Where the Wild Yeasts Are! by Dr

Home , Adjuncts, Beer in Belgium, Fruit beer, Lager, Lambic

Where the wild yeasts are! by Dr. Roger Mussche Belgium

1. Introduction

What means Wild Yeasts? My yeast is not wild, yours is wild. 6000-5000BC people were already brewing (fermented beverages) in the Middle East with only the yeast of the house; they called it like the place they lived at, e.g. Susa.

Wild fermentation is not so strange and done all over the world : (see table 1), but unique for the Belgian type of wild fermentation is, that only the wind is helping?

Even with our fruitbeers the wind and the fruits are the only tools to innoculate our wort or young lambic.

2. Our History

Between Tigris and Eufrat Rivers, the first brew was made by the Sumerians in 5500BC, with grinded cereals, herbs as aniseed, cinnamon and brewers own mouthamylases (always done by women). In 1300BC in Egypt, Ramses III took more than 10.000 stone cans of beer with him to heaven. Only the Greeks and Romans were against real beer drinking because of the unpredictable effect; they thought it was good for the German barbarians, so that they would easily loose the wars.

Till 800 beermaking was a ladies job, and from the early middle age (about 900) the real brewing as a commerical fact is starting. The monks were the stimulators for quality beers by reinocculation by the previous brew at more or less stable room temperatures; the top fermented beers were born.

About 768 hop was introduced to Europe by the monks and in 1402 the first dark-lager was made by Mr. Naburg in Bavaria. About 1840 a more pale lager was created in Pilsen. It happened by accident, because the malt was bleached by the SO2 and CO from the brown charcoal. The real pale lager was brewed in 1920 in Belgium at the Alken Brewery.

Besides the wild beers, known in Belgium as lambic since 1400, there were also: top fermented beers, bottom fermented beers, red-sour beers, white beers and so on. (Sheet 1 - Our History)

3. Lambic and Gueuze

Our spontaneously fermented lambic is made within a radius of 20km around Brussels. The unique natural combination of Senne-valley (river), small hills with a lot of cherry trees, and small farms with own hops, cereals, wheat and wooden kegs with fermentation-liquids give the region a micro-flora that is kept in the air and buildings of the brewery-farms for more than 500 years.

The composition of the raw materials makes the beer a unique broth (wort) for wild inocculation and fermentation.

By law we must use: • min. 30% unmalted wheat • 70% malt (1/3 winter; 2/3 summer cereal) • wort-strength: 11-12 degrees plato • acidity of the lambic: min. 30mili. eq. NaOH • volatile acidity: min. 2 milli. eq. NaOH • spontaneous inocculation from open coolship is requisite

(Sheet 2: comparison of raw materials)

R. Mussche (1994) Where the wild yeasts are ! 1 / 8 The brewing method of lambic is the turbid mash method with the pecularity that the brewer intends to obtain a highly dextrinous wort, more appropriate for sustaining a long fermentation by a mixed flora of microorganisms and leading to the typical lambic flavor. (See Sheet 3 - brewing method)

To avoid the bitter taste of hop in these acid beers, high doses of aged hop are used during the long wort boiling period. After wort boiling, it is cooled overnight in large shallow trays. During this cooling period microorganisms are introduced (Table 2,3), cooled and infected wort is then pumped into wooden casks of 700 liters. The spontaneous fermentation then starts and may last for two years.

(See sheet 4 - evolution of micro flora)

The fermentation is introduced by the development of wort enterobacteria. Their numbers in cooled wort may vary from brewery to brewery, but largely exceed yeast counts. These fastly growing bacteria obtain their maximal concentration (about 108 cells/ml) after one week; but they persist only for about one month because of pH lowering and ethanol production.

(Sheet 5 - Evolution of ethanol, pH and ethanol)

The wort enterobacteria are replaced by yeasts responsible for the main or primary alcoholic fermentation. This main fermentation is mainly done by the non-actidione-resistent yeasts (like Saccharomyces species). The real attenuation increases to 50-60% and the pH falls to about 4.0. Ethanol and normal by-products like esters are formed.

The yeast population is a succession of species with increasing fermentative capacity.

