Lactic Acid Beverages:
sour beer, (milk), & soda
Raj B Apte
Matadero Creek Brewery
–sour ale art/science
–in search of ginger beer plants
Chemistry of Flavor
Lactobacillus
Pediococcus
Brettanomyces
Lactic acid
4-ethyl phenol
ethyl lactate
4-ethyl guaiacol
Acetobacter
Enterobacteriaceae
ethyl acetate
acetic acid
Ethanol
Saccharomyces
Cast of characters
Lactobacillus
basis of yoghurt, cheese
lactic acid
salt resistant; hop sensitive?
slime-producing
Pediococcus
anaerobic
lactic acid
hop resistant
slime-producing
Acetobacter
aerobic
pellicle forming
Saccharomyces
inhibited by salt
sugar fungus
aerobic or anaerobic
produces alcohol
bakers, brewers, vintners; hundreds of strains
Brettanomyces
british fungus; british flavor
aerobic or anaerobic
Bordeaux v. Davis
produces alcohol or acetic acid
several species; many strains
(all photos stolen from websites without permission)
General Fermentation
salt
Alcohol
tolerance
Saccharomyces
-
20%, P
Brettanomyces
-
15%, P
8%
8%
Lactobacillus
Pediococcus
Acetobacter
Enterobacteriaceae
molds
Lactic
acid
Acetic
acid
starch
pH 4.5
0.5%
-
1% P
6-18%
oxygen
+
40-95
P
4-100
+
40-95
-
4
+
-
100
--
8% P
30
++
2%, P
pH 4.4,
P
0.5%,
P
2
+
-
-
--
5
++
P
--
temp,
F
2
2% P,
pH 3.4
-
time
produces
toxic
++ required
-, + minor effect
60-150
70-110
<100
We are not in Kansas anymore
glucose
Entner Doudoroff Pathway
There are many pathways, not
just
glucose → 2 x ethanol + 2 x CO2
Heterolactic
Pathway
ethanol
CO2
Embden Meyerhof
Pathway
We have no way of determining
alcohol accurately.
lactic acid
pyruvic acid
6-phosphogluconic acid
2,3-butanediol
acetaldehyde
CO2
lactic acid
formic acid
2CO2
S CoA
CO2
ethanol
acetic acid
ethanol
CO2 , H2, H2S
succinic acid
acetic acid
no bubbles
lactic acid, formic acid, succinic acid, acetic acid
H2 and CO2 bubbles
acetic acid
CO2 bubbles
ethanol + lactic acid, ethanol
Titratable acidity (TA) and pH, defined
pH
TA
10,000ppm = 1%
like temperature
like heat
-log10([H+])
how many molecules of acid,
by weight
measures strength of acid
measures quantity of acid
determines biological activity
determines taste
use pH paper or meter
titrate with base to neutrality
“how much drano do you add
to your beer to get pH 7.0”
Acid
pKa1
pKa2
Mass/
Mole
Tartaric
3.02
4.54
150
Citric
3.03
4.74
192
Malic
3.40
5.05
134
Lactic
3.86
--
90
Ascorbic
4.04
11.8
176
Acetic
4.76
--
60
1ml of 0.1N NaOH is equivalent to 9mg lactic acid
fiction
TA
tartaric
w%
%
abv
pH
Leinenkugel lager
4.9
4.1
Preston Merlot, 2000
13.5
3.5
0.6
Landskroon Chardonnay, 2003
13.0
3.0
0.7
Verhaeghe Duchesse
6.2
3.1
1.5
Petrus Aged Pale
7.3
3.4
1.0
Cantillon Iris
5.0
3.4
1.4
Hanssens Oudbeitje
6.0
2.8
1.5
Morte Subite Peche
4.3
3.6
0.7
Kriek de Ranke
7.0
3.5
0.7
Matadero Creek Cask Porter
(me)
6.0
3.3
0.6
Matadero Creek Kriek (me)
6.0
3.5
0.8
Furniture Legs and the Role of O2
Oxygen Permeability
cc-mil/m2-day-Bar
200L barrels admit 20cc/L of oxygen per year = 100cc/L air.
