Analiza Berii Fara Alcool

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Review A review of methods of low alcohol and alcohol-free beer production Tomáš Brányik a,, Daniel P. Silva b , Martin Baszczyn ˇ ski a , Radek Lehnert a , João B. Almeida e Silva c a Department of Fermentation Chemistry and Bioengineering, Institute of Chemical Technology Prague, Technická 5, 166 28 Prague, Czech Republic b Institute of Technology and Research, Tiradentes University, Campus Farolândia, Sector ITP, 49032-490 Aracaju, Sergipe, Brazil c Department of Biotechnology, Engineering School of Lorena, University of São Paulo, P.O. Box 116, 12602-810 Lorena, São Paulo, Brazil article info Article history: Received 29 June 2011 Received in revised form 14 September 2011 Accepted 25 September 2011 Available online 1 October 2011 Keywords: Beer Brewing Low-alcohol beer Alcohol-free beer Dealcoholization Limited fermentation abstract The increasing interest of consumers in health and alcohol abuse issues motivates breweries to expand the assortment of products with low alcohol content. The goal of producing beers with low alcohol con- tent can be achieved by two main strategies; namely by gentle removal of alcohol from regular beer and by limited ethanol formation during the beer fermentation. Within these two basic strategies, there are a number of techniques that vary in performance, efficiency and usability. This paper presents an overview and comparison of these techniques and provides an evaluation of sensorial properties of low-alcohol and an alcohol-free beer produced as well as suggests possibilities for their additional improvement. Ó 2011 Elsevier Ltd. All rights reserved. Contents 1. Introduction ......................................................................................................... 494 2. Beer and health ...................................................................................................... 494 3. Methods of the alcohol-free beer production ............................................................................... 494 4. Production of alcohol-free beer by ethanol removal methods ................................................................. 495 4.1. Thermal processes ............................................................................................... 495 4.1.1. Vacuum rectification plant................................................................................. 495 4.1.2. Thin film evaporators ..................................................................................... 496 4.2. Membrane processes ............................................................................................. 498 4.2.1. Dialysis ................................................................................................ 498 4.2.2. Reverse osmosis ......................................................................................... 498 5. Production of alcohol-free beer by methods of restricted ethanol formation ..................................................... 499 5.1. Changed mashing process ......................................................................................... 499 5.2. Arrested or limited fermentation process ............................................................................ 500 5.3. Use of special yeast .............................................................................................. 501 5.4. Continuous fermentation ......................................................................................... 502 6. Sensorial properties and additional improvements of alcohol-free beer ......................................................... 503 6.1. Post-treatments and blending ..................................................................................... 504 6.2. Additives ...................................................................................................... 504 7. Cost evaluation and conclusions ......................................................................................... 504 Acknowledgements ................................................................................................... 504 References .......................................................................................................... 505 0260-8774/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.09.020 Abbreviations: ABV, alcohol by volume; ADH, alcohol dehydrogenase; AF, arrested fermentation; AFB, alcohol-free beer; CCP, cold contact process; D, dialysis; DMS, dimethyl sulfide; EAA, esters of acetic acids; EBC, European Brewery Convention; ES, esters; FA, short-chain fatty acids; FF, falling film; FUM, fumarase; HA, higher alcohols; HAA, higher aliphatic alcohols; KGD, 2-ketoglutarate dehydrogenase; LAB, low-alcohol beer; RO, reverse osmosis; SCC, spinning cone column; VR, vacuum rectification. Corresponding author. E-mail address: [email protected] (T. Brányik). Journal of Food Engineering 108 (2012) 493–506 Contents lists available at SciVerse ScienceDirect Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng

Transcript of Analiza Berii Fara Alcool

Page 1: Analiza Berii Fara Alcool

Journal of Food Engineering 108 (2012) 493–506

Contents lists available at SciVerse ScienceDirect

Journal of Food Engineering

journal homepage: www.elsevier .com/ locate / j foodeng

Review

A review of methods of low alcohol and alcohol-free beer production

Tomáš Brányik a,⇑, Daniel P. Silva b, Martin Baszczynski a, Radek Lehnert a, João B. Almeida e Silva c

a Department of Fermentation Chemistry and Bioengineering, Institute of Chemical Technology Prague, Technická 5, 166 28 Prague, Czech Republicb Institute of Technology and Research, Tiradentes University, Campus Farolândia, Sector ITP, 49032-490 Aracaju, Sergipe, Brazilc Department of Biotechnology, Engineering School of Lorena, University of São Paulo, P.O. Box 116, 12602-810 Lorena, São Paulo, Brazil

a r t i c l e i n f o

Article history:Received 29 June 2011Received in revised form 14 September 2011Accepted 25 September 2011Available online 1 October 2011

Keywords:BeerBrewingLow-alcohol beerAlcohol-free beerDealcoholizationLimited fermentation

0260-8774/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.jfoodeng.2011.09.020

Abbreviations: ABV, alcohol by volume; ADH, alcodimethyl sulfide; EAA, esters of acetic acids; EBC, EuroHAA, higher aliphatic alcohols; KGD, 2-ketoglutarate⇑ Corresponding author.

E-mail address: [email protected] (T. Brányi

a b s t r a c t

The increasing interest of consumers in health and alcohol abuse issues motivates breweries to expandthe assortment of products with low alcohol content. The goal of producing beers with low alcohol con-tent can be achieved by two main strategies; namely by gentle removal of alcohol from regular beer andby limited ethanol formation during the beer fermentation. Within these two basic strategies, there are anumber of techniques that vary in performance, efficiency and usability. This paper presents an overviewand comparison of these techniques and provides an evaluation of sensorial properties of low-alcohol andan alcohol-free beer produced as well as suggests possibilities for their additional improvement.

� 2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4942. Beer and health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4943. Methods of the alcohol-free beer production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4944. Production of alcohol-free beer by ethanol removal methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495

4.1. Thermal processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495

4.1.1. Vacuum rectification plant. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4954.1.2. Thin film evaporators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496

4.2. Membrane processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498

4.2.1. Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4984.2.2. Reverse osmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498

5. Production of alcohol-free beer by methods of restricted ethanol formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499

5.1. Changed mashing process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4995.2. Arrested or limited fermentation process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5005.3. Use of special yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5015.4. Continuous fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502

6. Sensorial properties and additional improvements of alcohol-free beer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503

6.1. Post-treatments and blending . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5046.2. Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504

7. Cost evaluation and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505

ll rights reserved.

hol dehydrogenase; AF, arrested fermentation; AFB, alcohol-free beer; CCP, cold contact process; D, dialysis; DMS,pean Brewery Convention; ES, esters; FA, short-chain fatty acids; FF, falling film; FUM, fumarase; HA, higher alcohols;

dehydrogenase; LAB, low-alcohol beer; RO, reverse osmosis; SCC, spinning cone column; VR, vacuum rectification.

k).

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1. Introduction

The production of beers with low alcohol content had differenthistorical reasons in the past century. For instance, during WorldWars (1914–1918 and 1939–1945) it was the shortage of rawmaterials, which was leading to the production of beers with loworiginal extract (often with a high proportion of adjuncts) and thusof low alcohol content. On the other hand, in the years between1919 and 1933 it was the prohibition to manufacture, sale and con-sume alcohol, which increased the production of low alcohol con-tent beers in the United States of America (Meussdoerffer, 2009;Silva et al., 2010). In the late 20th century efforts of breweries toexpand the assortment of products with beers with low alcoholcontent was motivated mainly by the following reasons:

� Increase in the overall production by bringing out new productsin countries with highly competitive markets.� Provide beer consumers with alternative products prior or dur-

ing their activities (driving motor vehicles or operating machin-ery, engagement in sports) or under conditions (pregnancy,medication) irreconcilable with alcohol consumption.� Penetrate beverage markets in countries, where alcohol con-

sumption is forbidden for religious reasons.

Although the sales of beers with low alcohol content did not ful-fill the initial optimistic expectations and the market with theseproducts has been, for a long time, just a drop in the sea of theoverall beer production, nowadays it is a fast growing segment ofthe beer market worldwide. In the last five years, the average salesin Europe climbed by 50%. Spain is now the largest consumer ofbeers with low alcohol content in the European Union (EU), 9.5%of the beer sold there in 2010 were alcohol-free, while in Germany,the largest European beer market, the share of beers with low alco-hol content varies between 4% and 5%. Probably the most signifi-cant reasons for the annual increase in alcohol-free beer (AFB)sales in the EU countries are the legislative interventions restrict-ing the alcohol consumption and the increasing awareness of con-sumers about the benefits of moderate beer drinking (Silva et al.,2010; Informe, 2010).

In most of the EU countries beers with low alcohol content aredivided into alcohol free beers (AFBs) containing 60.5% alcohol byvolume (ABV), and to low-alcohol beers (LABs) with no more than1.2% ABV. In the United States alcohol-free beer means that there isno alcohol present, while the upper limit of 0.5% ABV correspondsto so-called non-alcoholic beer or ‘‘near-beer’’ (Montanari et al.,2009). In countries that enforce religious prohibition, the alcoholcontent in beverages must not exceed 0.05% by volume.

