Alcohol Beer: the CERB Experience

Brewing process study for high quality low-alcohol beer: the CERB experience

Prof. Giuseppe PERRETTI and Prof. Paolo FANTOZZI Italian Brewing Research Centre, University of Perugia

EBC Brewing Science Group 10th Technical Meeting 2014, Wien, 9-11th September

The importance of making an improved low- alcohol beer

 Drink-drive awareness  Diet  Health  Volume sales increasing (Germany, +12 % in 2013)  Total volume (Spain, 13% total)  Lower tax  New market opportunities  …

GIUSEPPE PERRETTI “Low-alcohol and ”Alcohol-free”. Main production methods

Biological: Physical: Cold contact Vacuum rectification (thermal) High gravity brewing Falling film evaporation (thermal) High temperature mashing Dialysis (membrane) Use of special yeasts (NCYs) Reverse osmosis (membrane) … Osmotic distillation (membrane) … Branyik T., 2012 (ICT). A review of methods of low alcohol and alcohol-free beer production. Journal of Food Engineering 108 493-506. GIUSEPPE PERRETTI Biological

The state of art: Cold contact process Muller R., 1990 (BRI). Review. The production of low-alcohol and alcohol-free beers by limited fermentations. Ferment 3 (4), 224–230. Perpète P., Collin S., 1999 (UCL). State of the art in low-alcohol beer production. Cerevisia 24, 27–33.

It is the only biological method to make alcohol-free beer with less than 0.05 v/v ethanol. Relatively simple to manage.

In comparison with other methods of production, the CCP is characterized by one of the lowest aldehyde reduction capacity, worty off-flavor. Long refrigeration needed.

GIUSEPPE PERRETTI Biological

The state of art: High gravity brewing Muller R., 1990 (BRI). Review. The production of low-alcohol and alcohol-free beers by limited fermentations. Ferment 3 (4), 224–230.

Relatively simple to manage. Good stability because of low level of fermentable sugars at the end of process. Good level of esters and higher alcohols.

Head retention very low. The foam is poor. More suitable to beers with 2 % v/v ethanol. Beer with 1 % v/v are lack of body and tasteless.

GIUSEPPE PERRETTI Biological

The state of art: High temperature mashing Muller R., 1990 (BRI). Review. The production of low-alcohol and alcohol-free beers by limited fermentations. Ferment 3 (4), 224–230.

Good flavor. No problem to achieve beer with less than 1.2 % v/v ethanol.

High pH of beer. Stability problem. Pasteurization needed. Risk of worty off-flavor.

GIUSEPPE PERRETTI Biological

The state of art: Use of special yeasts (NCYs) Narziss L., Miedaner H., Kern E., Leibhard M., 1992 (TUM). Technology and composition of nonalcoholic beers, Brauwelt International 4:396–410.

The process is easy to handle and does not require continuous monitoring of extract reduction. Difficulty in controlling the microbial activity. There is a remaining slight worty off-ﬂavor. Diacetyl level above the taste threshold. Risk of excessive sweetness of beer. High pH.

GIUSEPPE PERRETTI Physical The state of art: Vacuum rectification Narziss L., Back W., Stich S., 1993 (TUM). Alcohol removal from beer by countercurrent distillation in combination with rectification (written in German). Brauwelt 133 (38), 1806–1820. Kern M., 1994. Alcohol removal from beer using continuous vacuum reﬁnement. Alimentacion Equipos y Tecnologia 13 (5), 37–41. Zürcher A. (Feldschlösschen Getränke AG), Jakob M., Back W., 2005 (TUM). Improvements in ﬂavor and colloidal stability of alcohol free beers. In: Proceedings of the European Brewing Convention Congress, Prague, Czech Republic.

Potential to remove alcohol from beer completely, possibility to commercialize the separated alcohol, thermal stress very low. Elevated running and plant costs, excessive loss of volatile compounds. Taste empty, sour. Carbonation needed. Beer re- dilution after treatment.

GIUSEPPE PERRETTI Physical The state of art: Falling film evaporation Zufall C., Wackerbauer K., (VLB) 2000. Process engineering parameters for the dealcoholization of beer by means of falling ﬁlm evaporation and its inﬂuence on beer quality. Monatsschrift fuer Brauwissenschaft 53 (7/8), 124–137.

