Starches, Sugars, Gases, and Nitrogen

Brett Taubman Appalachian State University

A.R. Smith Department of Chemistry Fermentation Sciences

28 May 2013

Starch • Makes up greatest

proportion of malt, ~58% dry weight

• Breakdown products make up most of the extract in wort

• Major starch components are amylose (22-26%) and amylopectin (74-78%)

• Chains of D-glucopyranose residues linked α-(1,4) in chains and α-(1,6) at branch points

Starch Degradation

• Occurs during mashing • “Diastase” is the mixture of malt enzymes that

catalyze hydrolytic breakdown of starch • Still uncertainties about roles of some of the

enzymes • Primary enzymes are α- and β-amylases, but

other enzymes play significant roles • Only β-amylase correlates well with diastatic

power as it is normally measured

Starch Degradation

• α-amylase is a mixture of isoenzymes primarily formed during malting

• α-Amylase-I occurs in small amounts, is resistant to pH, but inhibited by heavy metal ions, e.g., copper ions

• α-Amylase-II is “classical” malt enzyme, resistant to heat (particularly in presence of Ca2+), heavy metal ions, but inhibited by calcium binding agents (e.g., phytic acid) and has a pH optimum of 5.3 (unstable below 4.9)

• α-Amylase mix attacks α-(1,4) links in starch, slower at chain ends and none near α-(1,6) branch points

• Products include glucose, maltose, and mix of branched and unbranched oligosaccharides and dextrins

• Dextrins produced are substrate for β-Amylase

Starch Degradation

• β-amylase occurs in barley, but proportion of free enzyme increases during malting

• Comprises multiple forms also • Relatively sensitive to heat (dependent on barley) and

heavy metal ions, but resistant to pH (5.0-5.3 optimum) and chelating agents

• Catalyzes hydrolysis of penultimate α-(1,4) link of non-reducing chain ends of amylose and amylopectin, releases disaccharide maltose

• Will NOT hydrolyze α-(1,4) bonds near branch points in amylopectin or dextrins

• α-(1,6) links may be broken by limit dextrinase, releasing maltose or maltotriose

Starch Degradation

Courtesy of: How to Brew, John Palmer, 2006.

Measuring Starch Iodine Test • Amylose and amylopectin adopt helical shapes into which

iodine can fit • Resulting complex appears blue-black (amylose), red-violet

(amylopectin) – longer starch chains give more intense color (< 9 molecules for amylose and < 60 for branched chains don't show a reaction with iodine anymore)

• Only works with cold mash/wort samples • Iodine is not very water soluble, so: I2 + I− ⇌ I3

−

• Can use iodophor or iodine from pharmacy (best to dilute ~10X with alcohol) on white dish, chalk, drywall (not paper!) – don’t test in presence of grain/husks

• Lab analysis only slightly different

Sugars

• Yeast take up sugar in the following order: glucose, fructose, sucrose, maltose, maltotriose

• Yeast use glucose so easily that the presence of glucose suppresses yeast’s ability to use maltose and maltotriose

Sugars

• Testing for fermentable carbohydrates in sweet wort (mono-, di-, and tri-saccharides)

• Two main ways to do this are measuring yeast fermentable extract or chromatography (not an in-house option for most small breweries)

• Don’t give exactly the same values – yeast fermentable extract is yeast and environment specific

• Two chromatographic options – GC and HPLC

GC-FID Method

• Must make mono-, di-, and tri-saccharides volatile

• Do so by forming trimethylsilyl derivatives

• Fructose, glucose, sucrose, maltose, and maltotriose are separated from higher molecular weight, nonfermentable carbohydrates

• Can separate individual sugars • Figure shows sample

chromatogram of trimethylsilyl derivatives of fermentable mono-, di-, and trisaccharides of wort with approximate retention times

HPLC Method

• Can inject dilute, centrifuged/filtered sample onto column directly

• Easier, but separates sugars based on degree of polymerization

• Maltose recorded as “DP2”, maltotriose as “DP3”, etc.

• Can also quantify total carbohydrates to asses fermentable vs. total carbs

Alcohol 1. By distillation: distill alcohol from beer, mix with

distilled water and measure specific gravity 2. By GC: specific for ethanol, uses n-propanol as

internal standard 3. By NIR: determines alcohol content using absorbance

at near-infrared wavelengths a. Anton Paar Alcolyzer/Density meter beer analyzer b. Density determined via built-in density meter c. Also gives apparent extract, real extract, original extract,

real degree of fermentation, calories, etc. d. May be an issue with heavily dry-hopped beers…? e. Optional measurements of turbidity, color, and pH

Carbohydrate Content Can be measured by spectrophotometry or HPLC Spectrophotometry • dextrin (primary carbohydrate in beer) is hydrolyzed to

dextrose (glucose) by sulfuric acid • dextrose and phenol, in presence of sulfuric acid, react to

produce colored complex HPLC • Cation exchange resin-based column with refractive index

detector Both of the above methods give lower values than the “calculated carbohydrate” content – this should be used for labeling purposes

