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

Journal of Food Engineering 108 (2012) 493–506

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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng

Review

A review of methods of low alcohol and alcohol-free beer production Tomáš Brányik a,⇑, Daniel P. Silva b, Martin Baszczynˇski 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 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 b

a r t i c l e

i n f o

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

a b s t r a c t 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 content 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. 2. 3. 4.

5.

6.

7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Beer and health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods of the alcohol-free beer production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Production of alcohol-free beer by ethanol removal methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Thermal processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Vacuum rectification plant. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Thin film evaporators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Membrane processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Reverse osmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Production of alcohol-free beer by methods of restricted ethanol formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Changed mashing process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Arrested or limited fermentation process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Use of special yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Continuous fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sensorial properties and additional improvements of alcohol-free beer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Post-treatments and blending . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cost evaluation and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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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). 0260-8774/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.09.020

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1. Introduction The production of beers with low alcohol content had different historical reasons in the past century. For instance, during World Wars (1914–1918 and 1939–1945) it was the shortage of raw materials, which was leading to the production of beers with low original extract (often with a high proportion of adjuncts) and thus of low alcohol content. On the other hand, in the years between 1919 and 1933 it was the prohibition to manufacture, sale and consume alcohol, which increased the production of low alcohol content beers in the United States of America (Meussdoerffer, 2009; Silva et al., 2010). In the late 20th century efforts of breweries to expand the assortment of products with beers with low alcohol content was motivated mainly by the following reasons:  Increase in the overall production by bringing out new products in countries with highly competitive markets.  Provide beer consumers with alternative products prior or during their activities (driving motor vehicles or operating machinery, engagement in sports) or under conditions (pregnancy, medication) irreconcilable with alcohol consumption.  Penetrate beverage markets in countries, where alcohol consumption is forbidden for religious reasons. Although the sales of beers with low alcohol content did not fulfill the initial optimistic expectations and the market with these products has been, for a long time, just a drop in the sea of the overall beer production, nowadays it is a fast growing segment of the beer market worldwide. In the last five years, the average sales in Europe climbed by 50%. Spain is now the largest consumer of beers 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 alcohol content varies between 4% and 5%. Probably the most significant reasons for the annual increase in alcohol-free beer (AFB) sales in the EU countries are the legislative interventions restricting the alcohol consumption and the increasing awareness of consumers about the benefits of moderate beer drinking (Silva et al., 2010; Informe, 2010). In most of the EU countries beers with low alcohol content are divided into alcohol free beers (AFBs) containing 60.5% alcohol by volume (ABV), and to low-alcohol beers (LABs) with no more than 1.2% ABV. In the United States alcohol-free beer means that there is no alcohol present, while the upper limit of 0.5% ABV corresponds to so-called non-alcoholic beer or ‘‘near-beer’’ (Montanari et al., 2009). In countries that enforce religious prohibition, the alcohol content in beverages must not exceed 0.05% by volume. The terminology of this article in the following chapters will be governed by the aforementioned European legislation. However, while the methods to produce both LAB and AFB are from practical point of view identical, the AFBs market share prevails over the LABs one. Hence, in this article the beverages with low alcohol content produced from malt will be generally termed alcohol-free beers (AFBs).

and binds cellular constituents generating harmful acetaldehyde adducts (Rajendram and Preedy, 2009). Simultaneously, there are strong evidences that moderate alcohol consumption has not only a better long-term health outcome than excessive alcohol consumption, but even better than abstaining. Moderate beer drinking has shown to be, at least, as effective as wine drinking at reducing risks of coronary diseases, heart attack, diabetes, and overall mortality (Mukamal and Rimm, 2008; Ferreira and Willoughby, 2008). Besides alcohol, which is probably the most important component of beer that counters atherosclerosis (Li and Mukamal, 2004; Tolstrup and Groenbaek, 2007), these positive effects may be attributed to a whole range of other properties and valuable cereal and hop-related substances found in beer such as no fat or cholesterol content, low energy and free sugar content, high antioxidant (e.g. polyphenols, flavonoids), magnesium and soluble fiber content. In addition, beer provides essential vitamins and minerals and is thus contributing to a healthy balanced diet (Bamforth, 2002). The alcohol-free beers also claim beneficial effects of healthy beer components with a simultaneous effect of the lower energy intake and complete absence of negative impacts of alcohol consumption.

3. Methods of the alcohol-free beer production The strategies to produce AFBs can be divided into two main groups (physical and biological processes), which can be further broken down as shown here (Fig. 1). The so called physical methods are based on gentle removal of alcohol from regular beer and require considerable investments into the special equipment for alcohol removal. After the removal process has been optimized, the sensorial quality of produced AFBs is usually good. Their further advantage is that they can remove ethanol from beers to vanishingly low levels. The most widespread biological approaches are based on limited ethanol formation during the beer fermentation. They are usually performed in traditional brewery equipment and hence do not require additional investments, but their products are often characterized by worty off-flavors. Improvements taking advantage of special yeast increase the costs by the purchase, selection, or construction of the production organisms as well as by the need their propagation have to be separated. However, suitable tailor-made or selected microorganisms can contribute significantly to the product sensorial quality improvement. There are also AFB production processes (continuous fermentation with immobilized yeast) based on limited alcohol formation, which require special equipment and material (continuously operating bioreactor, carrier for cell immobilization). In this case, the higher investment costs have to be justified by the higher productivity of continuous processes. In general, the ethanol formation, which is intrinsic to the biological methods, makes impossible the production of AFBs with alcohol content close to zero.

2. Beer and health Alcohol abuse has been on the public agenda for many years since it carries risks of violent crime, traffic accidents, public disorder, and health damage. Ethanol is one of the most commonly used recreational drugs worldwide and it is often ingested as a component of beer. When beer is consumed, ethanol is absorbed from the gastrointestinal tract by diffusion and is swiftly distributed in the blood before entering tissues. Ethanol is metabolized to acetaldehyde mainly in the stomach and liver. Acetaldehyde is highly toxic

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

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4. Production of alcohol-free beer by ethanol removal methods The technologies applied for complete or partial ethanol removal from regular beers can be classified into two groups based on the principle of the separation process such as thermal and membrane processes (Fig. 1). Besides the industrially applied methods of beer dealcoholization (vacuum rectification and evaporation, dialysis, and reverse osmosis) there have been several other methods studied under laboratory conditions such as membrane extraction (Matson, 1987; Etuk and Murray, 1990), supercritical CO2 extraction (Mori, 2004), pervaporation (Magalhaes Mendes 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 distillation under atmospheric pressure, which revealed significant temperature damage to the beer taste, were soon replaced by vacuum distillation (Zufall and Wackerbauer, 2000a). If the pressure is reduced, alcohol can be drawn off at much lower temperature. All thermal processes to produce AFBs are therefore performed at an absolute pressure of 4–20 kPa, whereby evaporation temperatures of 30–60 °C are achieved. Even so, a great loss of beer flavor and liveliness can occur during thermal processes. The deterioration of beer quality by thermal dealcoholization depends mainly on the evaporation temperature and the period of exposure, which depends on the thermal separator construction. AFB production at industrial 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 film evaporators. Generally, advantages of thermal processes are: the potential to remove alcohol from beer completely, the possibility to commercialize the separated alcohol, the continuous and automatic operation with a short start-up period, and the flexibility in terms of volumetric performance and the input beer composition. Conversely, the purchase of these systems requires significant investment as well as there is considerable running costs (energy consumption) and some risks of thermal damage or loss of volatiles from beer. At the end of all thermal processes the concentrated alcohol-free beer has to be diluted with oxygen-free water and carbonized (Zufall and Wackerbauer, 2000a).

4.1.1. Vacuum rectification plant This 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 liberation of volatile compounds in a vacuum degasser, dealcoholization in a vacuum column (usually a packed-bed rectifying column), recovering the aroma components from CO2 by spraying with dealcoholized beer or water, and redirecting them into dealcoholized beer (Regan, 1990; Narziss et al., 1993; API Schmidt-Bretten, 2004). In the rectification column the fluid flows down at a temperature between 42 and 46 °C. In counter flow the product contacts rising vapors, generated from alcohol-free beer in an evaporator, which brings about the selective separation of alcohol from the product. The dealcoholized product (less than 0.05% ABV is achievable) is then cooled. The production capacity of these systems is usually given in the range 4–200 hl of alcohol-free beer per hour. The system further contains an aroma recovery unit, where the aroma components are recovered and their redirection into the beer can be made under pressure (Koerner, 1996). The alcohol-rich vapors can be concentrated to 75% ABV in a rectification section and marketed immediately (Narziss et al., 1993; API Schmidt-Bretten, 2004). Without concentrating, the alcoholic by-product produced has about 8–9% ABV. This by-product can be sold for acetification to produce vinegar (Regan, 1990). An alcohol removal system (Sigmatec) from beer by countercurrent distillation in combination with rectification was used for dealcoholization of both top and bottom fermented beers. In the case 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 ethanol removal from the original 5.57 vol.% to 0.46, 0.22, and 0.12 vol.%, respectively. This means, that in dealcoholized wheat beer with 0.46 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 volatiles to a higher degree. Approximately 78% of total higher alcohols (104.7 mg/l), and almost 100% of total esters (19.6 mg/l) were removed already at 0.48% ABV (Table 1). In addition, the total higher alcohols in dealcoholized top- and bottom-fermented beers were represented almost exclusively by 2-phenylethanol (73 and 97%), a compound with floral odor (Narziss et al., 1993). This unbalanced content of volatiles shows the importance of adding them back, particularly into dealcoholized bottom fermented beer, otherwise the sensory properties of AFBs are significantly changed