Considered the cheapest thermal process. Easy to clean. Beer has a short product contact time, generally a few seconds.

Loss of volatile compounds. Re-dilution with degassed water needed. Re-addition of carbon dioxide required.

GIUSEPPE PERRETTI Physical

The state of art: Dialysis Donhauser S., Glas K., Mueller O., 1991 (TUM). Behavior of beer components during the manufacture of alcohol-reduced products by dialysis. Brauwelt International 2, 139–144. Zufall C., Wackerbauer K., (VLB). The dealcoholization of beer by dialysis – influencing beer quality by process engineering. Monatsschrift fuer Brauwissenschaft 53 (9/10), 164–179.

Low running temperature and pressure. No thermal and mechanical stress, no worthy off-flavor. A selective removal of ethanol cannot still be achieved. Excessive loss of volatile compounds. Carbonation is necessary at the end of dealcoholization. High plant costs.

GIUSEPPE PERRETTI Physical

The state of art: Reverse osmosis Kavanagh T. (Foster’s), Clarke B.J., Miles M., Nicholson B. 1991. Volatile flavor compounds in low alcohol beers. Tech. Q. Master Brew. Assoc. Am. 1991, 28, 111−118. Pilipovik M.V., Riverol C., 2005 (University of Trinidad and Tobago). Assessing dealcoholization systems based on reverse osmosis. Journal of Food Engineering, 69 (4), 437-441.

Starting from beers with normal composition, beers with alcohol contents very low (< 0.05 % vol.) can be obtained. Elevated running costs (energy). Carbonation is necessary ended the dealcoholization. Excessive loss of volatile compounds. It is not economically feasible to large-scale.

GIUSEPPE PERRETTI Physical

The state of art: Osmotic distillation, evaporative pertraction Diban N., Arruti A., Barceló A., Puxeu M., Urtiaga A., Ortiz I., 2013 (University of Cantabria). Membrane dealcoholization of different wine varieties reducing aroma losses. Modeling and experimental validation, Innovative Food Science and Emerging Technologies, 20, 259-268.

Low running temperature and pressure. Low energy consumption. No thermal and mechanical stress. Easy to manage. Low aroma losses.

It is necessary carbonation. Excessive loss of volatile compounds. Membrane life-time. Membrane replacement costs.

GIUSEPPE PERRETTI

the CERB experience

I experimental section: II experimental section: Biological process Physical process

Screening of a new strain of NCYs Beer dealcoholization using for low-alcohol beer production osmotic distillation

GIUSEPPE PERRETTI Italian Brewing Research Center

GIUSEPPE PERRETTI Interdisciplinary work on NCYs

Department of Agricultural, Food and Environmental

Italian Brewing Research Center Industrial Yeasts Collection

GIUSEPPE PERRETTI Interdisciplinary work on OD Department of Agricultural, Food and Environmental Sciences

Scale-Up

University of Salerno, Italy

GIUSEPPE PERRETTI NCYs: the literature

There are few detailed studies on the behavior of those yeasts.

1. Huige, N.J., Leidig, A.R. and Sanchez, G.W. Process for preparing a nonalcoholic (less to 0.5 volume percent alcohol) malt beverage. Patent, 1990. 2. Narziss, H. Miedaner, E. Kern and M. Leibhard. Technology and composition of non-alcoholic beers. 1992. 3. Mohammadi A., Razavi S.H., Mousavi S.M., Rezaei K. A comparison between sugar consumption and ethanol production in wort by immobilized Saccharomyces cerevisiae, Saccharomyces ludwigii and Saccharomyces rouxii on brewer’s spent grain. University oh Tehran, 2011.

Zygosaccharomyces rouxii Saccharomycodes ludwigii

GIUSEPPE PERRETTI Use of non-conventional yeasts (NCYs)

S. ludwigii is unable to use maltose, while Z. rouxii is able to partially use maltose. Boundy-Mills K., Stratford M., Miller M. W. (2011) The yeasts: a taxonomic study, fifth edition, (Kurtzman C. P., Fell J. W., Boekhout T. eds.), vol. 1, cap. 62, Elsevier.