The terminology of this article in the following chapters will begoverned by the aforementioned European legislation. However,while the methods to produce both LAB and AFB are from practicalpoint of view identical, the AFBs market share prevails over theLABs one. Hence, in this article the beverages with low alcohol con-tent produced from malt will be generally termed alcohol-freebeers (AFBs).

Fig. 1. The scheme of most common alcohol-free beer production methods.

2. Beer and health

Alcohol abuse has been on the public agenda for many yearssince it carries risks of violent crime, traffic accidents, public disor-der, and health damage. Ethanol is one of the most commonly usedrecreational drugs worldwide and it is often ingested as a compo-nent of beer. When beer is consumed, ethanol is absorbed from thegastrointestinal tract by diffusion and is swiftly distributed in theblood before entering tissues. Ethanol is metabolized to acetalde-hyde mainly in the stomach and liver. Acetaldehyde is highly toxic

and binds cellular constituents generating harmful acetaldehydeadducts (Rajendram and Preedy, 2009).

Simultaneously, there are strong evidences that moderatealcohol consumption has not only a better long-term health out-come than excessive alcohol consumption, but even better thanabstaining. Moderate beer drinking has shown to be, at least,as effective as wine drinking at reducing risks of coronary dis-eases, heart attack, diabetes, and overall mortality (Mukamaland Rimm, 2008; Ferreira and Willoughby, 2008). Besides alco-hol, which is probably the most important component of beerthat counters atherosclerosis (Li and Mukamal, 2004; Tolstrupand Groenbaek, 2007), these positive effects may be attributedto a whole range of other properties and valuable cereal andhop-related substances found in beer such as no fat or choles-terol content, low energy and free sugar content, high antioxi-dant (e.g. polyphenols, flavonoids), magnesium and solublefiber content. In addition, beer provides essential vitamins andminerals and is thus contributing to a healthy balanced diet(Bamforth, 2002). The alcohol-free beers also claim beneficialeffects of healthy beer components with a simultaneous effectof the lower energy intake and complete absence of negativeimpacts of alcohol consumption.

3. Methods of the alcohol-free beer production

The strategies to produce AFBs can be divided into two maingroups (physical and biological processes), which can be furtherbroken down as shown here (Fig. 1). The so called physical meth-ods are based on gentle removal of alcohol from regular beer andrequire considerable investments into the special equipment foralcohol removal. After the removal process has been optimized,the sensorial quality of produced AFBs is usually good. Their fur-ther advantage is that they can remove ethanol from beers to van-ishingly low levels. The most widespread biological approachesare based on limited ethanol formation during the beer fermenta-tion. They are usually performed in traditional brewery equip-ment and hence do not require additional investments, but theirproducts are often characterized by worty off-flavors. Improve-ments taking advantage of special yeast increase the costs bythe purchase, selection, or construction of the production organ-isms as well as by the need their propagation have to be sepa-rated. However, suitable tailor-made or selected microorganismscan contribute significantly to the product sensorial qualityimprovement. There are also AFB production processes (continu-ous fermentation with immobilized yeast) based on limited alco-hol formation, which require special equipment and material(continuously operating bioreactor, carrier for cell immobiliza-tion). In this case, the higher investment costs have to be justifiedby the higher productivity of continuous processes. In general, theethanol formation, which is intrinsic to the biological methods,makes impossible the production of AFBs with alcohol contentclose to zero.

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4. Production of alcohol-free beer by ethanol removal methods

The technologies applied for complete or partial ethanolremoval from regular beers can be classified into two groups basedon the principle of the separation process such as thermal andmembrane processes (Fig. 1). Besides the industrially appliedmethods of beer dealcoholization (vacuum rectification and evap-oration, dialysis, and reverse osmosis) there have been severalother methods studied under laboratory conditions such as mem-brane extraction (Matson, 1987; Etuk and Murray, 1990), super-critical CO2 extraction (Mori, 2004), pervaporation (MagalhaesMendes et al., 2008), adsorption on hydrophobic zeolites (Anglerot,1994), and freeze concentration (Von Hodenberg, 1991).

4.1. Thermal processes

The early attempts to dealcoholize beer by evaporation or distil-lation under atmospheric pressure, which revealed significant tem-perature damage to the beer taste, were soon replaced by vacuumdistillation (Zufall and Wackerbauer, 2000a). If the pressure isreduced, alcohol can be drawn off at much lower temperature.All thermal processes to produce AFBs are therefore performed atan absolute pressure of 4–20 kPa, whereby evaporation tempera-tures of 30–60 �C are achieved. Even so, a great loss of beer flavorand liveliness can occur during thermal processes. The deteriora-tion of beer quality by thermal dealcoholization depends mainlyon the evaporation temperature and the period of exposure, whichdepends on the thermal separator construction. AFB production atindustrial scale has been implemented using vacuum distillation(rectification) plants or vacuum evaporators (single or multi-stage)of two main construction variants i.e. centrifugal and falling filmevaporators.

Generally, advantages of thermal processes are: the potential toremove alcohol from beer completely, the possibility to commer-cialize the separated alcohol, the continuous and automatic opera-tion with a short start-up period, and the flexibility in terms ofvolumetric performance and the input beer composition. Con-versely, the purchase of these systems requires significant invest-ment as well as there is considerable running costs (energyconsumption) and some risks of thermal damage or loss of volatilesfrom beer. At the end of all thermal processes the concentratedalcohol-free beer has to be diluted with oxygen-free water and car-bonized (Zufall and Wackerbauer, 2000a).

Table 1Selected properties of original input beer and alcohol-free beers obtained by vacuum rect

Sample Original 1/Original 2 Dealcoholized 1a/Dealc

Original gravity (wt.%) 11.59/na 5.16/naEthanol (% ABV) 4.99/5.3 0.48/0.03Color (EBC) 8.4/na 9.5/napH 4.75/na 4.71/naBitterness (EBC) 24.9/na 25.5/na1-Propanol (mg/l) 6.7/23.4 0.8/nd2-Methylpropanol (mg/l) 11.2/24.3 nd/nd2-Methyl-1-butanol (mg/l) 15.2/22.4 nd/0.13-Methyl-1-butanol (mg/l) 52.8/64.1 nd/0.32-Phenylethanol (mg/l) 18.6/35.9 22.4/35.1Furfuryl alcohol (mg/l) 0.07/3.2 nd/2.8Ethyl acetate (mg/l) 16.9/23.1 nd/ndIsoamyl acetate (mg/l) 1.9/2.8 nd/nd2-Phenyl ethyl acetate (mg/l) 0.4/na 0.03/naTotal HAA (mg/l) 104.7/173.3 23.2/38.3Total EAA (mg/l) 19.6/25.9 0.04/nd

na, Data not available; nd, not detectable.a Dealcoholization of original 1 to 0.48% ABV (Narziss et al., 1993).b Dealcoholization of original 2 to 0.03% ABV (Zürcher et al., 2005).c Dealcoholization of original 1 to 0.1% ABV (Narziss et al., 1993).

4.1.1. Vacuum rectification plantThis process arrangement consists of the main steps as follows:

preheating of the filtered alcoholic beer in a plate heat exchanger,degassing of beer (loss of liveliness) and the simultaneous libera-tion of volatile compounds in a vacuum degasser, dealcoholizationin a vacuum column (usually a packed-bed rectifying column),recovering the aroma components from CO2 by spraying with deal-coholized beer or water, and redirecting them into dealcoholizedbeer (Regan, 1990; Narziss et al., 1993; API Schmidt-Bretten,2004). In the rectification column the fluid flows down at a tem-perature between 42 and 46 �C. In counter flow the product con-tacts rising vapors, generated from alcohol-free beer in anevaporator, which brings about the selective separation of alcoholfrom the product. The dealcoholized product (less than 0.05% ABVis achievable) is then cooled. The production capacity of these sys-tems is usually given in the range 4–200 hl of alcohol-free beer perhour. The system further contains an aroma recovery unit, wherethe aroma components are recovered and their redirection intothe beer can be made under pressure (Koerner, 1996). The alco-hol-rich vapors can be concentrated to 75% ABV in a rectificationsection and marketed immediately (Narziss et al., 1993; APISchmidt-Bretten, 2004). Without concentrating, the alcoholicby-product produced has about 8–9% ABV. This by-product canbe sold for acetification to produce vinegar (Regan, 1990).

