Where the Wild Yeasts are!

Transcribed by Mike Sharp

A few days ago I was digging through my piles of brewing related papers to find some grain analyses and I found a photocopy of the Homebrew U III notes from Dr. Roger Mussche's talk on lambic. There is some interesting information in the tables at the end of the talk as well as a few intersting points in the body of the text.

I remember receiving this (though I honestly don't have a clue as to who sent it ) and hearing rumor of a possible conference proceeding being published. At that time I didn't want to compromise any attempt at publishing the proceeding by posting the notes I had. Well, since the proceedings haven't ever been published, and since loosing this presentation doesn't do anyone any good, I've reproduced the photocopies here as well as I could.

Before we get to the meat of the article, I'd like to put in a few good words for Charlie Finkel of Merchant Du Vin (Importers of Lindemans as well as a host of other beers), and Liberty Malt Supply Co. of Seattle WA. Through their efforts to hold "Homebrew U", the homebrew community is able to benefit from talks such as this. I would also like to thank Dr. Mussche for his effort at presenting in English. Though the notes may not flow well in some places, I commend him and I must say that I doubt I could have done anywhere near as well if I where to try to make such a presentation in French.


Where the Wild Yeasts are!

By Dr. Roger Mussche

Table of Contents:

Introduction History Lambic and Gueuze Gueuze - Bottle Refermented Lambic Fruit Beers on a Lambic Base Uniqueness of Lambic & Gueuze

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 talbe 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 mouth amylases (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:

(Sheet 2: comparison of raw materials)

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 brewry, but largely exceed yeast counts. These fastly growing bacteria obtain their maximal concentration (about 10^8 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 Br. Bruxellensis and Br. 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 10^5 c.f.u./ml, developed during the first month as the counts on the day of bottling were only around 10^2 c.f.u./ml. [c.f.u. is an abbreviation for "colony forming units" -MDS]

Lactic acid bacteria at a level of around 10^6 c.f.u./ml remain consistently present after 5 months. At the day of the bottling their numbers were around 10^2/ml. Acetic acid bacteria disappeared after 3 to 4 months. At the day of the bottling their numbers were around 10^2/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 (10^2/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.

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
Lambic/Gueuze  Barley/wheat   Enterobacteria,      Van Oevelen, Mussche,
                              yeasts, Pediococcus  et.al. 1976, 1977
Acid Ales      Barley,rice,   Saccharomyces
               corn           Lactobacillus
Soy sauce      Soybeans/wheat Aspergillus,         Young & Wood, 1976
               EASTERN EUROPE
Kefir          Milk           Yeasts, Lactic
Tea Fungus     Tea Leaves,    Acetobacter, yeasts  Hesseltine, 1965
Sonti          Rice           Rhizopus, Yeasts     Hesseltine, 1965
Sake           Rice           Aspergillus,         Kodama & Yoshizawa
                              Lactic acid bact.
Pulgue         Agave          Zymomonas, Yeasts,
                              Lactic acid bact.
Merissa        Sorghum        Saccharomyces,
                              Lactic acid bact.
Bourbon        Maize/Rice/    Lactobacillis delbrueckii,
               Barley         Saccharomyces
Kwass          Rye/Barley     Yeasts,              Hesseltine, 1965
                              Lactic acid bact.
Weissbier      Wheat,Barley   Saccharomyces,
                              Lactic acid bact.
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

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

Table 4. Comparison between Saccharomyces and Brettanomcyes yeasts. [I've changed the table layout so it can be reproduced here. Theinformation 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,

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,
   Fresh hop preparates            Annuated hops
      as whole hops,
b. Brew-method
   Decoctie-method                 Turbid mash 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
     after propagation
f. Fermentation-maturation
     +- 8C - 6 days                6 months and 24 months
     +- 2C - 1 month

Malt                Wheat
Milling            Milling          500L at 55C
100 kg                 |                  |
   |                   |                  |
   +-------------Mash at 45C--------------+
                       | <-------- addition H2O of 90C
                 Mash at 52C
Taking of   <----------+
turbid mash            |
                       | <-------- addition H2O of 90C
                 Mash at 65C
Taking of   <----------+
turbid mash            |
                       | <-------- addition H2O of 90C
                 Mash at 72C
Taking of   <----------+
turbid mash            |
   |                   |
Heating to 85C ------->+
               Filtration at 78C
                       | <-------- wash with H2O of 95C
                       |           (in lauter tun)
               5-6h boiling  <-------- annuated hops 3kg/500L
               Hop - sieve - filter in coolship
               Cooling and air-inocculation
               Fermentation in wooden barrels or
               wood-coated tanks

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]

Sheet 8 - Belnding procedure for gueuze
Refermented in the bottle                      Filtered
(Brut, Fond, Artisanal)
               Blend of 2 parts of young lambic
                        1 part of old lambic
          |                                        |
          |                                     +sugar
          |                                    filtrated
          |                                       +CO2
          |                                        |
       Bottled                                  Bottled
          |                                        |
   Refermentation and                              |
   maturation in the                               |
   bottles                                         |
          |                                        |
        Sales                                    Sales

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
+125kg                 +25 to 35%                +aromas
sour-cherries          single juice              and concentrates
per 600L lambic              |                        |
or                           |                        |
+275kg                       |                        |
raspberries                  |                        |
per 600L lambic              |                        |
or                           |                        |
+250kg peaches               |                        |
per 600L lambic              |                        |
   |                         |                        |
Fermantation                 |                        |
   |                         |                        |
Bottling                 Filtration+             Filtration+
   |                      bottling                bottling
or refermentation            |                        |
in bottle                    |                        |
   |                      Bottling                    |
or filtration before     maturation                   |
bottling                     |                        |
   |                         |                        |
Sales                      Sales                    Sales

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]

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Last Updated: 16 August, 1995
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