FERMENTABLE S. ludwigii Z. rouxii SUGARS Glucose + +

Galactose - -

Sucrose + v Maltose - +/w Lactose - -

Raffinose + -

Trehalose - w/-

Melibiose - -

GIUSEPPE PERRETTI Use of non-conventional yeasts (NCYs) 11 strains were selected from DBVPG (International Industrial Yeast Collection – 4,000str, 500 Spp.)

STRAINS SPECIES Isolation source Isolation area DBVPG 4084 Dates Iraq

DBVPG 6187 Grape must Italy DBVPG 6424 Z. rouxii Honey The Netherlands DBVPG 6463 Pickle USA

DBVPG 6921 Honey Canada

DBVPG 3010 Grape must Italy

DBVPG 3304 N.A. Italy

DBVPG 3398 Tea beer N.A. S. ludwigii DBVPG 3931 Grape must Italy

DBVPG 4116 Grape must Slovenia

DBVPG 6721 N.A. N.A.

GIUSEPPE PERRETTI Use of non-conventional yeasts (NCYs)

The wort used to run micro-fermentation trials was produced by a specific mashing program with the CERB pilot plant.

GIUSEPPE PERRETTI

Specific mashing program

° Temperature ( Temperature

Time (min)

High temperature mashing method

GIUSEPPE PERRETTI Specific mashing program

Specific Target wort Parameters EBC method wort (12 °P)* ANALYTICA EBC 8.3 Original Gravity (wt%) 12.01 ± 0.10 12.25 ± 0.10 / 2004 ANALYTICA EBC pH 5.57 ± 0.08 5.49 ± 0.08 8.17 / 1999 ANALYTICA EBC 8.5 Colour (EBC units) 7.20 ± 0.8 9.40 ± 2.1 / 2000 > Dextrins ANALYTICA EBC 8.4 Viscosity (mPa*s) 1.82 ± 0.08 1.67 ± 0.04 / 2004 Turbidity (EBC units) IP/01/2007 67.60 ± 2.50 69.60 ± 1.50 ANALYTICA EBC Total Nitrogen (mg/L) 1090 ± 68.00 847 ± 58 8.9.1 / 2000 ANALYTICA EBC FAN (mg/L) 217.00 ± 7.00 161.00 ± 17 8.10 / 2002 ANALYTICA EBC Fermentability (%) 8.6.1 / 2004 65 ± 2 83 ± 2 ANALYTICA EBC β-glucan (mg/L) 231.00 ± 42.00 167.00 ÷ 39.00 4.16.3 / 2005

* Mashing: 52 °C for 10 min., 65 °C for 45 min., 72°C for 20 min., 76°C for 10 min.

GIUSEPPE PERRETTI Use of non-conventional yeasts (NCYs)

Yeasts suspended Suspensions transferred in in sterile water. standard wort for yeast to grow for 24 h at 25 °C under aerobic conditions on a rotary shaker.

GIUSEPPE PERRETTI Use of non-conventional yeasts (NCYs)

micro-fermentations (50 mL of specific wort, 20 °C, monitored every 24 hours, n=3).

Volatile profile analyzed by Gas-Chromatography

GIUSEPPE PERRETTI Results PCA scores (a) and loading plots (b) for all strains.

Samples/Scores Plot of "All the studied strains" Variables/Loadings Plot for "All the studied strains" 5 0.5 Scores on PC 2 (15.26%) PC 2 (15.26%) Saccharomycodes ludwigii Aldehyde Diacetyl 4 Zygosaccharomyces rouxii 0.4 Ester DBVPG 6187 95% Confidence Level Hexanal Ethanol 3 Higher Alcohol 0.3 Acetaldehyde DBVPG 6424 Vicinal Diketone 2 3-Methylpropionaldehyde DBVPG 6921 Isobutanol 0.2 n-Propanol 1 DBVPG 4084 Furfuryl Alcohol 0.1 2-Methyl-1-Butanol 0 DBVPG 3931 Ethyl Octanoate DBVPG 4116 DBVPG 3398 0