An alcohol removal system (Sigmatec) from beer by counter-current distillation in combination with rectification was used fordealcoholization of both top and bottom fermented beers. In thecase of top-fermented wheat beer the original total higher alcohol(182 mg/l) and ester (51.5 mg/l) contents decreased by 75.2/89.3%,77.3/91.9%, and 80.2/98%, respectively, along with the ethanolremoval from the original 5.57 vol.% to 0.46, 0.22, and 0.12 vol.%,respectively. This means, that in dealcoholized wheat beer with0.46 vol.%, there was still a considerable amount of volatiles(ca. 45 mg/l of higher alcohols and 5.5 mg/l of esters). Conversely,the bottom fermented beer (4.99% ABV) was depleted of volatilesto a higher degree. Approximately 78% of total higher alcohols(104.7 mg/l), and almost 100% of total esters (19.6 mg/l) wereremoved already at 0.48% ABV (Table 1). In addition, the total high-er alcohols in dealcoholized top- and bottom-fermented beerswere represented almost exclusively by 2-phenylethanol (73 and97%), a compound with floral odor (Narziss et al., 1993). Thisunbalanced content of volatiles shows the importance of addingthem back, particularly into dealcoholized bottom fermented beer,otherwise the sensory properties of AFBs are significantly changed

ification and post-treated with aroma redirection and blending with 6% krausen.

oholized 2b Dealcoholized 1a + aroma Dealcoholized 1c + 6% Krausen

4.98 5.220.51 0.548.7 9.54.78 4.6927.5 29.31.0 1.30.7 1.74.3 2.83.0 1020.0 23.00.01 0.023.3 5.20.5 0.5– 0.3429.0 38.823.83 6.13

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as compared to original beer (Zürcher et al., 2005). The recovery ofaromatic volatiles from the CO2 liberated during degassing andtheir addition into dealcoholized beer, returned about 6% and20% of the originally present higher alcohols and esters (Table 1),respectively (Narziss et al., 1993). The thermal stress exhibitedby the dealcoholization system was considered negligible. Therewas no increase in hydroxymethylfurfural or furfural observed inalcohol-free beers (Kern, 1994). The content of medium-chain fattyacids decreased during the dealcoholization by 20–40% as com-pared to original beer, with the exception of dealcoholized unfil-tered beer, where the thermal lysis of cells is blamed for theincreased decanoic acid content (Narziss et al., 1993).

4.1.2. Thin film evaporatorsIn order to shorten the ethanol removal, regular beer flows

through these vacuum devices as a thin film with large surface areain an extremely short residence time, which results in an improvedproduct quality. Examples of thin film evaporators, which producea thin liquid film in a mechanical (rotational movement) way, arethe Centritherm and spinning cone column (SCC) systems (sup-plied by Flavourtech, www.ft-tech.net). On the contrary, the fallingfilm evaporator does not contain moving parts and the liquid filmis created by gravity-induced downward movement of beer on theinner surface of heating tubes.

The Centritherm system structure resembles that of a plate cen-trifuge (Fig. 2). The centrifugal evaporator operates under vacuumat low temperatures (35–60 �C) and uses steam as the heatingmedium. The regular beer to be dealcoholized enters the evapora-tor through a feed tube and injection nozzles (one for each cone),which distribute it to the underside of the hollow rotating cone.Centrifugal force instantaneously spreads the beer over the entireheating surface in an extremely thin layer (approximately

Fig. 2. Rotating thin film evaporator (Centritherm system) with one rotating cone:(1) feed tube and injection nozzle, (2) product tube, (3) hollow cone, (4) vapors, (5)exhaust pipe, (6) steam, (7) condensate.

0.1 mm). The beer passes across the heating surface in less thanone second. The concentrated and dealcoholized beer collects atthe outer edge of the cones and then exits the evaporator througha stationary product tube. The vapors removed from the beer risethrough the center of the cone and enter an exhaust pipe thattransfers them to an external condenser. The Centritherm evapora-tors are designed with 1–12 hollow cones, which correspond toproduction capacities of AFB from 0.5 to 100 hl/h, respectively.Steam is supplied to the evaporator through a hollow spindle tothe steam chamber of each cone. As the steam condenses, the con-densate is immediately projected to the upper wall of the hollowsteam chamber. The condensate exits the steam chamber througha channel and is removed from the evaporator (Fig. 2). The Centri-therm is claimed to have minimal thermal impact and easy opera-tion, while the oxygen penetration through the seals of movingparts is considered as a potential risk (Zufall and Wackerbauer,2000a).

The spinning cone column (SCC) is a counter-current liquid–gascontacting device using gentle mechanical forces to enhance inter-phase contact (Fig. 3). This allows rapid and efficient separation ofvolatile compounds such as ethanol from a thin liquid (beer) film.The SCC contains two series of inverted cones. A series of fixed

Fig. 3. Vapor and liquid flow through the spinning cone column (SCC): (1) rotatingshaft, (2) fixed cone, (3) rotating cone, (4) fin, (5) downward liquid (beer) flow, (6)upward vapor flow, (7) external wall of the SCC.

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Fig. 4. Falling film evaporator: (1) feed beer, (2) dealcoholized beer, (3) heatingsteam inlet, (4) condensate, (5) head, (6) vapor separator, (7) vapor flow, (8) heatingsteam, (9) steam condensate film, (10) heating tube wall, (11) beer film.

T. Brányik et al. / Journal of Food Engineering 108 (2012) 493–506 497

cones is attached to the inside wall of the column. Another series ofcones is attached to the rotating shaft, in parallel to the fixed cones.The fixed and rotating cones alternate vertically. The full strengthbeer is fed into the column top. Pulled by gravity, it flows downthe upper surface of the first fixed cone and drops onto the firstrotating cone (300–500 rpm), which spins the beer into a thin, tur-bulent film. The centrifugal force induces an upward liquid flow tothe rim of the spinning cone where the beer is dropping onto thenext stationary cone below. In this manner, the beer flows to thebottom of the column. The stripping medium, steam producedfrom deaerated water, is fed into the column bottom and flowsupward, passing over the surface of the liquid thin film, collectingethanol and other volatile compounds as it rises. Fins on the under-side of the rotating cones induce a high degree of turbulence and apumping effect to the rising vapor stream. The turbulent liquid andvapor flow leads to highly efficient mass transfer of volatiles fromliquid to the vapor stream (Fig. 2). The vapor flows out of the col-umn top and passes through a condenser system, which capturesthe volatiles in a concentrated liquid form. The dealcoholized beeris pumped out of the column bottom (Craig, 1986). A low pressuredrop in the SCC allows a low operating temperature of 40–55 �Cunder vacuum. The residence time of beer in the SCC is approxi-mately 20 s, which is enough to reduce the original alcohol level(5% ABV) to 0.01–0.03% ABV in a single pass. Residual CO2 in thefeed beer shows no negative impact (overfoaming) and no oxygenpickup was found in the beer that passed the SCC (Moreira da Silvaand De Wit, 2008). Several different production strategies using theSCC system have been tested. The best flavor recovery wasachieved by a two stage process involving the flavor removal fol-lowed by dealcoholization. In the first passage through the SCC(highest temperature in the column 53.7 �C) the feed beer(4.8% ABV) looses practically all esters and 57% of total higher alco-hols, while the alcohol content of the beer decreases by 1% ABV.This beer with a reduced alcohol content (3.8% ABV) is then furtherdealcoholized to 0.17% ABV during the second passage through theSCC (highest temperature in the column 57.2 �C), which simulta-neously leads to the loss of remaining volatiles. The re-combina-tion of the dealcoholized beer stream with the volatile-richcondensate (75% ABV) captured after the first flavor-removal stageresulted in an AFB (0.5% ABV) with 25% and 30% of total esters andhigher alcohols retained, respectively, relative to 4.8% ABV beer(Badcock et al., 1994).

In contrast to centrifugal evaporators, the falling film evaporatorcontains no moving parts, which results in great benefits. The sys-tem is not only cheaper in construction, but also easier to cleanand there is practically no danger of oxygen transfer across the var-ious seals of moving parts. Overall, the acquisition and operationcosts of the falling film evaporation are considered the lowest ofall thermal dealcoholization systems (Stein, 1993; Zufall and Wack-erbauer, 2000a). Further energy savings can be achieved using amulti-stage design of falling film evaporators since the alcohol con-taining vapors from the first evaporator can be used as heatingsteam to the second one, while the vapor from the second onecan heat the third evaporator. Certain disadvantage of this multi-stage arrangement is the need to operate the first stage at relativelyhigh temperature (60 �C), so that the vapor temperature in the finalstage is sufficiently high for the alcohol removal (35–40 �C) (Hoch-berg, 1986). In falling film evaporators the original beer is pre-heated to the evaporation temperature (30–60 �C at 3.5–20 kPa)and enters the evaporator column through a distributor device,which ensures the formation of an even liquid film on inner wallsof the tubes. The beer flows downward at boiling temperatureand is partially evaporated (Fig. 4). The downward movement isinduced both by gravity and high speed co-current vapor flow(20–80 m/s). Thus the beer stays in the evaporator for only a fewseconds. Process steam (saturated steam) is used to heat the

evaporator tubes. The alcohol-rich vapor is separated from the deal-coholized beer concentrate in a vapor separator connected to thefalling film evaporator outlet and is finally condensed in a con-denser. The heaters, falling film evaporator, separator, and con-denser are connected to a common vacuum pump. As the beerpassing through the falling film evaporator was not only dealcoho-lized, but also concentrated, it must be re-diluted with degassedwater to the original extract content as well as it is necessary to car-bonize it. The main process parameters controlling the dealcoholi-zation degree in the falling film evaporator are the heating steamsupply and the evaporation temperature adjustable by the vacuumpump. However, it turned out that independently on the testedevaporation degree (30–55 kg of vapors from 100 kg inlet beer)there was a significant loss of total higher alcohols (91–97%), whileesters were practically completely removed. In terms of the alcoholcontent there was an evaporation degree of 40/100 kg necessary toachieve 0.5% ABV. In order to re-direct some volatiles into the deal-coholized beer, while not exceeding the alcohol limit for AFBs eitherthe condensed vapor flow or preferably the volatiles separated fromvapor condensate by rectification can be used. The overall materialbalance of input (original beer) and output streams (dealcoholizedbeer concentrate and condensed vapor) from the falling film evap-orator unit showed a loss of esters (�36%) and higher alcohols(�8%), and an overall accumulation of acetaldehyde (+17%),explained by thermal decomposition of acetaldehyde-bisulphitecomplex (Zufall and Wackerbauer, 2000a).

