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