-1 2(15.26%) PC 3-Methyl-1-Butanol DBVPG 3054 3-Methylbutanal Scores Scores on PC 2 (15.26%) DBVPG 3304 2-Methylbutanal -2 DBVPG 3010 2,3-Pentanedione -0.1 Ethanol Ethyl Acetate -3 Isoamyl Acetate DBVPG 6463 -0.2 2-Phenylacetaldehyde -4 Ethyl Butanoate Furfuraldehyde (a) (b) Ethyl Hexanoate -0.3 2-Phenylethanol -5

-8 -6 -4 -2 0 2 4 6 8 10 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 Scores on PC 1 (48.90%) PC 1 (48.90%)

De Francesco G., Turchetti B., Sileoni V., Marconi O., Perretti G. Screening of strains of Saccharomycodes ludwigii and Zygosaccharomyces rouxii to produce Low-Alcohol Beer. Proceedings of 8th International CIGR Technical Symposium on Advanced Food Processing and Quality Management, Guangzhou, 2013. GIUSEPPE PERRETTI Results Ethanol content after fermentation by Z. rouxii and S. ludwigii

2 Low-Alcohol Ethanol(% v/v) Beer in Italy 1

Yeast strain (Z. rouxii; S.ludwigii)

GIUSEPPE PERRETTI Total esters content after fermentation by Z. rouxii and S. ludwigii

80 Common 70 level in

60 beer

) 1 - 50

40 (mg L 30

20 Esters 10 0

Yeast strain (Z. rouxii; S.ludwigii)

Ethyl acetate De Francesco et alii, Guangzhou, 2013.

GIUSEPPE PERRETTI Total higher alcohols level after fermentation by Z. rouxii and S. ludwigii

200 180

160

) 1 - 140

120 (mg L 100 Common level in 80 beer

60 alcohols 40

20 Higher 0

Yeast strain (Z.rouxii; S.ludwigii)

2-phenylethanol De Francesco et alii, Guangzhou, 2013.

GIUSEPPE PERRETTI Diacetyl content after fermentation by Z. rouxii and S. ludwigii

1000 900

800

- 700 600 500 400

300 Diacetyl Diacetyl (ug L 200 Threshold 100 0

Yeast strain (Z.rouxii; S.ludwigii)

De Francesco et alii, Guangzhou, 2013.

GIUSEPPE PERRETTI Methional content after fermentation by Z. rouxii and S. ludwigii

250 Threshold

200

- 150

100

50 Methional Methional (ug L 0

Yeast strain (Z.rouxii; S.ludwigii)

methional De Francesco et alii, Guangzhou, 2013.

GIUSEPPE PERRETTI Osmotic Distillation OD: usually microporous PP hydrophobic membrane, involves the transport of volatile components PES dialysis involves transport of solutes (whether volatile or non-volatile) between two miscible liquid SS phases

Hogan P.A., Canning R.P., Peterson P.A., Johnson R.A., Michaels A.S. A New Option: Osmotic Distillation. Chemical Engineering Progress. 1998

BEER

GIUSEPPE PERRETTI

Osmotic Distillation

Scheme of the dealcoholization lab-scale plant

FEED, beer tank; P1 and P2 peristaltic pumps; MC, membrane contactor; CS, cooling system; SS, stripping solution tank; V1 to V6; manual valves; T, temperature controllers; P1 to P6, pressure gauges.

GIUSEPPE PERRETTI Osmotic Distillation

Dealcoholization lab-scale plant made at BRI

Membrane contactor MiniModule 1.7x5.5 x50 (Membrana GmbH, Wuppertal, Germany)

Material: Hydrophobic Polypropylene

Typical membrane surface area: 0.54 m2

Average pore size: 0,04 µm

Membrane porosity: 40 %

Beer inlet pressure 0,2 bar, beer outlet 0.1 bar. Water inlet pressure 0,2 bar, water outlet 0.1 bar.