Table 1 Selected properties of original input beer and alcohol-free beers obtained by vacuum rectification and post-treated with aroma redirection and blending with 6% krausen. Sample

Original 1/Original 2

Dealcoholized 1a/Dealcoholized 2b

Dealcoholized 1a + aroma

Dealcoholized 1c + 6% Krausen

Original gravity (wt.%) Ethanol (% ABV) Color (EBC) pH Bitterness (EBC) 1-Propanol (mg/l) 2-Methylpropanol (mg/l) 2-Methyl-1-butanol (mg/l) 3-Methyl-1-butanol (mg/l) 2-Phenylethanol (mg/l) Furfuryl alcohol (mg/l) Ethyl acetate (mg/l) Isoamyl acetate (mg/l) 2-Phenyl ethyl acetate (mg/l) Total HAA (mg/l) Total EAA (mg/l)

11.59/na 4.99/5.3 8.4/na 4.75/na 24.9/na 6.7/23.4 11.2/24.3 15.2/22.4 52.8/64.1 18.6/35.9 0.07/3.2 16.9/23.1 1.9/2.8 0.4/na 104.7/173.3 19.6/25.9

5.16/na 0.48/0.03 9.5/na 4.71/na 25.5/na 0.8/nd nd/nd nd/0.1 nd/0.3 22.4/35.1 nd/2.8 nd/nd nd/nd 0.03/na 23.2/38.3 0.04/nd

4.98 0.51 8.7 4.78 27.5 1.0 0.7 4.3 3.0 20.0 0.01 3.3 0.5 – 29.0 3.83

5.22 0.54 9.5 4.69 29.3 1.3 1.7 2.8 10 23.0 0.02 5.2 0.5 0.34 38.82 6.13

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).

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as compared to original beer (Zürcher et al., 2005). The recovery of aromatic volatiles from the CO2 liberated during degassing and their addition into dealcoholized beer, returned about 6% and 20% of the originally present higher alcohols and esters (Table 1), respectively (Narziss et al., 1993). The thermal stress exhibited by the dealcoholization system was considered negligible. There was no increase in hydroxymethylfurfural or furfural observed in alcohol-free beers (Kern, 1994). The content of medium-chain fatty acids decreased during the dealcoholization by 20–40% as compared to original beer, with the exception of dealcoholized unfiltered beer, where the thermal lysis of cells is blamed for the increased decanoic acid content (Narziss et al., 1993). 4.1.2. Thin film evaporators In order to shorten the ethanol removal, regular beer flows through these vacuum devices as a thin film with large surface area in an extremely short residence time, which results in an improved product quality. Examples of thin film evaporators, which produce a thin liquid film in a mechanical (rotational movement) way, are the Centritherm and spinning cone column (SCC) systems (supplied by Flavourtech, www.ft-tech.net). On the contrary, the falling film evaporator does not contain moving parts and the liquid film is created by gravity-induced downward movement of beer on the inner surface of heating tubes. The Centritherm system structure resembles that of a plate centrifuge (Fig. 2). The centrifugal evaporator operates under vacuum at low temperatures (35–60 °C) and uses steam as the heating medium. The regular beer to be dealcoholized enters the evaporator 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 entire heating 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 than one second. The concentrated and dealcoholized beer collects at the outer edge of the cones and then exits the evaporator through a stationary product tube. The vapors removed from the beer rise through the center of the cone and enter an exhaust pipe that transfers them to an external condenser. The Centritherm evaporators are designed with 1–12 hollow cones, which correspond to production capacities of AFB from 0.5 to 100 hl/h, respectively. Steam is supplied to the evaporator through a hollow spindle to the steam chamber of each cone. As the steam condenses, the condensate is immediately projected to the upper wall of the hollow steam chamber. The condensate exits the steam chamber through a channel and is removed from the evaporator (Fig. 2). The Centritherm is claimed to have minimal thermal impact and easy operation, while the oxygen penetration through the seals of moving parts is considered as a potential risk (Zufall and Wackerbauer, 2000a). The spinning cone column (SCC) is a counter-current liquid–gas contacting device using gentle mechanical forces to enhance interphase contact (Fig. 3). This allows rapid and efficient separation of volatile 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) rotating shaft, (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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cones is attached to the inside wall of the column. Another series of cones is attached to the rotating shaft, in parallel to the fixed cones. The fixed and rotating cones alternate vertically. The full strength beer is fed into the column top. Pulled by gravity, it flows down the upper surface of the first fixed cone and drops onto the first rotating cone (300–500 rpm), which spins the beer into a thin, turbulent film. The centrifugal force induces an upward liquid flow to the rim of the spinning cone where the beer is dropping onto the next stationary cone below. In this manner, the beer flows to the bottom of the column. The stripping medium, steam produced from deaerated water, is fed into the column bottom and flows upward, passing over the surface of the liquid thin film, collecting ethanol and other volatile compounds as it rises. Fins on the underside of the rotating cones induce a high degree of turbulence and a pumping effect to the rising vapor stream. The turbulent liquid and vapor flow leads to highly efficient mass transfer of volatiles from liquid to the vapor stream (Fig. 2). The vapor flows out of the column top and passes through a condenser system, which captures the volatiles in a concentrated liquid form. The dealcoholized beer is pumped out of the column bottom (Craig, 1986). A low pressure drop in the SCC allows a low operating temperature of 40–55 °C under vacuum. The residence time of beer in the SCC is approximately 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 the feed beer shows no negative impact (overfoaming) and no oxygen pickup was found in the beer that passed the SCC (Moreira da Silva and De Wit, 2008). Several different production strategies using the SCC system have been tested. The best flavor recovery was achieved by a two stage process involving the flavor removal followed 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 alcohols, while the alcohol content of the beer decreases by 1% ABV. This beer with a reduced alcohol content (3.8% ABV) is then further dealcoholized to 0.17% ABV during the second passage through the SCC (highest temperature in the column 57.2 °C), which simultaneously leads to the loss of remaining volatiles. The re-combination of the dealcoholized beer stream with the volatile-rich condensate (75% ABV) captured after the first flavor-removal stage resulted in an AFB (0.5% ABV) with 25% and 30% of total esters and higher alcohols retained, respectively, relative to 4.8% ABV beer (Badcock et al., 1994). In contrast to centrifugal evaporators, the falling film evaporator contains no moving parts, which results in great benefits. The system is not only cheaper in construction, but also easier to clean and there is practically no danger of oxygen transfer across the various seals of moving parts. Overall, the acquisition and operation costs of the falling film evaporation are considered the lowest of all thermal dealcoholization systems (Stein, 1993; Zufall and Wackerbauer, 2000a). Further energy savings can be achieved using a multi-stage design of falling film evaporators since the alcohol containing vapors from the first evaporator can be used as heating steam to the second one, while the vapor from the second one can heat the third evaporator. Certain disadvantage of this multistage arrangement is the need to operate the first stage at relatively high temperature (60 °C), so that the vapor temperature in the final stage is sufficiently high for the alcohol removal (35–40 °C) (Hochberg, 1986). In falling film evaporators the original beer is preheated 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 walls of the tubes. The beer flows downward at boiling temperature and is partially evaporated (Fig. 4). The downward movement is induced both by gravity and high speed co-current vapor flow (20–80 m/s). Thus the beer stays in the evaporator for only a few seconds. Process steam (saturated steam) is used to heat the

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

evaporator tubes. The alcohol-rich vapor is separated from the dealcoholized beer concentrate in a vapor separator connected to the falling film evaporator outlet and is finally condensed in a condenser. The heaters, falling film evaporator, separator, and condenser are connected to a common vacuum pump. As the beer passing through the falling film evaporator was not only dealcoholized, but also concentrated, it must be re-diluted with degassed water to the original extract content as well as it is necessary to carbonize it. The main process parameters controlling the dealcoholization degree in the falling film evaporator are the heating steam supply and the evaporation temperature adjustable by the vacuum pump. However, it turned out that independently on the tested evaporation degree (30–55 kg of vapors from 100 kg inlet beer) there was a significant loss of total higher alcohols (91–97%), while esters were practically completely removed. In terms of the alcohol content there was an evaporation degree of 40/100 kg necessary to achieve 0.5% ABV. In order to re-direct some volatiles into the dealcoholized beer, while not exceeding the alcohol limit for AFBs either the condensed vapor flow or preferably the volatiles separated from vapor condensate by rectification can be used. The overall material balance of input (original beer) and output streams (dealcoholized beer concentrate and condensed vapor) from the falling film evaporator unit showed a loss of esters ( 36%) and higher alcohols ( 8%), and an overall accumulation of acetaldehyde (+17%), explained by thermal decomposition of acetaldehyde-bisulphite complex (Zufall and Wackerbauer, 2000a).