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4.2. Membrane processes

These alcohol removal methods are based on the semiperme-able character of membranes, which separate only small moleculeslike ethanol and water from the beer to the permeate liquid. Twotypes of membrane processes used for beer dealcoholization canbe distinguished on the industrial scale: dialysis and reverse osmo-sis. They differ in applied pressures and temperatures, membranematerials and their structures. It is known that all of the membraneprocesses have less thermal impact on beer, they can be operatedautomatically and in flexible manner, but at the same time theyrequire significant capital and running costs. The economic feasi-bility of membrane processes for the production of beverages withan alcohol percentage lower than 0.45 vol.% was by some authorschallenged (Pilipovik and Riverol, 2005), while others stated thatthe energy requirement of a membrane system for alcohol purifica-tion (reverse osmosis) would be significantly lower than that of aconventional distillation system (Mehta, 1982). Membrane pro-cesses were suggested also as a part of a system for the continuousproduction of delacoholized beverages (Gresch, 1991).

4.2.1. DialysisThe driving force of the mass transfer across the semipermeable

dialysis membrane is the concentration gradient of compoundsbetween beer and dialysate. The semipermeable membrane actsas a molecular sieve permeable only to certain molecules, depend-ing on the pore size and surface properties of the membrane. Whendealcoholization by dialysis is performed into water, all beer ingre-dients tend to move from the area of the high concentration (beer)to the area of the low concentration (water), while some water willdiffuse from dialysate into beer. The prevailing mechanism of masstransfer in dialysis is molecular diffusion. When the transmem-brane pressure difference is applied (usually 10–60 kPa), in orderto suppress water diffusion into beer, the process is often calleddiafiltration and both diffusive and convective mass transfers takeplace (Leskosek and Mitrovic, 1994; Petkovska et al., 1997).

The process of dialysis is usually performed at 1–6 �C, eliminat-ing the thermal load of the product. Dialysis membranes are com-posed of either cellulose derivatives or various synthetic materials

Fig. 5. Flow diagram of beer dealcoholization by dialysis: (1A) principle of hollow fibeexchanger, (3) stripper column, (4) original beer, (5) dealcoholized beer, (6) dialysate, (7(11) stripping steam.

(e.g. polysulphone, polyethersulphone) and are generally arrangedin bundles of hollow fibres, known as modules. In hollow fibers thebeer passes along a dialysis membrane, while simultaneously analcohol-free dialysate liquid flows counter-currently along theother side of the membrane (Fig. 5). The principle of the counter-current flow guarantees a high concentration gradient betweenthe dialysate and the beer in terms of the alcohol content so thatan optimal diffusion can be obtained. To operate a dialysis moduleit is necessary to apply some pressure on both the beer side and thedialysate one, otherwise the diffusion can be disrupted by releaseof carbon dioxide. The applied pressure must be at least equal tothe saturation pressure of CO2 in beer at a given temperature. In or-der to further minimize the loss of CO2 it is recommended to add asmall amount of carbon dioxide into the dialysis water. This willalso eliminate the risk of oxygen transfer from dialysate to beer.Attention has to be paid also to the content of inorganic salts(sodium, calcium, nitrates), which can get concentrated in dialy-sate during rectification and then pass into beer (Moonen andNiefind, 1982; Attenborough, 1988; Donhauser et al., 1991).

Despite the optimization of membranes and process parametersa selective removal of ethanol cannot be achieved. Other compo-nents of beer, such as higher alcohols and esters, are thereforealmost completely removed from the beer by dialysis (Table 2).Losses of low-molecular-weight volatile compounds can be pre-vented by adding them into dialysate reducing thus their diffusionfrom beer. The extent and rate of dealcoholization, and also loss ofvolatiles, can be regulated mainly by the ratio of flow rates of dial-ysate and beer, which can be varied in a wide range from 0.4:1 to6.5:1. By increasing the ratio of dialysate to beer flow, the removalof alcohol and volatiles from beer becomes more pronounced.However, this ratio influences not only the rate of alcohol removalfrom beer but also the energy costs for the rectification of the dial-ysate (Donhauser et al., 1991; Leskosek et al., 1995; Zufall andWackerbauer, 2000b).

4.2.2. Reverse osmosisIn the reverse osmosis (RO) process, beer flows tangentially to the

membrane surface and ethanol (and water) permeates the mem-brane selectively when the transmembrane pressure substantially

r dialysis, (1B) schematic representation of capillary membrane module, (2) heat) make-up brewing water, (8) glycol, (9) dialysate pump, (10) alcoholic condensate,

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Table 2Selected properties of original input beer and alcohol-free beers obtained by dialysis and reverse osmosis.

Sample Original Dealcoholizeda Original Dealcoholizedb

Original gravity (wt.%) 11.16 4.53 10.83 2.48Ethanol (% ABV) 4.80 0.47 4.92 0.40Color (EBC) 7.25 7.5 – –pH 4.55 4.68 – –Bitterness (EBC) 30.7 29.7 24.6 12.31-Propanol (mg/l) 9.4 0.5 12.0 2.02-Methylpropanol (mg/l) 7.0 0.3 17.0 5.12-Methyl-1-butanol (mg/l) 9.9 0.4 4.3 2.83-Methyl-1-butanol (mg/l) 43.6 1.5 3.0 10Isoamyl alcohol (mg/l) – – 79.0 17.0Phenyl ethyl alcohol (mg/l) – – 40.0 3.7Total HA (mg/l) 69.9 2.7 148.0 27.9Ethyl acetate (mg/l) 12.1 <0.1 15.0 1.8Isoamyl acetate (mg/l) 2.2 <0.1 1.5 0.162-Phenyl ethyl acetate (mg/l) <0.1 <0.1 0.63 0.04Total ES (mg/l) 14.3 <0.1 17.6 2.0Iso-valeric acid (mg/l) 1.22 0.49 0.76 0.18Caproic acid (mg/l) 1.88 1.02 2.0 0.22Caprylic acid (mg/l) 4.61 2.55 3.6 0.35Capric acid (mg/l) 0.35 0.21 0.95 0.11Total FA (mg/l) 8.82 4.27 7.9 0.9

a Dealcoholization by dialysis (Zufall and Wackerbauer, 2000b).b Dealcoholization by reverse osmosis (Kavanagh et al., 1991).

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exceeds the osmotic pressure of beer. It is expected that other largemolecules, such as aroma and flavor compounds, will mostly remainat the retentate side of the membrane (Catarino et al., 2006). Reverseosmosis (RO) is usually carried out at transmembrane pressuresranging from 2 to 8 MPa generated by pressure pumps (e.g. pistonpump) and at temperatures below 15 �C achievable with, forinstance, a plate heat exchanger (Von Hodenberg, 1991; Catarinoet al., 2007). The membranes used for the alcohol removal from beerby RO are usually of asymmetric structure, with the active layermade of cellulose acetate, polyamide, or polyimide on polyester,polysulphone, or fiberglass support structures. An ideal membranefeatures the following characteristics:

� High permeability to ethanol and water.� Low permeability for other beer components (flavor, aroma and

bitter substances).� Temperature resistant.� Resistant to cleaning and disinfecting agents (pH 2–11).� Resistant to all kinds of fouling (inorganic, organic, colloidal,

and microbiological).� Chemically and mechanically resistant.� Shapable to high membrane area-to-volume ratio (packing

density).� Is inexpensive.

The membranes are usually placed in modules of different geo-metric arrangements (planar, tubular, spiral-wound) (Light et al.,1986).

In practice the RO is carried out in a so called diafiltration mode.The first phase is the concentration of the original beer by remov-ing permeates and not replacing it with demineralized water. Thisleads to an increase of the alcohol concentration and so does theflux of solute across the membrane increases, too. Subsequentlyduring the diafiltration phase the permeate removed from beer isquantitatively replaced by demineralized water. This continuesuntil a desired alcohol concentration is reached in beer. After thetarget alcohol content has been achieved, the retentate is madeup with demineralized water to the starting volume of beer andthe alcohol content is further lowered by this operation. Thediafiltration water applied in RO has to be sterile, completelydemineralized (conductivity < 50 lS) and deaerated (oxygen

content < 0.1 ppm). Carbonation of the product is necessary afterRO (Von Hodenberg, 1991).