Beer flow 500 mL min-1, water flow 500 mL min-1

The feed and stripping solutions were fed into the module by two peristaltic pumps

GIUSEPPE PERRETTI Osmotic Distillation

Different process parameters at lab-scale on commercial beers.

 stripping solution amount (3,4,5,6 L)  flow rate (500, 1000 mL min-1)  types of stripping solutions (normal and carbonated water)

GIUSEPPE PERRETTI Lab-scale results

Decrease in ethanol with different stripping solution (3 and 6 L)

5 a

b 100 4 87

90 76

80 3 70 60 2 a 50 b a a a

40 EtOHl content (% v/v) 1 b b b 30

Ethanol Ethanol Removed (%) 20 0 10 0 60 120 180 240 0 Time 1/3 1/6 B (1 L, 10°C); DW (3 L, 10°C) B (1L 10°C; DW (6 L, 10 °C) Ratio Beer/Stripping Solution (L)

Volatile compounds and quality parameters were slightly affected.

n:2. Values on the same column with different superscript letters are statistically different (P<0.05) De Francesco G., Freeman G., Lee E., Marconi O., Perretti G., Effect of operating conditions during low-alcohol beer production by osmotic distillation, Journal of Agricultural and Food Chemistry, 2014, 62 (14), pp 3279–3286 GIUSEPPE PERRETTI Lab-scale results

Decrease in ethanol with carbonated water as stripping solution. DW: deaerated water. CW: carbonated water

100 83 83 90 6

a 5 a 70 60 4 b 50 3

40 2 a b a

30 a a a a EthanolDecrease (%) EtOHl EtOHl (%contentv/v) 1 20 10 0 0 30 60 90 120 0 Time B (1 L, 10 °C); DW (5 L, 10 °C) B (1 L, 10 °C); CW (5 L, 10 °C) B (1 L, 10°C); DW (5 L, 10°C), beer in tubeside

B (1 L, 10°C); CW (5 L, 10°C) CW in tubeside

Volatile compounds and quality parameters were not affected also. Esters

were slightly influenced. CO2 ad pH also. n:2. Values on the same column with different superscript letters are statistically different (P<0.05)

De Francesco G. et alii, Journal of Agricultural and Food Chemistry, 2014

GIUSEPPE PERRETTI Lab-scale results

Decrease in ethanol with different stripping solution flow rate (500, 1000 mL min-1).

100

90 80 80 80 70 6 B (1 L, 10°C); DW (4 L, 10°C), a flow rate 500 mL/min 60 5 a 50 B (1 L, 10°C); DW (4 L, 10°C), 40 4 flow rate 1 L/min 30 3 20 b c Ethanol Removed (%) Removed Ethanol 2 10 d e 0 d e d e 1

500 1000 v/v) (% content EtOH 0 Flow Rate (mL min-1) 0 30 60 90 120 Time

Volatile compounds and quality parameters were not affected. Only iso- amylalcohol was slightly reduced at 1000 mL min-1

n:2. Values on the same column with different superscript letters are statistically different (P<0.05)

De Francesco G. et alii, Journal of Agricultural and Food Chemistry, 2014

GIUSEPPE PERRETTI Pilot plant scale-up

a&b tests • Mixture of malts: pilsner, caramel, chocolate and melanoidinic • English hop • CCV by using an English Ale yeast at 20 °C • Unfiltered beer

c test • CCV by using a Belgian Ale yeast at 24 °C

GIUSEPPE PERRETTI Pilot plant scale-up

Membrane contactor Extra-Flow 4x28x50 (Membrana GmbH, Wuppertal, Germany)

Material: Hydrophobic Polypropylene

Membrane porosity: 40 %

Membrane surface area: 20 m2

Average pore size: 0.04 µm

Tube inner diameter: 220 µm

Tube outer diameter: 300 µm

Beer inlet pressure 1.1 bar, beer outlet 0.3 bar. Water inlet pressure 1.1 bar, water outlet 0.3 bar. Beer flow 30 L min-1, water flow 30 L min-1.

The feed and stripping solutions were fed into the module by two centrifugal pumps (Lowara CEA 70/3/A, Q 30–80 L min-1, PZ 0.37 kW, Vicenza, Italy).

GIUSEPPE PERRETTI Pilot scale results

Ethanol decrease during dealcoholization test a.

4 e

2 d c b b Ethanol (% v/v) (% Ethanol 1 ab a a a a

0 0 30 60 90 120 150 180 210 240 270 Time (min)

n:3 technology replications De Francesco G., Marconi O., Sileoni V., Perretti G., Low-Alcohol Beer Production using Osmotic Distillation: scale-up on Pilot Plant. International Malting and Brewing Symposium “11th Trends in Brewing”, Ghent, 2014. GIUSEPPE PERRETTI Pilot scale results

Ethanol decrease during dealcoholization test b.