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4.2. Membrane processes These alcohol removal methods are based on the semipermeable character of membranes, which separate only small molecules like ethanol and water from the beer to the permeate liquid. Two types of membrane processes used for beer dealcoholization can be distinguished on the industrial scale: dialysis and reverse osmosis. They differ in applied pressures and temperatures, membrane materials and their structures. It is known that all of the membrane processes have less thermal impact on beer, they can be operated automatically and in flexible manner, but at the same time they require significant capital and running costs. The economic feasibility of membrane processes for the production of beverages with an alcohol percentage lower than 0.45 vol.% was by some authors challenged (Pilipovik and Riverol, 2005), while others stated that the energy requirement of a membrane system for alcohol purification (reverse osmosis) would be significantly lower than that of a conventional distillation system (Mehta, 1982). Membrane processes were suggested also as a part of a system for the continuous production of delacoholized beverages (Gresch, 1991). 4.2.1. Dialysis The driving force of the mass transfer across the semipermeable dialysis membrane is the concentration gradient of compounds between beer and dialysate. The semipermeable membrane acts as a molecular sieve permeable only to certain molecules, depending on the pore size and surface properties of the membrane. When dealcoholization by dialysis is performed into water, all beer ingredients tend to move from the area of the high concentration (beer) to the area of the low concentration (water), while some water will diffuse from dialysate into beer. The prevailing mechanism of mass transfer in dialysis is molecular diffusion. When the transmembrane pressure difference is applied (usually 10–60 kPa), in order to suppress water diffusion into beer, the process is often called diafiltration and both diffusive and convective mass transfers take place (Leskosek and Mitrovic, 1994; Petkovska et al., 1997). The process of dialysis is usually performed at 1–6 °C, eliminating the thermal load of the product. Dialysis membranes are composed of either cellulose derivatives or various synthetic materials

(e.g. polysulphone, polyethersulphone) and are generally arranged in bundles of hollow fibres, known as modules. In hollow fibers the beer passes along a dialysis membrane, while simultaneously an alcohol-free dialysate liquid flows counter-currently along the other side of the membrane (Fig. 5). The principle of the countercurrent flow guarantees a high concentration gradient between the dialysate and the beer in terms of the alcohol content so that an optimal diffusion can be obtained. To operate a dialysis module it is necessary to apply some pressure on both the beer side and the dialysate one, otherwise the diffusion can be disrupted by release of carbon dioxide. The applied pressure must be at least equal to the saturation pressure of CO2 in beer at a given temperature. In order to further minimize the loss of CO2 it is recommended to add a small amount of carbon dioxide into the dialysis water. This will also 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 dialysate during rectification and then pass into beer (Moonen and Niefind, 1982; Attenborough, 1988; Donhauser et al., 1991). Despite the optimization of membranes and process parameters a selective removal of ethanol cannot be achieved. Other components of beer, such as higher alcohols and esters, are therefore almost completely removed from the beer by dialysis (Table 2). Losses of low-molecular-weight volatile compounds can be prevented by adding them into dialysate reducing thus their diffusion from beer. The extent and rate of dealcoholization, and also loss of volatiles, can be regulated mainly by the ratio of flow rates of dialysate and beer, which can be varied in a wide range from 0.4:1 to 6.5:1. By increasing the ratio of dialysate to beer flow, the removal of alcohol and volatiles from beer becomes more pronounced. However, this ratio influences not only the rate of alcohol removal from beer but also the energy costs for the rectification of the dialysate (Donhauser et al., 1991; Leskosek et al., 1995; Zufall and Wackerbauer, 2000b). 4.2.2. Reverse osmosis In the reverse osmosis (RO) process, beer flows tangentially to the membrane surface and ethanol (and water) permeates the membrane selectively when the transmembrane pressure substantially

Fig. 5. Flow diagram of beer dealcoholization by dialysis: (1A) principle of hollow fiber dialysis, (1B) schematic representation of capillary membrane module, (2) heat exchanger, (3) stripper column, (4) original beer, (5) dealcoholized beer, (6) dialysate, (7) make-up brewing water, (8) glycol, (9) dialysate pump, (10) alcoholic condensate, (11) stripping steam.

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a b

Sample

Original

Dealcoholizeda

Original

Dealcoholizedb

Original gravity (wt.%) Ethanol (% ABV) Color (EBC) pH Bitterness (EBC) 1-Propanol (mg/l) 2-Methylpropanol (mg/l) 2-Methyl-1-butanol (mg/l) 3-Methyl-1-butanol (mg/l) Isoamyl alcohol (mg/l) Phenyl ethyl alcohol (mg/l) Total HA (mg/l) Ethyl acetate (mg/l) Isoamyl acetate (mg/l) 2-Phenyl ethyl acetate (mg/l) Total ES (mg/l) Iso-valeric acid (mg/l) Caproic acid (mg/l) Caprylic acid (mg/l) Capric acid (mg/l) Total FA (mg/l)

11.16 4.80 7.25 4.55 30.7 9.4 7.0 9.9 43.6 – – 69.9 12.1 2.2 <0.1 14.3 1.22 1.88 4.61 0.35 8.82

4.53 0.47 7.5 4.68 29.7 0.5 0.3 0.4 1.5 – – 2.7 <0.1 <0.1 <0.1 <0.1 0.49 1.02 2.55 0.21 4.27

10.83 4.92 – – 24.6 12.0 17.0 4.3 3.0 79.0 40.0 148.0 15.0 1.5 0.63 17.6 0.76 2.0 3.6 0.95 7.9

2.48 0.40 – – 12.3 2.0 5.1 2.8 10 17.0 3.7 27.9 1.8 0.16 0.04 2.0 0.18 0.22 0.35 0.11 0.9

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

exceeds the osmotic pressure of beer. It is expected that other large molecules, such as aroma and flavor compounds, will mostly remain at the retentate side of the membrane (Catarino et al., 2006). Reverse osmosis (RO) is usually carried out at transmembrane pressures ranging from 2 to 8 MPa generated by pressure pumps (e.g. piston pump) and at temperatures below 15 °C achievable with, for instance, a plate heat exchanger (Von Hodenberg, 1991; Catarino et al., 2007). The membranes used for the alcohol removal from beer by RO are usually of asymmetric structure, with the active layer made of cellulose acetate, polyamide, or polyimide on polyester, polysulphone, or fiberglass support structures. An ideal membrane features 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 geometric 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 removing permeates and not replacing it with demineralized water. This leads to an increase of the alcohol concentration and so does the flux of solute across the membrane increases, too. Subsequently during the diafiltration phase the permeate removed from beer is quantitatively replaced by demineralized water. This continues until a desired alcohol concentration is reached in beer. After the target alcohol content has been achieved, the retentate is made up with demineralized water to the starting volume of beer and the alcohol content is further lowered by this operation. The diafiltration water applied in RO has to be sterile, completely demineralized (conductivity < 50 lS) and deaerated (oxygen

content < 0.1 ppm). Carbonation of the product is necessary after RO (Von Hodenberg, 1991). Very few data are available on the composition of AFBs produced by RO. However, these report on significant losses of volatiles (70–80% of higher alcohols, 80–90% of esters) during the process (Table 2), which can be ascribed to imperfect selectivity of membranes (Kavanagh et al., 1991; Stein, 1993). Recently, several cellulose acetate and polyamide membranes have been tested in laboratory at different operation conditions (2–4 MPa transmembrane pressure, 5–20 °C, and different feed flow rates). It was found that higher transmembrane pressures resulted in higher permeate flux, higher rejection of ethanol and higher alcohols, but lower rejection of esters. Lower temperatures resulted in lower permeate flux but in higher rejection of aroma compounds (Catarino et al., 2007). Unfortunately, this study does not indicate the composition of beers dealcoholized by RO. 5. Production of alcohol-free beer by methods of restricted ethanol formation The methods of the alcohol-free beer (AFB) production based on limited alcohol formation can be divided according to the production equipment they require and further subdivided according to alterations in technology or use of special yeast (Fig. 1). The most exploited technologies are those requiring the equipment of a traditional brewery plant, while the continuous limited fermentation is a promising but marginal technology. The respective procedures applied 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 completely degrade starch to fermentable sugars and soluble dextrins. The spectrum of sugars formed depends on the actual enzyme activities present. b-Amylase (temperature optimum of 62–65 °C) produces the fermentable sugar of maltose, whereas a-amylase (temperature optimum of 72–75 °C) generates first non-fermentable sugars (dextrins) and at prolonged action also fermentable sugars (Kunze, 1996).

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The final content of fermentable sugars in wort then determines the alcohol level in beer. Therefore, by changing the mashing process, it is possible to modulate the profile of wort sugars in a way that their fermentability is limited and results in low alcohol content. A low wort sugar content can be achieved by different strategies as follows:  Inactivation of saccharifying b-amylase by high temperature mashing (75–80 °C). Under these conditions b-amylase is rapidly inactivated but sufficient a-amylase remains to digest and liquefy the starch. This procedure results in ca. 85% extraction of malt and a wort fermentable to 25%. The final ethanol content is influenced also by the original wort gravity, attenuation achieved during fermentation, and final dilution. According to the literature the flavor of these beers is very good, however, some problems with worty flavor have been reported. Concerns regarding colloidal stability of the product are also relevant (Muller, 1990, 1991, 2000):  Cold water malt extraction. It is based on extracting the maximum malt flavor compounds without increasing the wort gravity. The malt is extracted with water at temperatures insufficient (<60 °C) for starch gelatinization and subsequent enzymatic hydrolysis. The obtained wort contains some fermentable sugars, which result from barley modification during malting. In practice, difficulties with lautering could be expected while the remaining malt could be used in normal mashing. No information is available on the current use of this method (Muller, 1990).  Re-mashing of spent grains to produce a second extract with very little fermentable sugar. Two modifications of the original method are known: (i) extrusion cooking of the spent grains prior to the second extraction and (ii) acidic hydrolysis of spent grain to yield a secondary wort with a significant content of non-fermentable pentoses. The main advantage of these methods is that two beers can be produced from one dose of grain. Although it is not apparent that anyone is using this method, both difficulties with flavor and color formation were hypothesized and an increased investment costs, concerning the hydrolytic apparatus, would be inevitable (Muller, 1990; Zurcher and Gruss, 1991).  Barley varieties with wide variations of b-amylase thermostability as well as b-amylase deficient varieties have been reported (Kihara et al., 1998; Kihara et al., 1999). Although, it can be hypothesized that both thermolability and/or lack of b-amylase in special barley varieties could be advantageous to achieve low wort fermentability, no information on any research or industrial implementation of these barleys have been found so far. Used on their own, methods relying solely on the modified mashing are seldom successful for the production of AFBs and they