Very few data are available on the composition of AFBs pro-duced by RO. However, these report on significant losses of vola-tiles (70–80% of higher alcohols, 80–90% of esters) during theprocess (Table 2), which can be ascribed to imperfect selectivityof membranes (Kavanagh et al., 1991; Stein, 1993). Recently, sev-eral cellulose acetate and polyamide membranes have been testedin laboratory at different operation conditions (2–4 MPa trans-membrane pressure, 5–20 �C, and different feed flow rates). Itwas found that higher transmembrane pressures resulted in higherpermeate flux, higher rejection of ethanol and higher alcohols, butlower rejection of esters. Lower temperatures resulted in lowerpermeate flux but in higher rejection of aroma compounds(Catarino et al., 2007). Unfortunately, this study does not indicatethe composition of beers dealcoholized by RO.

5. Production of alcohol-free beer by methods of restrictedethanol formation

The methods of the alcohol-free beer (AFB) production based onlimited alcohol formation can be divided according to the produc-tion equipment they require and further subdivided according toalterations in technology or use of special yeast (Fig. 1). The mostexploited technologies are those requiring the equipment of a tra-ditional brewery plant, while the continuous limited fermentationis a promising but marginal technology. The respective proceduresapplied on the industrial scale are often combinations of strategies,which belong to technologies using traditional brewery facilities.

5.1. Changed mashing process

Mashing consists of complex physical, chemical, and biochemical(enzymatic) processes, the main purpose of which is to completelydegrade starch to fermentable sugars and soluble dextrins. The spec-trum of sugars formed depends on the actual enzyme activities pres-ent. b-Amylase (temperature optimum of 62–65 �C) produces thefermentable sugar of maltose, whereas a-amylase (temperatureoptimum of 72–75 �C) generates first non-fermentable sugars (dext-rins) and at prolonged action also fermentable sugars (Kunze, 1996).

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The final content of fermentable sugars in wort then determines thealcohol level in beer. Therefore, by changing the mashing process, itis possible to modulate the profile of wort sugars in a way that theirfermentability is limited and results in low alcohol content. A lowwort sugar content can be achieved by different strategies asfollows:

� Inactivation of saccharifying b-amylase by high temperaturemashing (75–80 �C). Under these conditions b-amylase is rap-idly inactivated but sufficient a-amylase remains to digestand liquefy the starch. This procedure results in ca. 85% extrac-tion of malt and a wort fermentable to 25%. The final ethanolcontent is influenced also by the original wort gravity, attenua-tion achieved during fermentation, and final dilution. Accordingto the literature the flavor of these beers is very good, however,some problems with worty flavor have been reported. Concernsregarding colloidal stability of the product are also relevant(Muller, 1990, 1991, 2000):� Cold water malt extraction. It is based on extracting the maxi-

mum malt flavor compounds without increasing the wort grav-ity. The malt is extracted with water at temperaturesinsufficient (<60 �C) for starch gelatinization and subsequentenzymatic hydrolysis. The obtained wort contains some fer-mentable sugars, which result from barley modification duringmalting. In practice, difficulties with lautering could beexpected while the remaining malt could be used in normalmashing. No information is available on the current use of thismethod (Muller, 1990).� Re-mashing of spent grains to produce a second extract with very

little fermentable sugar. Two modifications of the originalmethod are known: (i) extrusion cooking of the spent grains priorto the second extraction and (ii) acidic hydrolysis of spent grain toyield a secondary wort with a significant content of non-ferment-able pentoses. The main advantage of these methods is that twobeers can be produced from one dose of grain. Although it is notapparent that anyone is using this method, both difficulties withflavor and color formation were hypothesized and an increasedinvestment costs, concerning the hydrolytic apparatus, wouldbe inevitable (Muller, 1990; Zurcher and Gruss, 1991).� Barley varieties with wide variations of b-amylase thermostabil-

ity as well as b-amylase deficient varieties have been reported(Kihara et al., 1998; Kihara et al., 1999). Although, it can behypothesized that both thermolability and/or lack of b-amylasein special barley varieties could be advantageous to achieve lowwort fermentability, no information on any research or industrialimplementation of these barleys have been found so far.

Used on their own, methods relying solely on the modifiedmashing are seldom successful for the production of AFBs and they

Table 3The influence of yeast type and fermentation temperature on the composition of alcohol-

Fermentation yeast Bottom Bottom

Temperature (�C) 0 4Original gravity (wt.%) 11.4 7.5Ethanol (wt.%) 0.27 0.37Real extract (wt.%) 10.64 6.79Attenuation (wt.%) 8 12Fermentation time (h) 48 24pH 5.01 4.87Bitterness (EBC) 25.4 17.5DMS (lg/l) 30 22Total diacetyl (mg/l) 0.06 0.09Total HAA (mg/l) 2.2 6.8Total EAA (mg/l) 0.55 0.69Reduction of aldehydes (%) 85.8 77.6

* After 7 h at the initial temperature the wort was cooled to 0 �C until 24 h were comp

have to be combined with further measures such as vigorous wortboiling (lowering the level of aldehydes), wort acidification, limitedfermentation, color and bitterness adjustment, etc.

5.2. Arrested or limited fermentation process

In general, the major disadvantage of both stopped and limitedfermentation approach is that it is hardly feasible to achieve lowalcohol levels with adequate conversion of wort to beer. Therefore,the objective of these methods is keeping the ethanol content lowby removal of yeast before excessive attenuation (stopped fermen-tation) or creating conditions for restrained yeast metabolism (lim-ited fermentation) and simultaneously reducing the worty flavorimpression or limit it from the beginning (Muller, 1990). Theseproduction methods operate with traditional brewery equipmentand unit operations, but they require accurate and swift analyticalcontrol (Perpète and Collin, 1999a). These approaches representthe most usual way to produce alcohol-free or low-alcohol beers.When these techniques were carried out with worts of originalgravity from 9 to 13 wt.% the smell and taste of AFBs was charac-terized by a strong worty flavor impression due to the non-reducedwort aldehydes. It has been verified that for stopped or limited fer-mentation processes an original gravity from 4.0 to 7.5 wt.% isdesirable. However, brewing at high gravity (20 wt.%) increasesthe formation of higher alcohols and esters. This phenomenoncan be exploited to strengthen the flavor of reduced alcohol beersobtained after dilution of a higher gravity beer. Further adjust-ments of volatiles can be achieved by higher fermentation temper-ature (greater effects on lager yeasts) or reduced oxygen content ofwort, which increased dramatically the ester formation by aleyeasts (Muller et al., 1991).

The fermentation activity can be arrested (stopped or checked)quickly by temperature inactivation (rapid cooling to 0 �C, pasteur-ization) and/or by removal of yeast from fermenting wort (filtra-tion, centrifugation). The fermentation initial phase can becarried out at a relatively wide range of temperatures without asignificant impact on the formation of volatiles and the reductionof aldehydes (Table 3). However, at higher temperatures the fer-mentation has to be arrested, either by yeast separation or cooling,rather shortly after the wort was pitched, which requires a promptanalytical control and intervention (Attenborough, 1988; Narzisset al., 1992). The increasing fermentation temperature deepenedthe attenuation and simultaneously enhanced the formation of vol-atiles and diacetyl by bottom fermenting yeast. A comparison ofyeast types showed that the top fermenting yeast achieved a sig-nificantly higher aliphatic alcohol formation at lower attenuation,but the top fermented AFBs had also rather high diacetyl content(Table 3). After interrupting the fermentation at an alcohol contentless than 0.5 vol.%, the AFB is usually matured for at least 10 days

free beers.

Bottom Bottom Top Top

8 12 8 127.5 7.5 7.4 7.40.37 0.42 0.32 0.276.79 6.52 6.87 6.9412 16 9 87/24* 7/24* 24 7/24*

4.92 4.89 4.87 4.8917.7 17.4 17.7 17.830 32 35 450.08 0.14 0.51 0.356.7 8.6 15.8 14.20.60 0.84 0.90 0.9080.2 83.0 88.2 77.5

leted, source: Narziss et al. (1992).

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Table 4The influence of yeast type and wort acidification on the composition of alcohol-freebeers.

Fermentation yeast Bottom S. ludwigii S. ludwigii + acidification

Temperature (�C) 0 20 20Original gravity (wt.%) 11.5 11.5 11.5Ethanol (wt.%) 0.3 0.68* 0.68*

Real extract (wt.%) 10.7 10.34 10.34Attenuation (wt.%) 9 13 13Fermentation time (h) 48 120 120pH 5.15 4.98 4.18Bitter substances (EBC) 28.0 27.2 22.2Total diacetyl (mg/l) 0.04 0.14 0.13Total HAA (mg/l) 3.0 31.8 30.3Total EAA (mg/l) 0.79 1.88 2.31Reduction of aldehydes (%) 81.0 56.8 32.6

* Ethanol content higher than the legal limit for AFB. Source: Narziss et al. (1992)

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at 0 to 1 �C to avoid an overpowering sulfur flavor. Then the AFB isfiltered, carbonated, stabilized, and sterilized.