Saccharomyces globosus, S. dairensis, S. uvarum, S. bayanus and S. cerevisiae. The primary alcoholic fermentation takes 3-4 months.

The third phase in the lambic fermentation is a lactic acid fermentation by Pediococcus cerevisiae. The lactic acid increase to 5-6 g/l and the pH falls to pH 3.2. The pediococci dominate the flora from the 3rd to the 8th month.

During or after the lactic acid fermentation, the secondary alcoholic fermentation starts. This results in an increase of the real attenuation to 80%. The concentration of the ethyllactate shows a tenfold increase during this period. The yeast responsible (Sheet 6) for this phase belongs mainly to the species Brettanomyces bruxellensis and Brettanomyces lambicus.

(Sheet 6 - Evolution in ethylacetate and ethyl lactate).

After one year of fermentation, Brettanomyces spp and P. cerevisiae remain in suspension, but drastic changes in composition are no longer observed. The later part of the fermentation must be considered as a maturation during which the characteristic "old lambic flavour" develops.

Table 4: Comparison between Saccharomyces and Brettanomyces yeasts. Pictures 1 and 2 of Saccharomyces and Brettanomyces cells.

4. Gueuze, or refermentation of Lambic in the bottles

From the lambic in the wooden casks the gueuze is brewed. Whereas the lambic is always flat (no CO2), the gueuze is refermented in the bottles - method Champenoise - to obtain a sparkling sour-astringent beer. We blend 2/3 of a young lambic - full of yeasts - with 1/3 of overyears lambic - full of dextrinase - for a right amount of yeasts and available sugars or dextrines. Here is the reason for using the turbid mash method: we need enough dextrines in the lambic for the production of sugars as yeasts-substrate. The dextrinases are the hydrolysing factors for the dextrines.

Two groups of microorganisms seem important: the actidione- resistant yeasts (Brettanomyces) and the lactic acid bacteria, although the yeasts die after about 10 months. The yeasts at a level of about 105 c.f.u./ml,

R. Mussche (1994) Where the wild yeasts are ! 2 / 8 developed during the first month as the counts on the day of bottling were only around 102 c.f.u./ml. [c.f.u. is an abbreviation for "colony forming units" -MDS]

Lactic acid bacteria at a level of around 106 c.f.u./ml remain consistently present after 5 months. At the day of the bottling their numbers were around 102/ml. Acetic acid bacteria disappeared after 3 to 4 months. At the day of the bottling their numbers were around 102/ml. Acetic acid bacteria disappeared after 3 to 4 months. Non- actidione resistant yeasts were consistently present during the first 10 months. Their numbers were not much higher than at the time of bottling (102/ml). Many of these yeasts were isolated and identified. None was a Saccharomyces, but were of the genera Candida, Torulopsis, Hansenula, Pichia and Cryptococcus. Unexpectedly, the Pediococci, which are the most unconstantly growing organisms after isolation, remain the longest- living organisms in the gueuze.

5. Fruit-beers on lambic base

Nowadays lambic-fruit beers are extremely popular. The first fruit beer was made with sour-cherries. The cherries were growing around Brussels - "typical village Schaarbeek". In the year 1930 different farm breweries started cherry-kriek beer by adding 125kg crushed cherries per 600hl young lambic in the casks. The fruit-fermentation starts immediately in the casks because of the extra amount of sugar from the fruit and the high Brettanomyces counts on the fruit.

The best results are with sour-wild cherries; the meat of the cherries is very red-flavoured on a big stone. The fruit is added in the cherries-season (July). Since a 20 years the cherries-beer is made all over the year with single juice from crushed defrozen fruits. The last method gives much more colored, flavoured (like almond) cherries beer.

The beer with the fruits is well-balanced after another 6 months fermentation in the casks but only with sour- dark-red cherries; with a normal cherry-fruit the beer is light pink in color, and the taste- flavour is eaten by the yeast. Here again the Brettanomyces yeast is the most usefull. We obtain a refermented Kriek in the bottle by blending 2/3 of a young cherries-beer with 1/3 of an overyears cherries- beer.