have to be combined with further measures such as vigorous wort boiling (lowering the level of aldehydes), wort acidification, limited fermentation, color and bitterness adjustment, etc. 5.2. Arrested or limited fermentation process In general, the major disadvantage of both stopped and limited fermentation approach is that it is hardly feasible to achieve low alcohol levels with adequate conversion of wort to beer. Therefore, the objective of these methods is keeping the ethanol content low by removal of yeast before excessive attenuation (stopped fermentation) or creating conditions for restrained yeast metabolism (limited fermentation) and simultaneously reducing the worty flavor impression or limit it from the beginning (Muller, 1990). These production methods operate with traditional brewery equipment and unit operations, but they require accurate and swift analytical control (Perpète and Collin, 1999a). These approaches represent the most usual way to produce alcohol-free or low-alcohol beers. When these techniques were carried out with worts of original gravity from 9 to 13 wt.% the smell and taste of AFBs was characterized by a strong worty flavor impression due to the non-reduced wort aldehydes. It has been verified that for stopped or limited fermentation processes an original gravity from 4.0 to 7.5 wt.% is desirable. However, brewing at high gravity (20 wt.%) increases the formation of higher alcohols and esters. This phenomenon can be exploited to strengthen the flavor of reduced alcohol beers obtained after dilution of a higher gravity beer. Further adjustments of volatiles can be achieved by higher fermentation temperature (greater effects on lager yeasts) or reduced oxygen content of wort, which increased dramatically the ester formation by ale yeasts (Muller et al., 1991). The fermentation activity can be arrested (stopped or checked) quickly by temperature inactivation (rapid cooling to 0 °C, pasteurization) and/or by removal of yeast from fermenting wort (filtration, centrifugation). The fermentation initial phase can be carried out at a relatively wide range of temperatures without a significant impact on the formation of volatiles and the reduction of aldehydes (Table 3). However, at higher temperatures the fermentation has to be arrested, either by yeast separation or cooling, rather shortly after the wort was pitched, which requires a prompt analytical control and intervention (Attenborough, 1988; Narziss et al., 1992). The increasing fermentation temperature deepened the attenuation and simultaneously enhanced the formation of volatiles and diacetyl by bottom fermenting yeast. A comparison of yeast types showed that the top fermenting yeast achieved a significantly 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 content less than 0.5 vol.%, the AFB is usually matured for at least 10 days

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

*

Fermentation yeast

Bottom

Bottom

Bottom

Bottom

Top

Top

Temperature (°C) Original gravity (wt.%) Ethanol (wt.%) Real extract (wt.%) Attenuation (wt.%) Fermentation time (h) pH Bitterness (EBC) DMS (lg/l) Total diacetyl (mg/l) Total HAA (mg/l) Total EAA (mg/l) Reduction of aldehydes (%)

0 11.4 0.27 10.64 8 48 5.01 25.4 30 0.06 2.2 0.55 85.8

4 7.5 0.37 6.79 12 24 4.87 17.5 22 0.09 6.8 0.69 77.6

8 7.5 0.37 6.79 12 7/24* 4.92 17.7 30 0.08 6.7 0.60 80.2

12 7.5 0.42 6.52 16 7/24* 4.89 17.4 32 0.14 8.6 0.84 83.0

8 7.4 0.32 6.87 9 24 4.87 17.7 35 0.51 15.8 0.90 88.2

12 7.4 0.27 6.94 8 7/24* 4.89 17.8 45 0.35 14.2 0.90 77.5

After 7 h at the initial temperature the wort was cooled to 0 °C until 24 h were completed, source: Narziss et al. (1992).

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at 0 to 1 °C to avoid an overpowering sulfur flavor. Then the AFB is filtered, carbonated, stabilized, and sterilized. The most practical tool to suppress yeast metabolism is low temperature. The so-called ‘‘cold contact process’’ (CCP) is taking advantage of the fact that under these conditions the ethanol production is slow, but other biochemical processes, such as the formation of higher alcohols and esters and the reduction of carbonyls, may exhibit moderate activities. In an example of the CCP the alcohol-free beer is produced from wort (6 wt.%) cooled to 0–1 °C, acidified with lactic acid to pH 4, pitched with the yeast cell 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 pitching yeast slurry has a significant ethanol content (6 vol.%). In comparison with other methods of AFB production, the CCP was characterized by one of the highest volatile production and lowest aldehyde reduction capacity (Perpète and Collin, 1999a). Several carbonyl compounds present in wort are known to contribute to the worty off-flavor of AFBs produced by the CCP. Among them the branched aldehydes (3-methylbutanal, 2-methylbutanal, and 3-methylpropionaldehyde) with very low odor threshold values are less readily reduced both enzymatically and chemically. The total removal of branched Strecker aldehydes is under CCP conditions limited to approximately 65% of their initial concentration, remaining thus enough of them in AFBs to impair their sensorial profile. Therefore, there were two strategies suggested to improve the CCP, one being the use of genetically modified yeast at higher temperature (high temperature enhance chemical binding of aldehydes) or decreasing the wort polyphenol level (aldehydes bound to polyphenols resist enzymatic reduction) by using the polyvinylpolypyrrolidone (PVPP) just after wort cooling (Perpète and Collin, 1999b, 2000a). When using arrested or limited fermentation it is necessary to consider, besides fermentation temperature, wort gravity, and yeast type, also additional interventions into the production process in order to improve the products flavor characteristics:  The addition of dark (20%) or pale caramel malt (15%), compared to the brew with 100% pale malt, contributed positively to taste characteristics by masking the worty flavor impression with more beer flavor substances. In particular, pale caramel malt contributed the highest amount of substances resulting from Maillard reactions (e.g. furfural, 2-acetylpyrole) (Narziss et al., 1992).  Wort dilution after boiling (from 11.5 to 7.5 wt.% original gravity), instead of dilution before wort boiling, resulted in lower bitterness, increased ester and higher alcohol levels and the AFBs 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 impression. Therefore the effect of wort acidification with acid malt (5%) or lactic acid (10 min before the end of boiling to a pH of 4.3) was tested. The results showed that the acidification had a very favorable effect in suppressing the worty character of AFBs (Narziss et al., 1992).  The unpleasant bitter aftertaste of AFBs, probably due to oxidized malt substances, can be eliminated by adding ascorbic acid to the wort (approximately 20 mg/l to 7 wt.% wort) as long as it remains hot (Schur and Sauer, 1990).  Wort can be ’’hot‘‘ (Lommi et al., 1990) or ‘‘cold stripped’’ (Montanari et al., 2009), with sparging CO2 or N2 into the liquid, to wash out 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 vented through 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 to a low-alcohol beer (Barrell, 1979; Muller et al., 1991). However, the risk of contamination of the low gravity beer with undesirable 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 use of special yeast performing a limited fermentation process. The dissimilarity of these ‘‘special’’ yeasts compared to traditional brewing yeast lies mainly in their tendency to produce lower amounts of ethanol or no ethanol at all. This can be achieved by strategies such as selection of a proper microbial genus (strain) with specific properties or intentional modification of brewing yeast by random mutation or genetic engineering. The most common approach relies on the fact that the major fermentable sugar of all malt worts is maltose (ca. 75%) and some strains of the genus Saccharomyces (e.g. used in the wine fermentation) are unable to ferment this sugar. Thus the beer resultant from conversion of glucose, fructose, and sucrose will contain less than < 0.5% ethanol (Muller, 1993). Except the application of a special yeast strain this method of the AFB production is identical with the manufacturing of standard beer. However, due to limited yeast activity and high residual extract content this manufacturing process is vulnerable to microbial contamination. Therefore high standards of cleanliness and microbiological control are required (Muller, 1990). The most successful genus, other that Saccharomyces, used for the industrial production of alcohol-free beer is Saccharomycodes ludwigii. The controlled fermentation can be carried out by this yeast thanks to its disability to ferment maltose and maltotriose, the prevailing fermentable sugars of all malt worts. Although according to some authors the beer fermented by S. ludwigii tends to be sweet due to its high residual maltose and maltotriose content, the relative sweetness of these sugars is significantly lower than that of sucrose and glucose (Attenborough, 1988). The fermentation with S. ludwigii is characterized by slow attenuation even at 20 °C which implies that the process does not require continuous monitoring. A comparison of traditional bottom fermenting yeast with S. ludwigii with/without wort acidification showed a significantly higher formation of sensorially active by-products (higher alcohols and esters) by special yeast (Table 4). These volatiles, together with wort acidification, were found to mask the typical worty flavor of AFBs and contributed positively to fullness and pleasant liveliness of AFBs. However, in spite of the high volatile content there was a remaining slight worty off-flavor, which can be ascribed to lower aldehyde reduction by S. ludwigii. The AFBs Table 4 The influence of yeast type and wort acidification on the composition of alcohol-free beers.