The most practical tool to suppress yeast metabolism is lowtemperature. The so-called ‘‘cold contact process’’ (CCP) is takingadvantage of the fact that under these conditions the ethanol pro-duction is slow, but other biochemical processes, such as the for-mation of higher alcohols and esters and the reduction ofcarbonyls, may exhibit moderate activities. In an example of theCCP the alcohol-free beer is produced from wort (6 wt.%) cooledto 0–1 �C, acidified with lactic acid to pH 4, pitched with the yeastcell concentration of 30 � 106 cells/ml and kept at �0.5 �C for 48 h(Schur, 1988). However, when using a high yeast cell concentration(>108 cells/ml) it has to be taken into account that the pitchingyeast slurry has a significant ethanol content (�6 vol.%).

In comparison with other methods of AFB production, the CCPwas characterized by one of the highest volatile production andlowest aldehyde reduction capacity (Perpète and Collin, 1999a).Several carbonyl compounds present in wort are known to contrib-ute to the worty off-flavor of AFBs produced by the CCP. Amongthem the branched aldehydes (3-methylbutanal, 2-methylbutanal,and 3-methylpropionaldehyde) with very low odor threshold val-ues are less readily reduced both enzymatically and chemically.The total removal of branched Strecker aldehydes is under CCPconditions limited to approximately 65% of their initial concentra-tion, remaining thus enough of them in AFBs to impair their senso-rial profile. Therefore, there were two strategies suggested toimprove the CCP, one being the use of genetically modified yeastat higher temperature (high temperature enhance chemical bind-ing of aldehydes) or decreasing the wort polyphenol level (alde-hydes bound to polyphenols resist enzymatic reduction) by usingthe polyvinylpolypyrrolidone (PVPP) just after wort cooling (Per-pète and Collin, 1999b, 2000a).

When using arrested or limited fermentation it is necessary toconsider, besides fermentation temperature, wort gravity, andyeast type, also additional interventions into the production pro-cess in order to improve the products flavor characteristics:

� The addition of dark (20%) or pale caramel malt (15%), com-pared to the brew with 100% pale malt, contributed positivelyto taste characteristics by masking the worty flavor impressionwith more beer flavor substances. In particular, pale caramelmalt contributed the highest amount of substances resultingfrom Maillard reactions (e.g. furfural, 2-acetylpyrole) (Narzisset al., 1992).� Wort dilution after boiling (from 11.5 to 7.5 wt.% original grav-

ity), instead of dilution before wort boiling, resulted in lowerbitterness, increased ester and higher alcohol levels and theAFBs were characterized by purer, less worty smell and taste(Narziss et al., 1992).� An attenuation of about 10% will lead to pH of only 5.0, which

results in low liveliness emphasizing the worty flavor impres-sion. Therefore the effect of wort acidification with acid malt(5%) or lactic acid (10 min before the end of boiling to a pH of4.3) was tested. The results showed that the acidification hada very favorable effect in suppressing the worty character ofAFBs (Narziss et al., 1992).� The unpleasant bitter aftertaste of AFBs, probably due to oxi-

dized malt substances, can be eliminated by adding ascorbicacid to the wort (approximately 20 mg/l to 7 wt.% wort) as longas it remains hot (Schur and Sauer, 1990).� Wort can be ’’hot‘‘ (Lommi et al., 1990) or ‘‘cold stripped’’ (Monta-

nari et al., 2009), with sparging CO2 or N2 into the liquid, to washout undesirable volatiles (e.g. sulfur compounds, carbonyls).� The CO2 along with stripped volatiles produced in the primary

fermentation of a normal gravity beer (e.g. 10 wt.%) is ventedthrough the fermenting vessel of the low gravity beer (e.g. 3

wt.%) leading to a flavor enrichment of the later (Barrell patent).Finally, the two beers can be mixed in different ratios leading toa low-alcohol beer (Barrell, 1979; Muller et al., 1991). However,the risk of contamination of the low gravity beer with undesir-able flavor volatiles (H2S, carbonyls, terpenes) stripped by CO2

has been hypothesized (Muller, 1990).

5.3. Use of special yeast

This approach to the AFB production is associated with the useof special yeast performing a limited fermentation process. Thedissimilarity of these ‘‘special’’ yeasts compared to traditionalbrewing yeast lies mainly in their tendency to produce loweramounts of ethanol or no ethanol at all. This can be achieved bystrategies such as selection of a proper microbial genus (strain)with specific properties or intentional modification of brewingyeast by random mutation or genetic engineering.

The most common approach relies on the fact that the majorfermentable sugar of all malt worts is maltose (ca. 75%) and somestrains of the genus Saccharomyces (e.g. used in the wine fermenta-tion) are unable to ferment this sugar. Thus the beer resultant fromconversion of glucose, fructose, and sucrose will contain lessthan < 0.5% ethanol (Muller, 1993). Except the application of a spe-cial yeast strain this method of the AFB production is identical withthe manufacturing of standard beer. However, due to limited yeastactivity and high residual extract content this manufacturing pro-cess is vulnerable to microbial contamination. Therefore high stan-dards of cleanliness and microbiological control are required(Muller, 1990).

The most successful genus, other that Saccharomyces, used forthe industrial production of alcohol-free beer is Saccharomycodesludwigii. The controlled fermentation can be carried out by thisyeast thanks to its disability to ferment maltose and maltotriose,the prevailing fermentable sugars of all malt worts. Althoughaccording to some authors the beer fermented by S. ludwigii tendsto be sweet due to its high residual maltose and maltotriose con-tent, the relative sweetness of these sugars is significantly lowerthan that of sucrose and glucose (Attenborough, 1988). The fer-mentation with S. ludwigii is characterized by slow attenuationeven at 20 �C which implies that the process does not require con-tinuous monitoring. A comparison of traditional bottom ferment-ing yeast with S. ludwigii with/without wort acidification showeda significantly higher formation of sensorially active by-products(higher alcohols and esters) by special yeast (Table 4). These vola-tiles, together with wort acidification, were found to mask the typ-ical worty flavor of AFBs and contributed positively to fullness andpleasant liveliness of AFBs. However, in spite of the high volatilecontent there was a remaining slight worty off-flavor, which canbe ascribed to lower aldehyde reduction by S. ludwigii. The AFBs

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produced with S. ludwigii contained diacetyl slightly above thetaste threshold level, which was picked up in the tasting (Narzisset al., 1992).

Another invention suggests leading steam saturated air throughalcoholic beer in a sieve bottom column in order to desorb the alco-hol (Dziondziak and Seiffert, 1995). The involved loss of sensoriallyactive compounds by desorption was suggested to be compensatedeither by addition of a fermenting wort or by using the speciesSaccharomyces rouxii able to consume ethanol under aerobic condi-tions and at the same time to produce flavor active substances(Dziondziak, 1989a). However, the author does not suggest howto deal with the possible negative effect of oxygen from strippingair on flavor and colloidal stability of produced AFB.

A method for producing an alcohol-free beer-like fermentedbeverage employing a slow fermentation process by fungi fromthe genus Monascus has been proposed, too. According to theauthors the final product looks like beer, has a refreshing taste, glit-tering red color, low alcohol content, and has high anti-oxidationactivity (Lin et al., 2005). However, it is questionable whether thisbeverage can be considered beer.

Hand in hand with the growing consumption of AFBs increasesthe need for yeast that would fit the special requirements of theirproduction. Random mutagenesis by ultraviolet irradiation (themost convenient for food applications) followed by selection ofproper mutants has been applied for isolation of non-recombinantyeast strains with defects in citric acid cycle. The most acceptableAFBs were obtained after the fermentation with Saccharomycescerevisiae mutants lacking 2-ketoglutarate dehydrogenase (KGD)and fumarase (FUM) activity. These strains were studied in batchand continuous fermentations, both immobilized and free. In allstudied fermentation arrangements the AFBs produced by thesetwo strains were characterized by a low ethanol (up to0.21% wt.%) content and a high organic alcohols (up to 1.38 g/L)one. The production of total higher alcohol (45–75 mg/L) and es-ters (18–36 mg/L) were somewhat lower and higher compared toa reference beer (alcoholic), respectively. The organic acids pro-duced, especially lactic acid, had a strong protective effect on themicrobial stability of the final product and thus the usual additionof lactic acid could be omitted (Narvátil et al., 2002). The presentedparameters meet the criteria for AFBs; however, these results wereobtained with haploid or diploid laboratory strains. Since thebrewing yeast, which possess industrially important and stableproperties (fermentation rate, flavor formation, flocculation) arealloploids, it makes the approach of random mutagenesis lesseffective, in particular face to face the DNA repair mechanisms ofyeast (Petin et al., 2001; Brendel et al., 2003; Aylon and Kupiec,2004).