Calculated Carbohydrate Content

• Determine specific gravity, real extract, protein, and ash content of beer – Note: for ash, evaporate beer over water bath,

ignite evaporating dish to dull red until no more C, cool in desiccator and weigh to 0.0001 g

• Calculate carbohydrate content: carbohydrate/100 g beer = real extract – protein – ash carb/volume = carbohydrate/100 g x ((container vol (mL) x sp gr)/100) Note: beer volume should be measured at 20 °C

Oxygen Total Package Oxygen (TPO) = Head Space Oxygen (HSO) + Dissolved Oxygen (DO) • The only time oxygen should be intentionally injected into the process is

during transfer of sweet wort into fermenters, in line with heat exchange • Aeration of wort can be done with pure oxygen or filtered air – end result

~10 mg/L (ppm) • Packaging is major concern for introduction of oxygen – final product

specification for DO in beer typically < 0.2 mg/L (ppm), sometimes < 0.1 mg/L (ppm)

• During filling, oxygen pick-up must not exceed 0.02-0.03 mg/L (ppm) Several methods for measuring dissolved oxygen: • Membrane-covered oxygen electrode or cell • Optical dissolved oxygen sensors • Colorimetric determination

Dissolved Oxygen Colorimetric Method • Colorimetric determination of DO in beer by the reaction of oxygen

with reduced indigo carmine (disodium indigo disulfonate) • Beer is sampled in a test tube fitted with a self-sealing cap • Indigo carmine reduced with alkaline glucose is injected

(anaerobically) into the beer • The resulting blue color, formed by indigo carmine when completely

oxidized, is measured in a spectrophotometer • A calibration curve is prepared by adding varying amounts of indigo

carmine to samples of beer to cover the required range of oxygen concentrations

• This method is suitable for use in pale beers containing up to 2 mg/L (ppm) dissolved oxygen

• Recommended for calibration of DO analyzers

Total Package Oxygen

• Several instruments available for automated measurements of TPO

• Often combined with CO2 measurement • Samples can be taken directly from package –

don’t have to worry about introduction of oxygen during sampling

• Gives HSO and DO values separately as well

CO2 Pressure method for beer in tanks • Customary apparatus consists of a metal bottle with insulated handles and

means for measuring T and P (e.g., Zahm-Hartung CO2 Volume Meter) • Dependent on establishment by agitation of partial gas pressures in

headspace above beer in container at a particular temperature Pressure method for beer in bottles and cans • Similar to above, but uses piercing apparatus and absorption buret with

NaOH Volumetric method • Measures residual carbon dioxide in beer samples containing up to 1.6

volumes of carbon dioxide Volume expansion method • Determines dissolved CO2 in beer by an instrumental method using the

principle of volume expansion • Can be applied to all beers (in final package or from the tank) and covers

CO2 content from 2.47 to 6.20 g/L • Measurement unaffected by presence of other gases

Nitrogen

• Yeast assimilable nitrogen (YAN) = Free Amino Nitrogen (FAN) + Diammonium Phosphate (DAP, NH4

+) • N is the most important nutrient for healthy

fermentations • How to add nitrogen: DAP and Yeast Autolysate • DAP = NH4

+

• Yeast material = Amino Nitrogen, vitamins, minerals

Measuring Ammonium

Ammonium – Ion specific probe • Similar to pH probe; determines ion content based on activity of ions in solution • Ion Specific Probes can be purchased for any number of measurements, NH4

+ being one • Correlate electric potential produced through probe (ions passing through membrane) to concentration of that ion

Measuring FAN Based on reaction of alpha-amino acids (FAN) with ortho-phthalaldehyde (OPA) OPA binds to alpha amino acids, resulting in an increase in absorbance at 335 nm Proline – not an alpha-amino acid, is in higher concentrations and not utilized by yeast during fermentation Prepare a standard curve using iso-leucine (known quantities) Compare your sample to the standard curve to determine concentration Amino N + NH4

+ = YAN

Arginine Iso-Leucine Proline

Measuring FAN

• Ninhydrin method - similar to OPA method • Can be used to determine the amount of FAN in

wort or beer • The method measures amino acids, ammonia,

and, to some extent, end-group α-amino nitrogen in peptides and proteins

• The method is not specific for α-amino nitrogen since γ-aminobutyric acid, which is present in both wort and beer, yields substantial color with ninhydrin

• Measure color complex at 570 nm

Measuring Protein 1. Kjeldahl analysis 2. Combustion

a. Measures N formed during the combustion of the sample in pure oxygen

b. Gas is measured with a thermal conductivity detector c. Total N calculated based on the response from a known

chemically pure nitrogen standard d. Protein = N x 6.25

3. Spectrophotometric Method a. Applicable to beer stabilized with PVPP, beer not treated with

PVPP, and dark beer, defined as having ASBC color in the range of 15–50

b. Sample absorbance measurements are made at 215 and 225 nm

c. From these values, (plus the total polyphenol content for stabilized beer samples) the protein content can be determined