Other fruit beers of lambic brewers are mainly made by addition of the fresh juice to a young lambic. The problem with most of the fruits is the weakness of the flavour and color against the yeast-metabolism. Raspberries color and flavour is completely metabolished by the Brettanomyces yeast. Therefore in an artisanal lambic brewery the fruit beers are made by blending of lambic and fresh single juice before bottling. The maturation takes place in the bottle without fermentation.

6. Why are Lambic and Gueuze so unique?

The most important and distinguished factor for a good lambic- gueuze is the flavour and the taste.

Table 5. Substantial composition of gueuze

The combination of an acid taste (pH3.3) with a very dry mouth- feeling (from high tannin-content) makes the beer very drinkable.

The flavor is a combination of alcohols, esters, aldehydes and some hop-terpenoids.

It is strange that a beer with more than 5000ppm acids, over the 500ppm esters and at least 50.000ppm alcohol is so popular and gives the best base for a fruit beer... that's maybe the secret.

R. Mussche (1994) Where the wild yeasts are ! 3 / 8 Table 1. Fermented foods (beverages) containing mixed and/or wild cultures

[originally this was too wide to fit on the page. I removed columns of "Aspect" and "Use" since they didn't lend much additional information. -MDS]

Product/name Substrates Organisms References BELGIUM Lambic/Gueuze Barley/wheat Enterobacteria, Van Oevelen, Mussche, yeasts, Pediococcus et.al. 1976, 1977 Acid Ales Barley,rice, Saccharomyces corn Lactobacillus CHINA Soy sauce Soybeans/wheat Aspergillus, Young & Wood, 1976 Pediococcus, Saccharomyces EASTERN EUROPE Kefir Milk Yeasts, Lactic bacteria Tea Fungus Tea Leaves, Acetobacter, yeasts Hesseltine, 1965 Sugar INDIA Sonti Rice Rhizopus, Yeasts Hesseltine, 1965 JAPAN Sake Rice Aspergillus, Kodama & Yoshizawa Saccharomyces, Lactic acid bact. MEXICO Pulgue Agave Zymomonas, Yeasts, Lactic acid bact. SUDAN Merissa Sorghum Saccharomyces, Lactic acid bact. U.S.A. Bourbon Maize/Rice/ Lactobacillis delbrueckii, Barley Saccharomyces C.I.S.(U.S.S.R.) Kwass Rye/Barley Yeasts, Hesseltine, 1965 Lactic acid bact. West-Germany Weissbier Wheat,Barley Saccharomyces, Lactic acid bact. South-Africa Kaffir Sorghum,Maize Yeasts, Williamson, 1955 Lactic acid bact.

Table 2. Microorganisms Detected in Brewery Air (LAMBIC)

Yeasts Isolated from brewery air Total percentage outside inside Schizosaccharomyces 0 2 2.25 Kloeckera x (1) 0 3 3.37 Sacchromycodes 1 2 3.37 Sacchomyces (1) 8 18 29.21 Hansenula 0 1 1.12 Torulopsis 6 38 49.44 Candida 1 7 8.99 Brettanomyces (1) 1 1 2.25 TOTAL: 17 72

(1) Mainly during lambic fermentation

R. Mussche (1994) Where the wild yeasts are ! 4 / 8 Table 3. Degree of infection of lambic wort after 1 night cooling in open trays

Microbiol. species In lambic wort In Ringer solution after 1 night cooling after 1 night infection

Enterobacteria 1-50 CFU/ml 3 CFU/L Acetic acid bacteria not present in 100ml not present in 1L Lactic acid bacteria 1-10 CFU/L not found in 1L Total yeasts 10-150 CFU/ml 40 CFU/L Actidione-resistent 10-20 CFU/L 25 CFU/L yeasts

Table 4. Comparison between Saccharomyces and Brettanomcyes yeasts

[I've changed the table layout so it can be reproduced here. The information has not been altered -MDS]

Brettanomyces sp. Saccharomyces sp. Ascospores - + Acid from glucose + - Actidione Resistent + - Ferment: Glucose + + Maltose + + Raffinose - +1/3 Assimil: Glucose + + Maltose + + Raffinose - + Nitrate + -