*

Fermentation yeast

Bottom

S. ludwigii

S. ludwigii + acidification

Temperature (°C) Original gravity (wt.%) Ethanol (wt.%) Real extract (wt.%) Attenuation (wt.%) Fermentation time (h) pH Bitter substances (EBC) Total diacetyl (mg/l) Total HAA (mg/l) Total EAA (mg/l) Reduction of aldehydes (%)

0 11.5 0.3 10.7 9 48 5.15 28.0 0.04 3.0 0.79 81.0

20 11.5 0.68* 10.34 13 120 4.98 27.2 0.14 31.8 1.88 56.8

20 11.5 0.68* 10.34 13 120 4.18 22.2 0.13 30.3 2.31 32.6

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

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produced with S. ludwigii contained diacetyl slightly above the taste threshold level, which was picked up in the tasting (Narziss et al., 1992). Another invention suggests leading steam saturated air through alcoholic beer in a sieve bottom column in order to desorb the alcohol (Dziondziak and Seiffert, 1995). The involved loss of sensorially active compounds by desorption was suggested to be compensated either by addition of a fermenting wort or by using the species Saccharomyces rouxii able to consume ethanol under aerobic conditions and at the same time to produce flavor active substances (Dziondziak, 1989a). However, the author does not suggest how to deal with the possible negative effect of oxygen from stripping air on flavor and colloidal stability of produced AFB. A method for producing an alcohol-free beer-like fermented beverage employing a slow fermentation process by fungi from the genus Monascus has been proposed, too. According to the authors the final product looks like beer, has a refreshing taste, glittering red color, low alcohol content, and has high anti-oxidation activity (Lin et al., 2005). However, it is questionable whether this beverage can be considered beer. Hand in hand with the growing consumption of AFBs increases the need for yeast that would fit the special requirements of their production. Random mutagenesis by ultraviolet irradiation (the most convenient for food applications) followed by selection of proper mutants has been applied for isolation of non-recombinant yeast strains with defects in citric acid cycle. The most acceptable AFBs were obtained after the fermentation with Saccharomyces cerevisiae mutants lacking 2-ketoglutarate dehydrogenase (KGD) and fumarase (FUM) activity. These strains were studied in batch and continuous fermentations, both immobilized and free. In all studied fermentation arrangements the AFBs produced by these two strains were characterized by a low ethanol (up to 0.21% 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 esters (18–36 mg/L) were somewhat lower and higher compared to a reference beer (alcoholic), respectively. The organic acids produced, especially lactic acid, had a strong protective effect on the microbial stability of the final product and thus the usual addition of lactic acid could be omitted (Narvátil et al., 2002). The presented parameters meet the criteria for AFBs; however, these results were obtained with haploid or diploid laboratory strains. Since the brewing yeast, which possess industrially important and stable properties (fermentation rate, flavor formation, flocculation) are alloploids, it makes the approach of random mutagenesis less effective, in particular face to face the DNA repair mechanisms of yeast (Petin et al., 2001; Brendel et al., 2003; Aylon and Kupiec, 2004). Yeast strains with intentional gene deletions in citric acid cycle have 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 obtained with DKGD1, DKGD2 and DFUM1 strains. Thus it is no surprise that the composition of AFBs produced by yeast with a gene deletion (Selecky´ et al., 2008) was close to those produced by random mutation (Narvátil et al., 2002). Since the gene deletions were carried out only on diploid strains, the preparation of a hybrid between a brewing yeast and a laboratory strain, carrying all the genetic properties responsible for all the industrially important properties (taste and flavor formation, flocculation), and simultaneously deficient in the citric acid cycle enzyme genes, would be beneficial. Another example of the gene deletion strategy is the use of alcohol dehydrogenase-free (ADH) non-revertible mutant of S. cerevisiae to produce AFB having 0.3–2.0 vol.% glycerol content, which is reported to improve the body of the beer. The excessive accumulation 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 gassing on the content of beer volatiles (higher alcohols, esters) is not discussed in the patent (Dziondziak, 1989b). The beneficial effect of lacking ADH activity was demonstrated on haploid S. cerevisiae showing a double phenotype: low ethanol production and enhanced worty aldehyde reduction (Evellin et al., 1999). However, the elimination of each ADH locus in a polyploidy brewer’s yeast has not been published. Conversely to the gene deletion strategy, the overexpression of glycerol-3-phosphate dehydrogenase gene was performed in an industrial lager brewing yeast (Saccharomyces pastorianus) to reduce the ethanol content in beer. The results were not fully satisfactory since this transformation led to 5.6 times increased glycerol production and the ethanol production decreased only by 18% when compared to the wild-type. Although only minor changes in the concentration of higher alcohols, esters, and fatty acids could be observed, concentrations of several other by-products, particularly acetoin, 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 possible the future construction of strains tailor-made for the AFB production. However, as long as both legal obstacles and particularly the consumers’ negative attitude towards the use of genetically modified yeast persist, the breweries will not risk their industrial application. 5.4. Continuous fermentation Investigation on the continuous culture of free and immobilized yeast for the beer production has been motivated by the advantages comprising of lower capital, production, and manpower costs. Several reviews have been written recently on the state-ofthe-art of continuous beer fermentation systems and the flavor particularities of the continuously fermented and/or maturated beers (Brányik et al., 2005, 2008; Willaert and Nedovic, 2006). The potential advantages arise mostly out of the accelerated transformation of wort into beer driven by an increased biomass concentration (van Iersel et al., 1998). This is achieved by immobilization of biomass based on physical confinement of yeast inside a bioreactor. Various carrier types have been used for the beer fermentation by immobilized brewing yeast. Among them, inert carrier types with adsorption as the prevailing immobilization mechanism (DEAE-cellulose, wood chips, spent grains) have shown to be technically useful and economically affordable. For each cell immobilization technique a variety of reactor types can be selected and a careful matching of immobilization method, reactor configuration and process characteristics is important for a successful industrial implementation (Verbelen et al., 2006). Since the continuous alcohol-free beer (AFB) production is based on the limited fermentation strategy, the corresponding fermentation systems generally consists of one stage bioreactors equipped with additional apparatus for a continuous wort supply and process control. Although the continuous beer fermentation has been studied for several decades, the number of industrial applications is still limited. The major obstacle hindering the industrial exploitation of this technology is the difficulty in achieving the correct balance of sensory compounds in the final product (Pilkington et al., 1998; Brányik et al., 2008). Given the shifts in metabolism of cells grown in continuous culture, it has proven difficult to ‘‘translate’’ the traditional batch process into a continuous and immobilized process. The production of AFBs using immobilized yeast cell systems rank among limited fermentation methods using short contact (1–12 h residence time) between immobilized yeasts and wort (Van De Winkel 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 fermentation can outperform the rival technologies in productivity; however, it is essential that it produces a final product competitive in terms of sensorial quality. Alcohol-free beers are usually characterized by worty offflavors and lack of pleasant fruity (estery) aroma found in regular beers. Although the formation of higher alcohols and esters during continuous AFB production has already been studied, very few papers comparing flavor formation in the traditional batch fermentation and the continuous one are available. One reported a significantly lower formation of higher alcohol and acetic acid esters (Aivasidis et al., 1991), while more recent papers concluded the importance of process parameters and yeast strains for the formation of volatiles (Lehnert et al., 2008, 2009). Among process parameters it is aeration, which has perhaps the most important impact on the formation of volatiles in continuous systems (Virkajärvi et al., 1999). An optimal and constant flavor profile of the AFB can 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 fermented model medium under optimized oxygen supply was comparable with those found in three commercial alcohol-free beers (Lehnert et al., 2008). The interplay between the appropriate production strain, carrier material, and bioreactor design is very important in continuous immobilized cell reactors and their suitable combination can improve both the system performance and product quality. The importance of careful matching of the chosen yeast strain with an immobilization method and a suitable reactor arrangement was demonstrated. It was shown that the laboratory yeast strain with disruption in the KGD2 (2-ketoglutarate dehydrogenase) gene performed, in terms of the flavor formation, equally well in the batch and continuous packed-bed reactor. However, it was unable to form a biofilm around spent grain particles and therefore its use was not possible in the gas-lift reactor. Conversely, the bottom fermenting strain W96 adhered to the solid supports readily, but the formation of flavor active compounds was insufficient with the exception of the immobilization onto spent grains in the gas-lift reactor. This system arrangement proved that even a strain, which seems to be less suitable for the AFB production by the arrested fermentation, can under appropriate conditions produce an acceptable final product (Mota et al., 2011). Several studies have been carried out on the alcohol-free beer production by the limited fermentation with immobilized cells of S. cerevisiae at low temperature (0–4 °C) and nearly anaerobic conditions (Lommi et al., 1990; Aivasidis et al., 1991; van Iersel et al., 1995). Similarly to the cold contact process (CCP) these conditions lead to the suppressed cell growth, low ethanol formation, and stimulated production of higher alcohols and esters. The authors hypothesized that the increased production of volatiles can be ascribed to the effort of cells to maintain the redox balance under anaerobic conditions by reoxidation of NADH coupled with the reduction of carbonyl compounds to higher alcohols (van Iersel et al., 1995). However, some oxygen is essential for several yeast biosynthetic pathways (Snoek and Steensma, 2007) and thus for the long-term production of AFB with balanced flavor (van Iersel et al., 1999). Wort carbonyls were proposed to contribute to the unpleasant worty taste of AFBs (Perpète and Collin, 1999c). Although the reduction of wort aldehydes by yeast is relatively fast during batch fermentations, there was concern it may not be sufficient at the speed of the continuous AFB production. However, the carbonyl reducing capacity of continuous immobilized cells systems for the AFB production has been reported to be satisfactory (Debourg et al., 1995; van Iersel et al., 2000; Lehnert et al., 2008). This can be ascribed to either an increased alcohol dehydrogenase activity in

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immobilized yeast (van Iersel et al., 2000) and/or a wise compromise between the alcohol formation and the carbonyl reduction by optimizing the process parameters (biomass load, residence time, 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 pH drop during the continuous AFB production does not take place in the required extent, a continuous biological acidification for the direct adjustment of pH of mash and wort by immobilized lactic acid bacteria (DEAE cellulose) has been studied during test periods of a few months. The continuous acidification would suitably match with the subsequent continuous fermentation (Pittner and Back, 1995). Several industrial examples of testing (Aivasidis et al., 1991) and implementation of the continuous AFB production have been reported (Van Dieren, 1995; Mensour et al., 1997). However, information on the current industrial application of the continuous limited fermentation of AFBs is not available to the authors. It can be assumed that the continuous fermentation systems have not found widespread utilization in the AFB production mainly due to the need of special equipment (bioreactor and tools for its continuous feed and control), eventually additional methods (immobilization) and materials (carrier).