Yeast strains with intentional gene deletions in citric acid cyclehave been studied first related to sake (Magarifuchi et al., 1995;Yano et al., 2003) and later to AFB production (Selecky et al.,2008). Similarly to random mutants the best AFBs were obtainedwith DKGD1, DKGD2 and DFUM1 strains. Thus it is no surprisethat the composition of AFBs produced by yeast with a gene dele-tion (Selecky et al., 2008) was close to those produced by randommutation (Narvátil et al., 2002). Since the gene deletions were car-ried out only on diploid strains, the preparation of a hybridbetween a brewing yeast and a laboratory strain, carrying all thegenetic properties responsible for all the industrially importantproperties (taste and flavor formation, flocculation), and simulta-neously deficient in the citric acid cycle enzyme genes, would bebeneficial.

Another example of the gene deletion strategy is the use of alco-hol dehydrogenase-free (ADH) non-revertible mutant ofS. cerevisiaeto produce AFB having 0.3–2.0 vol.% glycerol content, which isreported to improve the body of the beer. The excessive accumula-tion of acetaldehyde, a fermentation by-product toxic to yeast, by

this recombinant yeast is prevented by daily gassing with CO2

(30 m3/hl/h) for 30 min into the fermenting tank. The effect of gas-sing on the content of beer volatiles (higher alcohols, esters) is notdiscussed in the patent (Dziondziak, 1989b). The beneficial effectof lacking ADH activity was demonstrated on haploid S. cerevisiaeshowing a double phenotype: low ethanol production andenhanced worty aldehyde reduction (Evellin et al., 1999). However,the elimination of each ADH locus in a polyploidy brewer’s yeast hasnot been published.

Conversely to the gene deletion strategy, the overexpression ofglycerol-3-phosphate dehydrogenase gene was performed in anindustrial lager brewing yeast (Saccharomyces pastorianus) toreduce the ethanol content in beer. The results were not fully satis-factory since this transformation led to 5.6 times increased glycerolproduction and the ethanol production decreased only by 18% whencompared to the wild-type. Although only minor changes in theconcentration of higher alcohols, esters, and fatty acids could beobserved, concentrations of several other by-products, particularlyacetoin, diacetyl, and acetaldehyde, were considerably increased(Nevoigt et al., 2002).

Despite of some controversy and lack of decisive breakthrough,the potential of genetic engineering is enormous, making possiblethe future construction of strains tailor-made for the AFB produc-tion. However, as long as both legal obstacles and particularlythe consumers’ negative attitude towards the use of geneticallymodified yeast persist, the breweries will not risk their industrialapplication.

5.4. Continuous fermentation

Investigation on the continuous culture of free and immobilizedyeast for the beer production has been motivated by the advanta-ges comprising of lower capital, production, and manpower costs.Several reviews have been written recently on the state-of-the-art of continuous beer fermentation systems and the flavorparticularities of the continuously fermented and/or maturatedbeers (Brányik et al., 2005, 2008; Willaert and Nedovic, 2006).The potential advantages arise mostly out of the accelerated trans-formation of wort into beer driven by an increased biomassconcentration (van Iersel et al., 1998). This is achieved by immobi-lization of biomass based on physical confinement of yeast inside abioreactor. Various carrier types have been used for the beer fer-mentation by immobilized brewing yeast. Among them, inertcarrier types with adsorption as the prevailing immobilizationmechanism (DEAE-cellulose, wood chips, spent grains) have shownto be technically useful and economically affordable. For each cellimmobilization technique a variety of reactor types can be selectedand a careful matching of immobilization method, reactor configu-ration and process characteristics is important for a successfulindustrial implementation (Verbelen et al., 2006). Since the contin-uous alcohol-free beer (AFB) production is based on the limited fer-mentation strategy, the corresponding fermentation systemsgenerally consists of one stage bioreactors equipped with addi-tional apparatus for a continuous wort supply and process control.

Although the continuous beer fermentation has been studiedfor several decades, the number of industrial applications is stilllimited. The major obstacle hindering the industrial exploitationof this technology is the difficulty in achieving the correct balanceof sensory compounds in the final product (Pilkington et al., 1998;Brányik et al., 2008). Given the shifts in metabolism of cells grownin continuous culture, it has proven difficult to ‘‘translate’’ the tra-ditional batch process into a continuous and immobilized process.The production of AFBs using immobilized yeast cell systems rankamong limited fermentation methods using short contact (1–12 hresidence time) between immobilized yeasts and wort (Van DeWinkel et al., 1991; Aivasidis et al., 1991; van Iersel et al., 1995;

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Debourg et al., 1995; Lehnert et al., 2008). The continuous AFB fer-mentation can outperform the rival technologies in productivity;however, it is essential that it produces a final product competitivein terms of sensorial quality.

Alcohol-free beers are usually characterized by worty off-flavors and lack of pleasant fruity (estery) aroma found in regularbeers. Although the formation of higher alcohols and esters duringcontinuous AFB production has already been studied, very fewpapers comparing flavor formation in the traditional batch fermen-tation and the continuous one are available. One reported a signif-icantly lower formation of higher alcohol and acetic acid esters(Aivasidis et al., 1991), while more recent papers concluded theimportance of process parameters and yeast strains for the forma-tion of volatiles (Lehnert et al., 2008, 2009). Among process param-eters it is aeration, which has perhaps the most important impacton the formation of volatiles in continuous systems (Virkajärviet al., 1999). An optimal and constant flavor profile of the AFBcan be achieved by the accurate oxygen supply (van Iersel et al.,1999). The concentration of total higher alcohols (HA) and ester(ES) as well as the HA/ES ratio found in continuously fermentedmodel medium under optimized oxygen supply was comparablewith those found in three commercial alcohol-free beers (Lehnertet al., 2008).

The interplay between the appropriate production strain, car-rier material, and bioreactor design is very important in continuousimmobilized cell reactors and their suitable combination canimprove both the system performance and product quality. Theimportance of careful matching of the chosen yeast strain withan immobilization method and a suitable reactor arrangementwas demonstrated. It was shown that the laboratory yeast strainwith disruption in the KGD2 (2-ketoglutarate dehydrogenase) geneperformed, in terms of the flavor formation, equally well in thebatch and continuous packed-bed reactor. However, it was unableto form a biofilm around spent grain particles and therefore its usewas not possible in the gas-lift reactor. Conversely, the bottom fer-menting strain W96 adhered to the solid supports readily, but theformation of flavor active compounds was insufficient with theexception of the immobilization onto spent grains in the gas-liftreactor. This system arrangement proved that even a strain, whichseems to be less suitable for the AFB production by the arrestedfermentation, can under appropriate conditions produce an accept-able final product (Mota et al., 2011).

Several studies have been carried out on the alcohol-free beerproduction by the limited fermentation with immobilized cells ofS. cerevisiae at low temperature (0–4 �C) and nearly anaerobic con-ditions (Lommi et al., 1990; Aivasidis et al., 1991; van Iersel et al.,1995). Similarly to the cold contact process (CCP) these conditionslead to the suppressed cell growth, low ethanol formation, andstimulated production of higher alcohols and esters. The authorshypothesized that the increased production of volatiles can beascribed to the effort of cells to maintain the redox balance underanaerobic conditions by reoxidation of NADH coupled with thereduction of carbonyl compounds to higher alcohols (van Ierselet al., 1995). However, some oxygen is essential for several yeastbiosynthetic pathways (Snoek and Steensma, 2007) and thus forthe long-term production of AFB with balanced flavor (van Ierselet al., 1999).

Wort carbonyls were proposed to contribute to the unpleasantworty taste of AFBs (Perpète and Collin, 1999c). Although thereduction of wort aldehydes by yeast is relatively fast during batchfermentations, there was concern it may not be sufficient at thespeed of the continuous AFB production. However, the carbonylreducing capacity of continuous immobilized cells systems forthe AFB production has been reported to be satisfactory (Debourget al., 1995; van Iersel et al., 2000; Lehnert et al., 2008). This can beascribed to either an increased alcohol dehydrogenase activity in

immobilized yeast (van Iersel et al., 2000) and/or a wise compro-mise between the alcohol formation and the carbonyl reductionby optimizing the process parameters (biomass load, residencetime, temperature, and aeration) of the continuous systems(Debourg et al., 1994; van Iersel et al., 1995; Lehnert et al., 2008).

As in the processes involving the limited fermentation, the pHdrop during the continuous AFB production does not take placein the required extent, a continuous biological acidification forthe direct adjustment of pH of mash and wort by immobilized lac-tic acid bacteria (DEAE cellulose) has been studied during test peri-ods of a few months. The continuous acidification would suitablymatch with the subsequent continuous fermentation (Pittner andBack, 1995).

