Lactic Acid Beverages:sour beer, (milk), & soda
Raj B ApteMatadero Creek Brewery
–sour ale art/science–in search of ginger beer plants
Chemistry of Flavor
Ethanol
Lactic acid
acetic acid
ethyl lactate
ethyl acetate
4-ethyl phenol
4-ethyl guaiacol
Saccharomyces
LactobacillusPediococcus
AcetobacterEnterobacteriaceae
Brettanomyces
Cast of charactersLactobacillus
basis of yoghurt, cheeselactic acidsalt resistant; hop sensitive?slime-producing
Pediococcusanaerobiclactic acidhop resistantslime-producing
Acetobacteraerobicpellicle forminginhibited by salt Saccharomyces
sugar fungusaerobic or anaerobicproduces alcoholbakers, brewers, vintners; hundreds of strains
Brettanomycesbritish fungus; british flavoraerobic or anaerobicBordeaux v. Davisproduces alcohol or acetic acidseveral species; many strains
(all photos stolen from websites without permission)
General Fermentation
+20.5%,P
pH 4.4, P
2%, PEnterobacteriaceae
<100++5----molds
70-110++308% P6-18%-Acetobacter
--100+2% P, pH 3.4
Pediococcus
60-1504-1% P8%8%Lactobacillus
40-95+4-100P15%, P-Brettanomyces
40-95+2-0.5%pH 4.520%, P-Saccharomyces
temp, F
oxygentimestarchAcetic acid
Lactic acid
Alcohol tolerance
salt
P produces ++ required-- toxic -, + minor effect
-
We are not in Kansas anymoreThere are many pathways, not just
glucose → 2 x ethanol + 2 x CO2
We have no way of determining alcohol accurately.
lactic acid
acetic acid
glucose
2,3-butanediol
succinic acid
formic acidacetaldehyde
S CoA
pyruvic acid
6-phosphogluconic acid
HeterolacticPathway
Embden MeyerhofPathway
lactic acid
CO2 , H2, H2S
2CO2
ethanol + lactic acid, ethanolCO2 bubbles
acetic acidH2 and CO2 bubbles
lactic acid, formic acid, succinic acid, acetic acidno bubblesacetic acid
ethanolethanol
CO2
Entner Doudoroff Pathway
CO2
ethanol CO2
60--4.76Acetic
17611.84.04Ascorbic
90--3.86Lactic
1345.053.40Malic
1924.743.03Citric
1504.543.02Tartaric
Mass/MolepKa2pKa1Acid
Titratable acidity (TA) and pH, defined10,000ppm = 1%
1ml of 0.1N NaOH is equivalent to 9mg lactic acid
like heatlike temperature
how many molecules of acid, by weight
-log10([H+])
titrate with base to neutrality“how much drano do you add to your beer to get pH 7.0”
use pH paper or meter
determines tastedetermines biological activity
measures quantity of acidmeasures strength of acid
TApH
4.1Leinenkugel lager
0.63.513.5Preston Merlot, 2000
0.73.013.0Landskroon Chardonnay, 2003
0.83.56.0Matadero Creek Kriek (me)
0.63.36.0Matadero Creek Cask Porter (me)
0.73.57.0Kriek de Ranke
0.73.64.3Morte Subite Peche
1.52.86.0Hanssens Oudbeitje
1.43.45.0Cantillon Iris
1.03.47.3Petrus Aged Pale
1.53.16.2Verhaeghe Duchesse
TA tartaric
w%pH%
abv
4.9
fiction
Furniture Legs and the Role of O2
2Saran
79Nylon (not oriented)
4650Polycarbonate PC
20Vinyl
400Polyethylene terephthalate copolymer PETG
8586Low-density polyethylene LDPE
2325High-density polyethylene HDPE
7200Wood, Oak
Oxygen Permeability cc-mil/m2-day-BarMaterial 200L barrels admit 20cc/L of oxygen per year = 100cc/L air.
For 20L carboy, that’s 2L of air.
Brett can grow but will not thrive without oxygen. If you use cultured Brett, the large cell population may overcome this.
negative Pasteur effect = Brett wants O2
Tank Volume [L] O2 cc/L.yearBurgundy barrel 300Rodenbach tank, wood, small 12000 0.86Rodenbach tank, wood, large 20000 0.53HDPE bucket 20Homebrew barrel 40Glass carboy, 30cm vinyl immersion tube 20 0.31Glass carboy, silicone stopper 20Glass carboy, wood stopper, cross grain 20 0.1Glass carboy, wood stopper, end grain 20
8.5
220 23
17
≈ 1.0Advantages of Furniture Legs v. Barrels—Oxygen diffusion—Habitat for Brettanomyces—Toast/vanillin—Autoclaveable—$1— (danger of refermentation)
$1 Homebrew Oak Barrel
Furniture leg:hardware removedsoaked, toastedteflon tapeautoclavedinoculated with B claussenii
AAR Fermentation Dynamics: Succession
Weeks
log
(CFU
/ml),
eth
anol
(abv
)
0
1000
2000
3000
4000
5000
6000
7000
0 12 24 36 48 60 72 84 96 108 120
Acid
(ppm
)
0
1
2
3
4
5
6
7
8
9
0
1000
2000
3000
4000
5000
6000
7000
0 12 24 36 48 60 72 84 96 108 1200
1
2
3
4
5
6
7
8
9
2
3
4
5
4
5
pH
enterobacteriatotal yeastactidione-resistant yeastacetic acid bacterialactic acid bacteria
ethanolpHacetic acidlactic acid
data taken from Martens
Recipe
1. Mash– Grist 100% pale (wheat, caramel, &c).– 67C, 75C– boil with aged or fresh hops– starch may be added to wort by late additions of adjunct
2. Primary fermentation (1wk): fruity ale– Chr. Hansen Harmony: S. cerevisiae, Torulaspora delbrueckii, Kluyveromyces thermotolerans
(www.thewinelab.com)3. Secondary (6wk): acidity
– Lactobacillus delbrueckii4. Tertiary (2yr): acidity and Brett
– any combination of lactic bacteria and Brettanomyces– wood stopper (pitched or carried) – topping off
5. Dryhop and blend
Ward, Philosophical Trasactions of the Royal Society of London. B, Vol. 183 (1892) 125-197
Ward 1892, cont'd
...
Recent HistoryBees 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] 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.
[Pidoux 1989] 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.
[Pidoux 1990] Lactobacilli isolated from sugary kefir grains capable of polysaccharide production and minicell formation. J. Appl. Bacteriology 1990, 69, 311-320.
[Leroi and Pidoux 1993] 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?
0 1 2 3 4 5 6 7 8 9 100
1
2
3
4
5
6
7
8
9
10
GBP Fermentation Dynamics
Lactic Acid, g/LBacteria CFUEthanol, g/LS. florentinus, CFU
CF/
ml,
g/L
data from Leroi & Pidoux 1993
day
Pidoux 1990: Ropiness
Recipe: get your kids fermenting1 L water120-180g sugar, white or raw, malt20ml lemon/lime juice and/or zest4g ground ginger root powder1g of cream of tartar50g 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