6. Sensorial properties and additional improvements of alcoholfree beer The aroma and taste of an AFB is usually rather different from its fully fermented counterpart. The AFBs often suffer from various flavor imperfections. For instance, the AFBs produced by the membrane processes have usually less body and a low aromatic profile, the thermally dealcoholized AFBs may suffer heat damages, while the beers obtained by biological methods have often a sweet and worty off-flavor (Montanari et al., 2009). It has been proved that ethanol significantly increases aldehyde retention, leading to lower perception of the worty character. In a usual 5% ABV beer the retention of aldehydes was 32–39% in comparison to 8–12% retention at 0.5% ABV. Similarly, higher levels of mono- and disaccharides in AFBs intensify such off-favors. Headspace extraction and sensorial analysis further showed that the aldehyde retention in AFBs can be enhanced by increasing the level of dextrins or glycerol (Perpète and Collin, 2000b). These findings suggest that the flavor perception of a regular beer cannot be mimicked simply by trying to get the volatile distribution in AFBs as close as possible to that one in a regular beer. Instead, promising results can be achieved by changing the degree of volatile retention in AFBs and/or by creating a balance of volatiles, different from that present in beers containing ethanol, but with similar flavor impression. Particular flavor balance can be produced by process adjustments as 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 comparison of beer properties before and after dealcoholization. These are summarized in Table 5 and presented as a percentage change of selected properties. It can be seen that the thermal processes tend to increase, while the membrane processes decrease the color of the AFB. The bitterness and foam stability were usually impaired by all dealcoholization processes. However, the most significant impact of the alcohol removal was observed on the loss of volatiles. All the technologies led to significant losses of volatiles, the smallest being observed in the case of the membrane processes. In the case of the arrested fermentation the difference in the volatile content was calculated for an average German AFB and a fullfermented pale beer as found in Narziss et al., 1992. The absence of volatiles in an AFB produced by the arrested fermentation is

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

a b c d e f

Difference (%)

VRa

Color (EBC) Bitterness (EBC) Foam (NIBEM) Esters Higher alcohols

+13 +2 – 99 78

VRb – – – 100 78

FFc 0 7 3 95 98

FFd +10 8 100 95

Dc 6 12 1 85 85

ROc

ROe

3 7 8 78 69

– 50 – 87 81

AFf – – – 87 80

Narziss et al. (1993). Zürcher et al. (2005). Stein (1993). Zufall and Wackerbauer (2000a). Kavanagh et al. (1991). Narziss et al. (1992).

comparable with that in AFBs produced by the alcohol removal (Table 5). The flavor imperfections of AFBs raised the need to correct them. Some strategies of the flavor improvement were discussed together with the technologies, while here are mentioned some additional possibilities.

a sweetener with an unpleasant bitter or metallic aftertaste at higher concentrations, is motivated by the desire to strengthen the body of the AFB. Besides these frequently used additives some producers indicate also the use of citric acid (acidity regulator, E330), potassium metabisulphite (preservative, E224), caramel (coloring, E150), and glucose-fructose syrup. Moreover, the addition of dextrins into beer has been reported to improve the flavor profile of LABs and AFBs through their action on the retention and/or perception of flavor active compounds (Louant and Dufour, 1991). However, an anonymous evaluation of Czech AFBs by a panel of 35 tasters (brew masters, brewing engineers and researchers) showed that the additives solely cannot substitute the use of high quality raw materials and an optimized production process. The distribution of beers with different additives and their combinations across the final ranking of AFBs was random and no tendency of improved evaluation based on the use of additives was found (Vecˇerková, 2010). It was found that the popularity of semi-dark AFBs (dark and caramel malts added) is on the rise (2nd and 3rd place in the contest), but at the same time the worst contestant was also a semi-dark AFB underlining the fact that solely the addition of special malts cannot improve the products sensorial quality.

6.1. Post-treatments and blending Both the thermal and membrane processes often use different post-treatment and blending techniques in order to improve the sensorial quality and colloidal stability of dealcoholized beers. Improvements can be achieved by the addition of fresh yeast followed by maturation or by blending with the original beer (Schedl et al., 1988; Moreira da Silva and De Wit, 2008), aromatic beer (beer fermented at elevated temperatures), or krausen (Narziss et al., 1993). Another possibility is to adopt the Barrell patent (Barrell, 1979) to gently dealcoholize beer by treating it with CO2 from fermenting green beer and finally add krausen followed by maturation and filtration process (Zürcher et al., 2005). The addition of 6 vol.% of krausen into a beer dealcoholized to 0.1 ABV returned about 15% and 31% of the higher alcohols and esters originally present in the alcoholic beer (Table 1), respectively (Narziss et al., 1993). Other studies have shown that the thresholds of the important aroma components in the AFBs are significantly lower than in the alcohol-containing beer. This means that even a partial replacement of aroma compounds by one of the above suggested methods can improve significantly the AFB flavor (Zufall and Wackerbauer, 2000a). 6.2. Additives The use of additives will be explained on the example of Czech alcohol-free beers. Currently there are 30 AFB brands commercialized in the Czech Republic (AFBs represented 2.92% of the Czech beer market in 2008). Among them 26 brands are produced by the arrested/limited fermentation (at least one uses also a changed mashing process), two are fermented with special yeast and one is produced by vacuum rectification. According to information on the labels, 10 brands from the whole group of AFBs are produced only using traditional brewing raw materials, 9 contain one additive, 9 contain two additives, and 2 contain three or more additives. The most frequently used additives are: saccharin (sweetener E954, 11 AFBs), ascorbic acid (antioxidant E300, 9 AFBs), lactic acid (preservative E270, 8 AFBs). In the case of AFBs produced by the arrested/limited fermentation, the use of lactic acid (preservative with antimicrobial and flavor effects) and ascorbic acid (antioxidant increasing flavor and colloidal stabilities) is justified. Besides the addition of lactic acid during the production process a biological acidification of wort with lactobacillary strains was tested as well (Narziss et al., 1991). The widespread addition of saccharin,

