsner wort  80% + caramel 20% Volatile compounds in Pilsner wort 313 continually, accelerating the yeast metabolism and in this way the yeast will be able to consume the sugars faster and complete. Also an evolution of higher alcohols values was detected: 3-methyl-butan-1-ol decreased from a concentration of 59.409 % in the 5th day to 58.052 % in the 6th day and 2-methyl-butan-1-ol increased from a concentration of 15.502 % to 21.432 %. These volatiles compounds give the characteristics of young beer.  Because the higher quantity of higher alcohols is form during riotously fermentation, their concentration decrease at the end of primary fermentation is explicable.  The evolution of vicinals diketones is very important for a good management of fermentation process. The objective of the fermentation process is to reduce the value of these compounds below the threshold of sensory perception: 3mg/l. The concentration of 2,3-pentandione decreased from 4.235 %  to 3.421 %, which means a good fermentation.  Regarding the esters evolution, was observed an increased of their concentration at the end of fermentation, being a marker of beer secondary fermentation. The concentration of 1-butanol-3-methyl-acetate increased from 2.380 % to 2.697 %, and the concentration of ethyl octanoate from 0.338 % to 1.015 %.  In Pilsner wort (80%) with caramel (20%), the peak area of 1-butanol-3-methyl-acetate was tripled in the 6th day of primary fermentation while the concentration of 2,3-pentandione decreased from 4.678 % to 1.178 %. To both types of wort was observed in the 6th day of analysis the appearance of new compounds and the disappearance of others.  CONCLUSIONS  The monitoring of volatile compounds in beer wort during primary fermentation, obtained from two types of malt: Pilsner malt (100%) and Pilsner malt (80%) with Caramel malt (20%), shows a higher concentration of flavor compounds founded in Pilsner beer wort samples.In Pilsner wort with caramel, the concentration of these compounds was low because the caramel malt used in the technological process decreases the fermentescible sugars amount.Volatile compounds spectre is much riche for Pilsner wort (80%) with caramel (20%) compared with Pilsner wort (100%), because of flavor compounds obtained from malt rousting.  REFERENCES  1. Bickham, Scott. (1997). An Introduction to Sensory Analysis. Brewing Techniques. 38. 149. 2. Edney, M. J. and M. S. Izydorczyk.. (2003). Malt Types and Products. Elsevier Science Ltd. All Rights Reserved. 3671. 3675. 3. Guido, L. F., P. G. Rodrigues, J. A. Rodrigues, C. R. Goncalves and A. A. Barros. (2004). The impact of the physiological condition of the pitching yeast on beer flavour stability: an industrial approach. Food Chemistry 87, 187. 4. Peacock, V., and M. Deinzer. (1981). The Chemistry of Hop Aroma in Beer. J. Am. Soc. Brew. Chem. 39:136. 5. Raymond, P. and W. Scott. (2003). Gas Chromatography Spectroscopy Combined Systems. Chrom-Ed Book Series. by Libraryforscience. 

Preliminary Research Concerning the Determination of Beer Wort Flavor Compounds During Primary Fermentation

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Preliminary Research Concerning the Determination of Beer Wort Flavor Compounds During Primary Fermentation

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