For 20L carboy, that’s 2L of air.
Wood, Oak
7200
High-density polyethylene HDPE
2325
Brett can grow but will not thrive without oxygen. If you use
cultured Brett, the large cell population may overcome this.
Low-density polyethylene LDPE
8586
Polyethylene terephthalate
copolymer PETG
400
Vinyl
20
Material
Polycarbonate PC
4650
Nylon (not oriented)
79
Saran
2
Advantages of Furniture Legs v. Barrels
— Oxygen diffusion
— Habitat for Brettanomyces
— Toast/vanillin
— Autoclaveable
— $1
— (danger of refermentation)
negative Pasteur effect = Brett wants O2
Tank
Burgundy barrel
Rodenbach tank, wood, small
Rodenbach tank, wood, large
HDPE bucket
Homebrew barrel
Glass carboy, 30cm vinyl immersion tube
Glass carboy, silicone stopper
Glass carboy, wood stopper, cross grain
Glass carboy, wood stopper, end grain
Volume [L] O2 cc/L.year
300
8.5 12000
0.86
20000
0.53
20
220 40
23 20
0.31
20
17 20
0.1
20
≈ 1.0
$1 Homebrew Oak Barrel
Furniture leg:
hardware removed
soaked, toasted
teflon tape
autoclaved
inoculated with B claussenii
AAR Fermentation Dynamics: Succession
7000
9
5
8
6000
Acid (ppm)
6
4000
5
3000
4
3
4
pH
5000
log (CFU/ml), ethanol (abv)
7
3
2000
2
1000
1
0
0
0
12
24
36
48
60
72
84
96
108
120
Weeks
enterobacteria
total yeast
actidione-resistant yeast
acetic acid bacteria
lactic acid bacteria
ethanol
pH
acetic acid
lactic acid
data taken from Martens
2
Recipe
1.
Mash
–
Grist 100% pale (wheat, caramel, &c).
–
67C, 75C
–
boil with aged or fresh hops
2.
starch may be added to wort by late additions of adjunct
Primary fermentation (1wk): fruity ale
3.
Chr. Hansen Harmony: S. cerevisiae, Torulaspora delbrueckii, Kluyveromyces thermotolerans
(www.thewinelab.com)
Secondary (6wk): acidity
4.
Lactobacillus delbrueckii
Tertiary (2yr): acidity and Brett
–
–
–
–
any combination of lactic bacteria and Brettanomyces
–
wood stopper (pitched or carried)
topping off
Dryhop and blend
–
5.
Ward, Philosophical Trasactions of the Royal Society of
London. B, Vol. 183 (1892) 125-197
Ward 1892, cont'd
...
Recent History
Bees Wine ... [is] a fermented drink which was most often produced in home kitchens and was
probably most popular in the 1920's to 1950's. The culture was usually kept in a glass container
by a window and grown in a mixture of water, brown sugar and black treacle (there are several
variations on the exact ingredients used). It was usually drained once a week and fresh water and
sugar were added. As the culture naturally multiplied any excess was either discarded or passed
on to others to begin new 'Bees wine'.
According to our old records 'Bees Wine' is a mixture of yeasts and bacteria. The bacteria are
Lactobacilli and an unknown Gram positive rod that forms a gelatinous sheath that coils and traps
the other cells in it. This is also responsible for a thick 'scum' which forms on the surface of the
liquid. The yeasts that have been isolated from the mixture include Saccharomyces cerevisiae,
Brettanomyces anomalus and Hansenula anomala.
The gelatinous lumps formed in the 'Bees Wine' rise and fall as carbon dioxide is produced and
released. Sugar, black treacle and ginger are fermented to produce a mildly alcoholic, rather
sweet drink. Lemon/orange peel is sometimes added ....