7. Cost evaluation and conclusions The available literature is poor in comparisons of processes and their impact on the product quality, but the comparison of economic aspects of processes producing LABs of AFBs is even scarcer. Nevertheless, it is clear that the arrested/limited fermentation process can be performed in a common brewery equipment but shorter production time and less raw materials are needed. Therefore, the production costs for such LAB/AFBs are the same or lower, than for the regular beer. Conversely, processes of the alcohol removal do require an extra equipment, relevant utilities and space, which mean additional investments and operating costs above the production costs of the regular beer to be dealcoholized. The advantage of alcohol separation processes is their flexibility (start-up within hours, high productivity) and possibility to produce zero alcohol beers, which is hardly achievable by fermentative processes, given by their nature. Some authors also state that the taste of the dealcoholized AFBs is dryer and closer to regular beer (Basarˇová et al., 2010). Somewhat contradictory to this is the fact the only Czech AFB produced by vacuum rectification was ranked 21st among 30 samples by the taste panel (Vecˇerková, 2010). Additional profit can be created also from the separated alcohol, which is obtained at different concentrations. The diluted alcohol solution can be further concentrated to a marketable content, used in the brewing process as blending water or sold for acetification to produce vinegar (Regan, 1990; Stein, 1993). From one rare cost comparison 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 reliable and comprehensive economic comparison of various methods of the LAB/AFB production is not available and therefore it is impossible to define the best process. Moreover, choosing the most appropriate process is further influenced by the available production capacity, expected sales, and marketing strategy of the product and hence it requires a detailed balance sheet reflecting the existing technology and the specifics of the local market. Acknowledgements The authors thank to the Ministry of Education, Youth and Sports 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 Innovation Foundation (FAPITEC/SE, Brazil) for their financial support. References Aivasidis, A., Wandrey, Ch., Ellis, H.G., Katzke, M., 1991. Continuous fermentation of alcohol-free beer with immobilized yeast in fluidized bed reactors. In: Proceedings of the European Brewing Convention Congress, Lisbon, Portugal. Anglerot, D., 1994. Process of making alcohol-free beer and beer aroma concentrates. US Patent 5308,631. API Schmidt-Bretten, 2004. Sigmatec Dealcoholization System, Nr. 372e, 10/2004. (downloaded 20.07. 10). Attenborough, W.M., 1988. Evaluation of processes for the production of low- and non-alcohol beer. Ferment 1 (2), 40–44. Aylon, Y., Kupiec, M., 2004. DSB repair: the yeast paradigm. DNA Repair 3 (8–9), 797–815. Badcock, R.J., Benwell, R.A., Gray, C., 1994. An evaluation of spinning cone column technology for low alcohol beer production. In: Proceedings of the 23rd Convention – Institute of Brewing (Asia Pacific Section), Sydney, Australia. Bamforth, C.W., 2002. Nutritional aspects of beer – a review. Nutrition Research 22 (1/2), 227–237. Barrell, G.W., 1979. Process for the production of fermented beverages. G.B. Patent 2033,424. Basarˇová, G., Šavel, J., Basarˇ, P., Lejsek, T., 2010. Brewing: Theory and Practice of Beer Production (written in Czech), first ed. Vydavatelsví VŠCHT Praha, Prague, pp. 571–572. Brányik, T., Vicente, A.A., Dostálek, P., Teixeira, J.A., 2005. Continuous beer fermentation using immobilized yeast cell bioreactor systems. Biotechnology Progress 21 (3), 653–663. Brányik, T., Vicente, A.A., Dostálek, P., Teixeira, J.A., 2008. A review of flavour formation in continuous beer fermentations. Journal of the Institute of Brewing 114, 3–13. Brendel, M., Bonatto, D., Strauss, M., Revers, L.F., Pungartnik, C., Saffi, J., Henriques, J.A.P., 2003. Role of PSO genes in repair of DNA damage of Saccharomyces cerevisiae. Mutation Research, Reviews in Mutation Research 544 (2–3), 179– 193. Catarino, M., Mendes, A., Madeira, L.M., Ferreira, A., 2006. Beer dealcoholization by reverse osmosis. Desalination 200 (1–3), 397–399. Catarino, M., Mendes, A., Madeira, L.M., Ferreira, A., 2007. Alcohol removal from beer by reverse osmosis. Separation Science and Technology 42, 3011–3027. Craig, A.J.M., 1986. Gas–liquid contacting device. European Patent 0 191 625 A2. Daenen, L., Saison, D., De Schutter, D.P., De Cooman, L., Verstrepen, K.J., Delvaux, F.R., Derdelinckx, G., Verachtert, H., 2009. Bioflavoring of beer through fermentation, refermentation and plant parts addition. In: Preedy, V.R. (Ed.), Beer in Health and Disease Prevention. Elsevier Inc., Burlington, MA, pp. e33– e49. Debourg, A., Laurent, M., Goossens, E., Borremans, E., Van de Winkel, L., Masschelein, C.A., 1994. Wort aldehyde reduction potential in free and immobilized yeast systems. Journal of the American Society of Brewing Chemists 52, 100–106. Debourg, A., Laurent, M., Verlinden, V., Van Winkel, L., Masschelein, C.A., Van Nedervelde, L., 1995. Yeast physiological state on carbonyl reduction. In: European Brewery Convention Monograph XXIV, EBC Symposium Immobilized Yeast Application in the Brewing Industry, Espoo, Finland. Donhauser, S., Glas, K., Mueller, O., 1991. Behavior of beer components during the manufacture of alcohol-reduced products by dialysis. Brauwelt International 2, 139–144. Dziondziak, K., 1989a. Method for the production of alcohol-free beer. US Patent 4882,177. Dziondziak, K., 1989b. Method for the production of low alcohol or alcohol-free beer. US Patent 4814,188. Dziondziak, K., Seiffert, T., 1995. Process for the continuous production of alcoholfree beer. In: Proceedings of the European Brewing Convention Congress, Brussels, Belgium. Etuk, B.R., Murray, K.R., 1990. Mechanism for the removal of alcohol from beer by emulsion liquid membranes. In: IChemE, Symposium Series, vol. 119, pp. 279– 298. Evellin, F., Perpète, P., Collin, S., 1999. Yeast ADHI disruption: a way to promote carbonyl compounds reduction in alcohol-free beer production. Journal of the American Society of Brewing Chemists 57 (3), 109–113. Ferreira, M.P., Willoughby, D., 2008. Alcohol consumption: the good, the bad, and the indifferent. Applied Physiology, Nutrition, and Metabolism 33 (1), 12–20. Gresch, W., 1991. Process for the production of dealcoholized beverages, as well as a unit and device for performing the process. US Patent 5014,612. Heymann, H., Goldberg, J.R., Wallin, C.E., Bamforth, C.W., 2010. A ‘‘beer’’ made from a bland alcohol base. Journal of the American Society of Brewing Chemists 68 (2), 75–76. Hochberg, U., 1986. Beer dealcoholization by evaporation (written in German). Brauindustrie 71 (8), pp. 480-2, 484-5. Informe socioeconómico del sector de la cerveza en España, 2010 (written in Spanish). (downloaded 12.9.11). Kavanagh, T.E., Clarke, B.J., Gee, P.S., Miles, M., Nicholson, B.N., 1991. Volatile flavor compounds in low alcohol beers. Technical Quarterly – Master Brewers Association of the Americas 28 (3), 111–118.

505

Kern, M., 1994. Alcohol removal from beer using continuous vacuum refinement (written in Spanish). Alimentacion Equipos y Tecnologia 13 (5), 37–41. Kihara, M., Kaneko, T., Ito, K., 1998. Genetic variation of b-amylase thermostability among varieties of barley, Hordeum vulgare, and relation to malting quality. Plant Breeding 117 (5), 425–428. Kihara, M., Kaneko, T., Ito, K., Ajda, Y., Takeda, K., 1999. Geographical variation of b-amylase thermostability among varieties of barley (Hordeum vulgare) and b-amylase deficiency. Plant Breeding 118 (5), 453–455. Koerner, R., 1996. Returning aroma compounds removed during thermal dealcoholization of beverages (written in Spanish). European Patent 0 713 911 A2. Kunze, W., 1996. Technology Brewing and Malting, Int ed. VLB Berlin, Berlin, pp. 190–224. Lehnert, R., Kurˇec, M., Brányik, T., Teixeira, J.A., 2008. Effect of oxygen supply on flavor formation during continuous alcohol-free beer production: a model study. Journal of the American Society of Brewing Chemists 66 (4), 233–238. Lehnert, R., Novak, P., Macieira, F., Kurec, M., Teixeira, J.A., Brányik, T., 2009. Optimisation of lab-scale continuous alcohol-free beer production. Czech Journal of Food Sciences 27 (4), 267–275. Leskosek, I.J., Mitrovic, M., 1994. Optimization of beer dialysis with cuprophane membranes. Journal of the Institute of Brewing 100 (4), 287–292. Leskosek, I., Mitrovic, M., Nedovic, V., 1995. Factors influencing alcohol and extract separation in beer dialysis. World Journal of Microbiology & Biotechnology 11 (5), 512–514. Li, J.M., Mukamal, K.J., 2004. An update on alcohol and atherosclerosis. Current Opinion in Lipidology 15 (6), 673–680. Light, W.G., Mooney, L.A., Chu, H.C., Wood, S.K., 1986. Alcohol removal from beer by reverse osmosis. AIChE Symposium Series 82, 1–8. Lin, T.F., Chen, Y.L., Wang, T.H., 2005. Method for producing beer-like and alcoholfree fermented beverage. UK Patent Application GB 2414486 A. Lommi, H., Swinkels, W., Van Dieren, B., 1990. A process for the production of nonalcoholic malt beverage. European Patent 0 424 794 A2. Louant, G., Dufour, J.-P. 1991. Effect of dextrins composition on flavor characteristics. In: Proceedings of the European Brewing Convention Congress, Lisbon, Portugal. Magalhaes Mendes, A.M., Palma Madeira, L.M., Dias Catarino, M., 2008. Process for enriching the aroma profile of a dealcoholized beverage. PCT International Patent Application WO, 2008/099325 A2. Magarifuchi, T., Goto, K., Iimura, Y., Tadenuma, M., Tamura, G., 1995. Effect of yeast fumarase gene (FUM1) disruption on production of malic, fumaric and succinic acids in sake mash. Journal of Fermentation and Bioengineering 80 (4), 355– 361. Matson, S.L., 1987. Production of low-ethanol beverages by membrane extraction. PCT International Patent Application WO 87/02380. Mehta, G.D., 1982. Comparison of membrane processes with distillation for alcohol/ water separation. Journal of Membrane Science 12, 1–26. Mensour, N.A., Margaritis, A., Briens, C.L., Pilkington, H., Russell, I., 1997. New developments in the brewing industry using immobilised yeast cell bioreactor systems. Journal of the Institute of Brewing 103, 363–370. Meussdoerffer, F.G., 2009. A comprehensive history of beer brewing. In: Esslinger, H.M. (Ed.), Handbook of Brewing. Wiley-VCH Verlag GmbH & Co., Weinheim, pp. 1–42. Montanari, L., Marconi, O., Mayer, H., Fantozzi, P., 2009. Production of alcohol-free beer. In: Preedy, V.R. (Ed.), Beer in Health and Disease Prevention. Elsevier Inc., Burlington, MA, pp. 61–75. Moonen, H., Niefind, H.J., 1982. Alcohol reduction in beer by means of dialysis. Desalination 41 (3), 327–335. Moreira da Silva, P., De Wit, B., 2008. Spinning cone column distillation – innovative technology for beer dealcoholisation. Cerevisia 33 (2), 91–95. Mori, A., 2004. Method for lowering alcohol content of ready-made beer (written in Japanese). Japanese Patent JP 2004215551. Mota, A., Novák, P., Macieira, F., Vicente, A.A., Teixeira, J.A., Šmogrovicˇová, D., Brányik, T., 2011. Formation of flavor active compounds during continuous alcohol-free beer production: the influence of yeast strain, reactor configuration and carrier type. Journal of the American Society of Brewing Chemists 69 (1), 1–7. Mukamal, K.J., Rimm, E.B., 2008. Alcohol consumption: risks and benefits. Current Atherosclerosis Reports 10 (6), 536–543. Muller, R., 1990. The production of low-alcohol and alcohol-free beers by limited fermentations. Ferment 3 (4), 224–230. Muller, R.E., 1991. The effects of mashing temperature and mash thickness on wort carbohydrate composition. Journal of the Institute of Brewing 97, 85–92. Muller, R., 1993. Flavour in low-alcohol beers. Chemistry Review 3 (1), 2–7. Muller, R., 2000. A mathematical model of the formation of fermentable sugars from starch hydrolysis during high-temperature mashing. Enzyme and Microbial Technology 27, 337–344. Muller, R.E., Barrett, J., Baxter, E.D., 1991. Enhancement of flavor in beers with reduced alcohol contents. In: Proceedings of the European Brewing Convention Congress, Lisbon, Portugal. Narvátil, M., Dömény, Z., Šturdík, E., Šmogrovicˇová, D., Gemeiner, P., 2002. Production of non-alcoholic beer using free and immobilized cells of Saccharomyces cerevisiae deficient in the tricarboxylic acid cycle. Biotechnology and Applied Biochemistry 35 (2), 133–140. Narziss, L., Back, W., Liebhard, M., 1991. Optimization of biological process for the production of alcoholfree beer using suitable cultures. Brauwelt International 1, 52–57.

