The relevance of

starch structure and

fermentable sugars The first studies combining:

* starch structure,

* gelatinisation temp

* diastatic power enzymes and

* fermentable sugars

@GlenFox9

Acknowledgements

• The University of Queensland

• QAAFI (Centre for Nutrition & Food Science)

• QDAF

• Prof Bob Gilbert

• Dr Ben Schulz (SCMB)

Past postgrads (barley starch)

• Peter Gous

• Shang Chu

• Sandra Balet (South Africa)

• Oi Wan Mo (Hons)

Current PhDs

• Wenwen Yu

• Sarah Osama (proteomics)

• Rex Quek (Hons)

Outline

Starch properties

Gelatinisation

Starch hydrolysis and fermentable sugars

Starch protein interaction

Starch (substrate) in grains

Starch = energy

Total starch (50-80% db) depending upon cereal type

Starch = Amylose:amylopectin; ratio approx. 25:75

Amylopectin most abundant component in grains (approx 30 to 40%, compared to 8 to 15% protein)

Starch = energy

Total starch (50-80% db) depending upon cereal type

Amylose (AM):amylopectin (AP); ratio approx. 25:75

Amylopectin most abundant component in grains (approx 30 to 40%, compared to 8 to 15% protein)

Granules: size, shape, ratios, AP content

Large granules have more AM

Impacts on gelatinisation temperatures ‒ higher GT - lower AM content and shorter AM chains

1. Know your substrate +

2. Know your enzymes

The mechanics of starch

hydrolysis during mashing?

Biochem 101: Substrate + Enzyme = Products

Virtually no research

A lot of research

Brewing 101: Starch + DP Enzymes = Fermentable sugars

Amylose LD Maltose

Amylopectin BA Maltotriose

AA Glucose

GA

Simple! Everything is under control, right

Raw materials

Malt (barley, wheat, sorghum)

Adjuncts (wheat, barley, rice, corn, ……) Liquid adjunct (sucrose)

All the solid material have variable starch structure

Starch in brewing

Range of starch in different cereals

Normal, high amylose, high amylopectin (waxy)

Major starch synthesis proteins (genes)

X

X

Ratios SS:BE:DBE

More BE to SS then

shorter chains and

increase in gel temp

Hierarchical levels of starch structure in cereal grain

Stationary phase (the columns)

Rubsam, H., M. Krottenthaler, M. Gastl and T. Becker (2012). "An overview of separation methods in starch analysis: The importance of size exclusion

chromatography and field flow fractionation." Starch-Starke 64(9): 683-695.

Size exclusion

chromatography

The separation of molecules a

Fully dissolve the starch in a solvent system which gives molecular dispersion

without degradation - DMSO/LiBr 80˚C

Measuring starch structure

Measuring starch

structure

Chain length – No

of glucose units

Size of the AP and

AM polymers

Environmental impacts on starch structure

-2.00E-01

0.00E+00

2.00E-01

4.00E-01

6.00E-01

8.00E-01

1.00E+00

1.20E+00

1 10 100 1000 10000 100000

w(l

og

X)

DP X

H Fitzroy Low N

H Fitzroy High N

H Grout Low N

H Grout High N

Y Fitzroy Low N

Y Fitzroy High N

Y Grout Low N

Y Grout High N

0.00E+00

2.00E-01

4.00E-01

6.00E-01

8.00E-01

1.00E+00

1.20E+00

1 10 100 1000 10000 100000

w(l

og

X)

DP X

Fitzroy Low N

Fitzroy High N

Grout Low N

Grout High N

Variation in amylose chain length (310 barleys and malts)

XAM – number of glucose units

0 1000 2000 3000 4000 5000 6000

A10

A7

A27

A3

A21

A28

A12

A20

A11

A24

B19

B14

A4

A25

B18

B13

B17

A22

B9

B15

B16

B6

B7

B5

B25

B29

B27

B31

B35

B24

B30

B33

S

a

m

p

l

e

s

Range in total starch – 47% to 64%

Range in total amylose – 22% to 32%

(25% published value)