Table 5. Substantial composition of gueuze

[I've changed the table layout so it can be reproduced here. The information has not been altered -MDS]

Traditional Gueuze Filtered Gueuze Lagers Ales BrewA BrewB BrewC BrewA BrewB BrewC

Density d20 1.011 1.01 1.01 1.017 1.016 1.014 Tannins ppm 350 375 360 340 335 345 150 210 Ethanol g% 4.95 4.89 4.58 4.71 3.84 4.49 pH 3.34 3.25 3.45 3.2 3.45 3.35 4.2 4 Lactic acid ppm 3434 3000 5277 2071 3510 2107 70-200 70-200 Acetic acid ppm 656 680 1238 1177 580 538 60-140 60-140 Ethyl Acetate ppm 61 72 82 68 34 34 08-20 06-23 Ethyl Lactate ppm 384 437 419 107 188 118 0.1 0.1 1-amyl acetate ppm 0.5 0.6 0.1 1.6 3.1 2.1 1.2-28 0.7-3.3 2,3 Butanediol ppm 716 406 618 318 274 196 10-51 42-128

Remark: Filtered Gueuze: - High content of 1-amylacetate means that the young lambic is blended with some top-fermented beers before filtration Traditional Gueuze: - Typically very high in tannins from hops, ethyllactate, 2,3-butanediol

R. Mussche (1994) Where the wild yeasts are ! 5 / 8 Sheet 1 - Our history

6000-5500BC Fermented cereal to a kind of beer (sikaru), in Mesopotamia later "shekar"

2500-1500BC Fermented cereals + dads to "tithum" = beer in Egypt

800-1000 Beer at monasteries with hops, cereals, top fermented yeast

1400 Dark lager beer by Naburg, Bavaria (cooling with ice)

1400 Lambic-recipe

1500 Lambic-name

1840 Light dark lager beer in Pilsen (CSR) with paler malt

Pasteur and Hansen found yeast cells in the fermentation

1890 Light dark lager beer in Bavaria

1893 Lambic in bottles - gueuze

1920 Light pale lager beer in Belgium (Alken) with pale malt

1930 Kriek - cherry beer

1978 Framboos - respberry beer

1983 Perzik - peaches beer

Sheet 2 - Comparison between a lager pils brewery and a lambic brewery

Lager Lambic a. Raw materials Malt min 50% Malt Adjuncts max 50% Unmalted wheat min 30% as rice, corn, starch Fresh hop preparates Annuated hops as whole hops, pellets, extracts b. Brew-method Decoctie-method Turbid mash method Infusion-method c. Boiling-copper 90 minutes 5-6 hours d. Cooling Aseptic - fast 8C Coolship open under the wind e. Inocculation Pure yeast amount From the brewery air

R. Mussche (1994) Where the wild yeasts are ! 6 / 8 after propagation f. Fermentation-maturation +- 8C - 6 days 6 months and 24 months +- 2C - 1 month

Sheet 3 - Brewing method

Malt Wheat

Sheet 4 - Evolution of microbiol. populations during the lambic fermentation

Sheet 5 - Evolution in attenuation, ethanol, and pH during lambic fermentation

Sheet 6 - Evolution in concentration of ethyllactate and ethylacetate during lambic fermentation

Sheet 7 - Microbiological profile of gueuze bottle fermentation

[sorry folks, I can't do these graphs in ASCII-graphics -MDS]

R. Mussche (1994) Where the wild yeasts are ! 7 / 8 Sheet 8 - Blending procedure for gueuze

Refermented in the bottle Filtered (Brut, Fond, Artisanal)

Blend of 2 parts of young lambic 1 part of old lambic

Sheet 9 - Lambic-fruitbeers with fruit with fresh single with concentrates juice and flavours (artisanal) (artisanal) (industrial) young lambic young lambic young beer (6 months old) (6 to 12 months old) in casks in casks

Remark: 1. and 2. are from real lambic breweries like Lindemans

[there are also 6 photomicrographs of various organisms, but I'm sure not going to reproduce these in ASCII graphics -MDS]

R. Mussche (1994) Where the wild yeasts are ! 8 / 8