'Bees Wine' has several variations and is also known as 'Ginger Beer Plant', 'Palestinian' or
'Californian' Bees or 'Balm of Gilead'.
The NCYC still keeps a culture of 'Bees Wine' in the laboratory although this is purely for scientific
interest and none of the present staff have tried to make the 'wine' itself from the culture.
http://www.ncyc.co.uk/beeswine.php
* Not a wild yeast technique *
Bibliography
[Ward 1892]
[Pidoux 1989]
[Pidoux 1990]
[Leroi and Pidoux 1993]
The Ginger-Beer Plant, and the Organisms Composing
it: A Contribution to the Study of Fermentation-Yeasts
and Bacteria. Philosophical Transactions of the Royal
Society of London, B, 183 (1892), 125-197.
The microbial flora of sugary kefir grain (the gingerbeer
plant): biosynthesis of the grain from Lactobacillus
hilgardii producing a polysaccharide gel. MIRCEN
Journal 1989, 5, 223-238.
Lactobacilli isolated from sugary kefir grains capable of
polysaccharide production and minicell formation. J.
Appl. Bacteriology 1990, 69, 311-320.
Detection of interactions between yeasts and lactic
acid bacteria isolated from sugary kefir grains. JAB
1993, 74, 48-53. Characterization of interactions
between Lactobacillus hilgardii and Saccharomyces
florentinus isolated from sugary kefir grains. JAB
1993, 74, 54-60.
Found in the back of a freezer in Germany....
•
Gingerbeer:
□
balm of Gilead
□
California wees wine
□
Palestinian bees wine
□
tibicos?
□
Tibetan mushroom
□
Japanese water crystals
water/sugary kefir grains
Lactobacillus hilgardii
□
•
□
Bacterium vermiforme
•
Betabacterium vermiforme
•
Lactobacillus brevis
□
heterofermentative
□
anaerobic
□
mesothermic
□
dextran-slime coat
dextran-slime gel
Saccharomyces florentinus
□
•
□
Saccharomyces pyriformis
□
Zygosaccharomyces florentinus)
Wondrous Animalcules
Ginger and Mary-Ann
1”
Ginger Beer Plant
Milk Kefir Grain
Pidoux 1989
Temperature: key to sweet, lactic soda
Pidoux
1990
Leroi and Pidoux 1993: Parasitism?
GBP Fermentation Dynamics
10
Lactic Acid, g/L
Bacteria CFU
Ethanol, g/L
S. florentinus, CFU
9
8
CF/ml, g/L
7
6
data from Leroi &
Pidoux 1993
5
4
3
2
1
0
0
1
2
3
4
5
day
6
7
8
9
10
Pidoux 1990: Ropiness
Recipe: get your kids fermenting
1 L water
120-180g sugar, white or raw, malt
20ml lemon/lime juice and/or zest
4g ground ginger root powder
1g of cream of tartar
50g ginger beer plants
No mash/boil. No sanitation
Primary: 25-30C, 1-3 days, lock optional (for Brett)
Secondary: strain all solids, retain plant. Correction of sugar &
acidity. 25-30C, 1-2 days, in bottle/keg/siphon.
Tertiary: lager 5-10C, >1wk.
— spices
◦ cinnamon, cassia, coca, cola, cloves, nutmeg,
vanilla, hibicus (jamaica), dried figs, mint,
syrup/molasses, lemon/lime juice/zest, corriander,
fennel, cardamom, star anise, rice, tamarind.
◦ dry powder, puree, whole spice, extract
◦ tisane (short or long), direct
plant
sources
—
◦ DSM 2484 - Ginger beer plant
◦ share with others: dried culture easily mailed
◦ http://health.groups.yahoo.com/group/GingerBeer
Plant/
◦ fermentedtreasures.com
Kegs, Bottles, or Siphons