Several industrial examples of testing (Aivasidis et al., 1991)and implementation of the continuous AFB production have beenreported (Van Dieren, 1995; Mensour et al., 1997). However, infor-mation on the current industrial application of the continuous lim-ited fermentation of AFBs is not available to the authors. It can beassumed that the continuous fermentation systems have not foundwidespread utilization in the AFB production mainly due to theneed of special equipment (bioreactor and tools for its continuousfeed and control), eventually additional methods (immobilization)and materials (carrier).

6. Sensorial properties and additional improvements of alcohol-free beer

The aroma and taste of an AFB is usually rather different fromits fully fermented counterpart. The AFBs often suffer from variousflavor imperfections. For instance, the AFBs produced by the mem-brane processes have usually less body and a low aromatic profile,the thermally dealcoholized AFBs may suffer heat damages, whilethe beers obtained by biological methods have often a sweet andworty off-flavor (Montanari et al., 2009). It has been proved thatethanol significantly increases aldehyde retention, leading to lowerperception of the worty character. In a usual 5% ABV beer theretention of aldehydes was 32–39% in comparison to 8–12% reten-tion at 0.5% ABV. Similarly, higher levels of mono- and disaccha-rides in AFBs intensify such off-favors. Headspace extraction andsensorial analysis further showed that the aldehyde retention inAFBs can be enhanced by increasing the level of dextrins or glyc-erol (Perpète and Collin, 2000b). These findings suggest that theflavor perception of a regular beer cannot be mimicked simply bytrying to get the volatile distribution in AFBs as close as possibleto that one in a regular beer. Instead, promising results can beachieved by changing the degree of volatile retention in AFBsand/or by creating a balance of volatiles, different from that pres-ent in beers containing ethanol, but with similar flavor impression.Particular flavor balance can be produced by process adjustmentsas well as by adding flavor active compounds into the final product(Daenen et al., 2009; Heymann et al., 2010).

There are solely a very few articles, which allow the comparisonof beer properties before and after dealcoholization. These aresummarized in Table 5 and presented as a percentage change ofselected properties. It can be seen that the thermal processes tendto increase, while the membrane processes decrease the color ofthe AFB. The bitterness and foam stability were usually impairedby all dealcoholization processes. However, the most significantimpact of the alcohol removal was observed on the loss of volatiles.All the technologies led to significant losses of volatiles, the small-est being observed in the case of the membrane processes. In thecase of the arrested fermentation the difference in the volatilecontent was calculated for an average German AFB and a full-fermented pale beer as found in Narziss et al., 1992. The absenceof volatiles in an AFB produced by the arrested fermentation is

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Table 5Alterations in the properties of alcohol-free beers as compared to original beersresulting from their dealcoholization or arrested fermentation.

Difference (%) VRa VRb FFc FFd Dc ROc ROe AFf

Color (EBC) +13 – 0 +10 �6 �3 – –Bitterness (EBC) +2 – �7 �8 �12 �7 �50 –Foam (NIBEM) – – �3 � �1 �8 – –Esters �99 �100 �95 �100 �85 �78 �87 �87Higher alcohols �78 �78 �98 �95 �85 �69 �81 �80

a Narziss et al. (1993).b Zürcher et al. (2005).c Stein (1993).d Zufall and Wackerbauer (2000a).e Kavanagh et al. (1991).f Narziss et al. (1992).

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comparable with that in AFBs produced by the alcohol removal(Table 5). The flavor imperfections of AFBs raised the need to cor-rect them. Some strategies of the flavor improvement were dis-cussed together with the technologies, while here are mentionedsome additional possibilities.

6.1. Post-treatments and blending

Both the thermal and membrane processes often use differentpost-treatment and blending techniques in order to improve thesensorial quality and colloidal stability of dealcoholized beers.Improvements can be achieved by the addition of fresh yeast fol-lowed by maturation or by blending with the original beer (Schedlet al., 1988; Moreira da Silva and De Wit, 2008), aromatic beer(beer fermented at elevated temperatures), or krausen (Narzisset al., 1993). Another possibility is to adopt the Barrell patent (Bar-rell, 1979) to gently dealcoholize beer by treating it with CO2 fromfermenting green beer and finally add krausen followed by matu-ration and filtration process (Zürcher et al., 2005). The additionof 6 vol.% of krausen into a beer dealcoholized to 0.1 ABV returnedabout 15% and 31% of the higher alcohols and esters originallypresent in the alcoholic beer (Table 1), respectively (Narzisset al., 1993). Other studies have shown that the thresholds of theimportant aroma components in the AFBs are significantly lowerthan in the alcohol-containing beer. This means that even a partialreplacement of aroma compounds by one of the above suggestedmethods can improve significantly the AFB flavor (Zufall andWackerbauer, 2000a).

6.2. Additives

The use of additives will be explained on the example of Czechalcohol-free beers. Currently there are 30 AFB brands commercial-ized in the Czech Republic (AFBs represented 2.92% of the Czechbeer market in 2008). Among them 26 brands are produced bythe arrested/limited fermentation (at least one uses also a changedmashing process), two are fermented with special yeast and one isproduced by vacuum rectification. According to information on thelabels, 10 brands from the whole group of AFBs are produced onlyusing traditional brewing raw materials, 9 contain one additive, 9contain two additives, and 2 contain three or more additives. Themost frequently used additives are: saccharin (sweetener E954,11 AFBs), ascorbic acid (antioxidant E300, 9 AFBs), lactic acid (pre-servative E270, 8 AFBs). In the case of AFBs produced by thearrested/limited fermentation, the use of lactic acid (preservativewith antimicrobial and flavor effects) and ascorbic acid (antioxi-dant increasing flavor and colloidal stabilities) is justified. Besidesthe addition of lactic acid during the production process a biolog-ical acidification of wort with lactobacillary strains was tested aswell (Narziss et al., 1991). The widespread addition of saccharin,

a sweetener with an unpleasant bitter or metallic aftertaste athigher concentrations, is motivated by the desire to strengthenthe body of the AFB. Besides these frequently used additives someproducers indicate also the use of citric acid (acidity regulator,E330), potassium metabisulphite (preservative, E224), caramel(coloring, E150), and glucose-fructose syrup. Moreover, the addi-tion of dextrins into beer has been reported to improve the flavorprofile of LABs and AFBs through their action on the retentionand/or perception of flavor active compounds (Louant and Dufour,1991). However, an anonymous evaluation of Czech AFBs by apanel of 35 tasters (brew masters, brewing engineers and research-ers) showed that the additives solely cannot substitute the use ofhigh quality raw materials and an optimized production process.The distribution of beers with different additives and their combi-nations across the final ranking of AFBs was random and no ten-dency of improved evaluation based on the use of additives wasfound (Vecerková, 2010). It was found that the popularity ofsemi-dark AFBs (dark and caramel malts added) is on the rise(2nd and 3rd place in the contest), but at the same time the worstcontestant was also a semi-dark AFB underlining the fact thatsolely the addition of special malts cannot improve the productssensorial quality.

7. Cost evaluation and conclusions

The available literature is poor in comparisons of processes andtheir impact on the product quality, but the comparison of eco-nomic aspects of processes producing LABs of AFBs is even scarcer.Nevertheless, it is clear that the arrested/limited fermentation pro-cess can be performed in a common brewery equipment but short-er production time and less raw materials are needed. Therefore,the production costs for such LAB/AFBs are the same or lower, thanfor the regular beer. Conversely, processes of the alcohol removaldo require an extra equipment, relevant utilities and space, whichmean additional investments and operating costs above the pro-duction costs of the regular beer to be dealcoholized. The advan-tage of alcohol separation processes is their flexibility (start-upwithin hours, high productivity) and possibility to produce zeroalcohol beers, which is hardly achievable by fermentative pro-cesses, given by their nature. Some authors also state that the tasteof the dealcoholized AFBs is dryer and closer to regular beer (Basar-ová et al., 2010). Somewhat contradictory to this is the fact the onlyCzech AFB produced by vacuum rectification was ranked 21stamong 30 samples by the taste panel (Vecerková, 2010). Additionalprofit can be created also from the separated alcohol, which isobtained at different concentrations. The diluted alcohol solutioncan be further concentrated to a marketable content, used in thebrewing process as blending water or sold for acetification to pro-duce vinegar (Regan, 1990; Stein, 1993). From one rare cost com-parison of four different processes it was the falling film system,which emerged victorious followed by the thin film evaporator,reverse osmosis, and, finally, dialysis (Stein, 1993). However, a reli-able and comprehensive economic comparison of various methodsof the LAB/AFB production is not available and therefore it isimpossible to define the best process. Moreover, choosing the mostappropriate process is further influenced by the available produc-tion capacity, expected sales, and marketing strategy of the prod-uct and hence it requires a detailed balance sheet reflecting theexisting technology and the specifics of the local market.

Acknowledgements

The authors thank to the Ministry of Education, Youth andSports of the Czech Republic (MSM 6046137305 and 1M0570),National Council for Scientific and Technological Development

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(CNPq/Brazil), and Sergipe State Research and Technological Inno-vation Foundation (FAPITEC/SE, Brazil) for their financial support.

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