Eight fold difference in average chain length

Over 8oC difference in gelatinisation temp

30% amylose

30% amylose

60% starch

60% starch

Starch and gelatinisation temperature in mashing

Factors influence gelatinisation temperature ‒ AP content (AM content)

‒ AP:AM

‒ AP and AM CLD

‒ Proteins

‒ Granule architecture| crystalline:amphorus

‒ Processing (malting and milling)

More AM increase GT

Shorter AM chains increases GT

Lower protein (higher KI) increases GT

14

Starch

Gelatinization

Fermentable sugars

Leached out starch chains

Starch structural effects on gelatinisation

Substrate + enzyme/s

=

products

Sweet (wort) spot

Gel temp

Enzyme activity (and life)

Starch to be solubilized prior to full enzyme activity

Longer to gel then less Enzyme, impact on FS profile

DP enzymes

Gelatinisation temperatures of different malts (low to high protein)

59.5a

64.5 fg

63.2 d

63.9e

61.5b

61.6b

62.6c

64.1ef

64.8 g

64.9 g

DSC curves

Changes to structure during mashing

unmalted (Black),

Malted (Red),

BSG (Blue) and

Wort (yellow)

• Coarse grist

• 1:4 G:L

• 65oC mash 60 min, ramp to

75 for 10 min

• Low protein (8.5% - top)

• High protein (12.0% - left)

(non-fermentable) Dextrins in wort

• Variation in residual

AP linkages and AM

chains

• Top - molecular size

• Bottom - oP

Brewers spent grains

• Coarse grist

• 1:4 G:L

• 65oC mash 60 min, ramp to

75 for 10 min

• Low protein (8.5% - left)

• High protein 12.0% - right)

What happens when we add an adjunct?

Brewing 101: Starch + DP Enzymes = Fermentable sugars

Amylose LD Maltose

Amylopectin BA Maltotriose

AA Glucose

GA

Brewing 101: Malt +adjunct + DP Enzymes = FS

Amylose LD Maltose

Amylopectin BA Maltotriose

AA Glucose

GA

Starch

Adjunct

LD

+ BA = FS

AA

GA

What happens when we add an adjunct?

Adjunct G:L ratio Gravity

(oPlato)

Maltose

(mg/kg)

Liquid 1:3 15.4 9.1

Liquid 1:4 12.6 7.9

Solid 1:3 17.7 8.3

Solid 1:4 13.3 6.9

Starch protein interactions

More than just starch

Protein matrix

Protein negatively correlated to starch

Large grain, more starch, less protein

Protein specs (9% to 12%)

Protein content and composition of fermentable sugars

Protein Glucose Maltose Maltotriose Total

Sugar

Total protein -0.3 -0.6 -0.73 -0.64

Malt protein -0.45 -0.79 -0.83 -0.79

Water- insoluble -0.33 -0.7 -0.77 -0.71

GLT0 -0.041 -0.353 -0.651 -0.401

GLT5 -0.445 -0.468 -0.697 -0.562

Hordein B -0.082 -0.144 -0.164 -0.147

Hordein D 0.026 0.066 -0.395 -0.071

Hordein C -0.558 -0.852 -0.754 -0.824

Hordein A -0.244 -0.572 -0.672 -0.575

• Coarse grist (12 different malts)

• 1:4 G:L

• 65oC mash 60 min, ramp to 75oC, hold for 10 min

• Proteomics on malt

• FS – sweet wort

Significantly negative correlations

Protein content and composition and fermentable sugars

Summary starch structure and quality

• Variation in starch structure (degree of branching and

length of AM and AP chains) from all cereals

• Shorter starch chains increase gelatinization temp

• Longer chains lower GT and increases maltose content

• Survival of DP enzymes temperature dependent

• Protein content negatively related to get temp.

• Knowing gel temp of malt may be helpful

• Can’t pre-program starch structure in barley (cereal) grain

• Currently investigating barley variety effects and flavour

• Sorghum for gluten-free beer

• Next step – fermentation tests (various yeast)

• What does the brewer want to know? GT; FS profile;?

Questions