4 d

Ethanol(% v/v) c c 1 bc a

0 0 30 60 90 120 150 Time (min)

n:3 technology replications De Francesco G. et alii, Ghent, 2014

GIUSEPPE PERRETTI Pilot scale results

Ethanol decrease during dealcoholization test c.

4 e

d 2

c Ethanol(% v/v) bc b 1 a

0 0 30 60 90 120 150 Time (min)

n:3 technology replications De Francesco G. et alii, Ghent, 2014

GIUSEPPE PERRETTI Comparison with other techniques

100

(%)

50 Dialysis

percentage Vacuum Distillation

40 Reverse Osmosis

Loss 30 OD 20

References Dialysis:Zufall, C., Wackerbauer, K., 2000. The dealcoholization of beer by dialysis – influencing beer quality by process engineering. Vacuum Distillation: Zurcher, A.; Jakob, M.; Back, W. 2005. Improvements in flavor and colloidal stability of alcohol free beers. Reverse osmosis: Kavanagh, T.; Clarke, B.; Gee, P.; Miles, M.; Nicholson, B. 1991. Volatile flavor compounds in low alcohol beers. Tech. Q. Master Brew. Assoc. Am. 1991, 28, 111−118.

GIUSEPPE PERRETTI Pilot scale results

Higher alcohols level of three dealcoholisation tests (a, b, c).

OB: original beer; DB: dealcoholized beer.

250

) 1

- 200 (mg L (mg 150

alcohols 100

Higher 50

0 OB test a DB test a OB test b DB test b OB test c DB test c N-propanol Isobuthanol Amyl alcohols 2-phenylethanol Total higher alcohols

De Francesco G. et alii, Ghent, 2014

GIUSEPPE PERRETTI Pilot scale results Trained panel 1-5 scores of beer before and after the dealcoholization treatment (test c).

global intensity (a-a) 2 linger (b-a) malty (a-a) 1

OB body (b-a) 0 hoppy (a-a) DB

bitter (a-a) estery (b-a)

sweet (b-a)

De Francesco G. et alii, Ghent, 2014

GIUSEPPE PERRETTI Conclusions of biological experimental section

The study confirmed the possibility to produce low-alcohol beer using NCYs. S. ludwigii resulted more appropriate to obtain low and alcohol-free beer, producing an ethanol content lower than 0.5 % v/v.

In particular, the beer obtained using DBVPG 3010 showed the lowest ethanol content (0.5 % v/v), an appreciable esters amount (about 15 mg L-1) and a noticeable higher alcohols amount (about 43 mg L-1).

GIUSEPPE PERRETTI Conclusions of physical experimental section

OD has been demonstrated to be a feasible process to produce low- alcohol beer. The process settings and control was relatively simple, as well as membrane and plant cleaning.

Furthermore, it is important to focus on the stripping solution, which is slightly alcoholic and flavored at the end of the process. It is completely reusable in other processes as high gravity dilution, distilled dilution, or as a new innovative product, e.g. flavored water with small amount of alcohol.

GIUSEPPE PERRETTI Conclusions of physical experimental section

However, there are a number of problems that limit its growth

 operating costs  investment and membrane replacement costs  excessive loss of volatile compounds  beer degassing

GIUSEPPE PERRETTI Future…

The use of NCYs confirmed as useful method of production. Use of aromatic ingredients (malts, hops, spices) to increase the taste. Test on plant-scale the selected strains. Catch on new interesting strains or species.

Osmotic distillation resulted an interesting process. The beer with malty profile (stout, porter) is more appropriate to this treatment. Special brewing techniques (e.g. dry-hopping) could be useful to improve the aroma.

Zurcher A. “Optimization of NAB quality”. EBC-BSG Copenhagen 2012: “The disadvantages of methods can be compensated by combining production methods”

GIUSEPPE PERRETTI GIUSEPPE PERRETTI THANK YOU FOR YOUR ATTENTION

GIUSEPPE PERRETTI