506

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

Narziss, L., Miedaner, H., Kern, E., Leibhard, M., 1992. Technology and composition of non-alcoholic beers. Processes using arrested fermentation. Brauwelt International 4, 396–410. Narziss, L., Back, W., Stich, S., 1993. Alcohol removal from beer by countercurrent distillation in combination with rectification (written in German). Brauwelt 133 (38), 1806–1820. Nevoigt, E., Pilger, R., Mast-Gerlach, E., Schmidt, U., Freihammer, S., Eschenbrenner, M., Garbe, L., Stahl, U., 2002. Genetic engineering of brewing yeast to reduce the content of ethanol in beer. FEMS Yeast Research 2 (2), 225–232. Perpète, P., Collin, S., 1999a. State of the art in low-alcohol beer production. Cerevisia 24, 27–33. Perpète, P., Collin, S., 1999b. Fate of the worty flavours in a cold contact fermentation. Food Chemistry 66, 359–363. Perpète, P., Collin, S., 1999c. Contribution of 3-methylthiopropionaldehyde to the worty flavor of alcohol-free beers. Journal of Agricultural and Food Chemistry 47, 2374–2378. Perpète, P., Collin, S., 2000a. How to improve the enzymatic worty flavor reduction in a cold contact fermentation. Food Chemistry 70, 457–462. Perpète, P., Collin, S., 2000b. Influence of beer ethanol content on the wort flavour perception. Food Chemistry 71, 379–385. Petin, V.G., Kim, J.K., Rassokhina, A.V., Zhurakovskaya, G.P., 2001. Mitotic recombination and inactivation in Saccharomyces cerevisiae induced by UVradiation (254 nm) and hyperthermia depend on UV fluence rate. Mutation Research, Fundamental and Molecular Mechanisms of Mutagenesis 478 (1-2), 169–176. Petkovska, M., Leskosek, I., Nedovic, V., 1997. Analysis of mass transfer in beer diafiltration with cellulose-based and polysulfone membranes. Food and Bioproducts Processing 75, 247–252. Pilipovik, M.V., Riverol, C., 2005. Assessing dealcoholization systems based on reverse osmosis. Journal of Food Engineering 69, 437–441. Pilkington, P.H., Margaritis, A., Mensour, N.A., Russell, I., 1998. Fundamentals of immobilized yeast cells for continuous beer fermentation: a review. Journal of the Institute of Brewing 104, 19–31. Pittner, H., Back, W., 1995. Continuous production of acidified wort for alcohol free beer using immobilized lactic acid bacteria. Technical Quarterly – Master Brewers Association of the Americas 32 (3), 163–168. Rajendram, R., Preedy, V.R., 2009. Ethanol in beer: production, absorption and metabolism. In: Preedy, V.R. (Ed.), Beer in Health and Disease Prevention. Elsevier Inc., Burlington, Mass, pp. 431–440. Regan, J., 1990. Production of alcohol-free and low alcohol beers by vacuum distillation and dialysis. Ferment 3 (4), 235–237. Schedl, S., Eppinger, H., Schuler, V., 1988. Process for the production of alcohol-free, yeast white beer. US. Patent 4790,993. Schur, F., 1988. Process for the preparation of alcohol-free drinks with a yeast aroma. US Patent 4746,518. Schur, F., Sauer, P., 1990. Process for the preparation of beer with a low alcohol content. US Patent 4971,807. Selecky´, R., Šmogrovicˇová, D., Sulo, P., 2008. Beer with reduced ethanol content produced using Saccharomyces cerevisiae yeasts deficient in various tricarboxylic acid cycle enzymes. Journal of the Institute of Brewing 114 (2), 97–101. Silva, D.P., Brányik, T., Teixeira, J.A., Almeida E Silva, J.B., 2010. Alcohol-free beer (written in Portuguese). In: Venturini Filho, W.G. (Ed.), Bebidas Alcoólicas: Ciência e Tecnologia, vol.1. Edgard Blucher Ltda, São Paulo, pp. 69–83. Snoek, I.S.I., Steensma, H.Y., 2007. Factors involved in anaerobic growth of Saccharomyces cerevisiae. Yeast 24, 1–10.

Stein, W., 1993. Dealcoholization of beer. Technical Quarterly – Master Brewers Association of the Americas 30 (2), 54–57. Tolstrup, J., Groenbaek, M., 2007. Alcohol and atherosclerosis: recent insights. Current Atherosclerosis Reports 9 (2), 116–124. Van De Winkel, L., Van Beveren, P.C., Masschelein, C.A., 1991. The application of an immobilized yeast loop reactor to the continuous production of alcohol-free beer. In: Proceedings of the European Brewing Convention Congress, Lisbon, Portugal. Van Dieren, B., 1995. Yeast metabolism and the production of alcohol-free beer. In: European Brewery Convention Monograph XXIV, EBC Symposium Immobilized Yeast Application in the Brewing Industry, Espoo, Finland. van Iersel, M.F.M., Meersman, E., Swinkels, W., Abee, T., Rombouts, F.M., 1995. Continous production of non-alcohol beer by immobilized yeast at low temperature. Journal of Industrial Microbiology 14, 495–501. van Iersel, M.F.M., Meersman, E., Arntz, M., Rombouts, F.M., Abee, T., 1998. Effect of environmental conditions on flocculation and immobilization of brewer’s yeast during production of alcohol-free beer. Journal of the Institute of Brewing 104, 131–136. van Iersel, M.F.M., Van Dieren, B., Rombouts, F.M., Abee, T., 1999. Flavour formation and cell physiology during the production of alcohol-free beer with immobilized Saccharomyces cerevisiae. Enzyme and Microbial Technology 24, 407–411. van Iersel, M.F.M., Brouwer-Post, E., Rombouts, F.M., Abee, T., 2000. Influence of yeast immobilization on fermentation and aldehyde reduction during the production of alcohol-free beer. Enzyme and Microbial Technology 26, 602– 607. Vecˇerková, H., 2010. Most liked beer Platan (written in Czech), MF Dnes Ekonomika – Test 06.10.2010. pp. 8–9. (downloaded 10.2.11). Verbelen, P.J., De Schutter, D.P., Delvaux, F., Verstrepen, K.J., Delvaux, F.R., 2006. Immobilized yeast cell systems for continuous fermentation applications. Biotechnology Letters 28, 1515–1525. Virkajärvi, I., Lindborg, K., Kronlöf, J., Pajunen, E., 1999. Effects of aeration on flavor compounds in immobilized primary fermentation. Monatsschrift fuer Brauwissenschaft 52 (9–12), 25–28. Von Hodenberg, G.W., 1991. Production of alcoholfree beers using reverse osmosis. Brauwelt International 2, 145–149. Willaert, R., Nedovic, V.A., 2006. Primary beer fermentation by immobilized yeast – a review on flavour formation and control strategies. Journal of Chemical Technology & Biotechnology 81, 1353–1367. Yano, S., Asano, T., Kurose, N., Hiramatsu, J., Shimoi, H., Ito, K., 2003. Characterization of an a-ketoglutarate-resistant sake yeast mutant with high organic acid productivity. Journal of Bioscience and Bioengineering 96 (4), 332– 336. Zufall, C., Wackerbauer, K., 2000a. Process engineering parameters for the dealcoholization of beer by means of falling film evaporation and its influence on beer quality (written in German). Monatsschrift fuer Brauwissenschaft 53 (7/ 8), 124–137. Zufall, C., Wackerbauer, K., 2000b. The dealcoholization of beer by dialysis – influencing beer quality by process engineering (written in German). Monatsschrift fuer Brauwissenschaft 53 (9/10), 164–179. Zurcher, Ch., Gruss, R., 1991. Method of making alcohol-free or nearly alcohol-free beer. US Patent 5077,061. Zürcher, A., Jakob, M., Back, W., 2005. Improvements in flavor and colloidal stability of alcohol free beers. In: Proceedings of the European Brewing Convention Congress, Prague, Czech Republic.