biopolymers in solution - mitweb.mit.edu/nnf/people/clasen/harvard2002_presentation.pdf · –...

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Biopolymers in Solution – Rheo-mechanical and Rheo-optical Detection of Gels in Beer

Biopolymers in Solution

Rheo-mechanical and Rheo-optical Detection of Gels in Beer


Biopolymers are ofsubstantial industrial interestNearly unlimited resourcesMore likely to be bio-compatible and biodegradableGood mechanical propertiesAlready Designed and Optimized by nature to fulfill a certain task Easy to make derivatives with desired property profiles“Natural” name on ingredient lists

2


Application of Celluloseethers

* CM =carboxmethyl, HE = hydroxyethyl, HP = hydroxypropyl, M = methyl, SE = sulphoethyl, C = cellulose

thickeners, binding agents, stabilizers and emulsifiers

CMC, HPMC, MCFoodstuffs (sauces, milkshakes, bakery products)

thickeners, binding, emulsifying and stabilizing agents, film formation, tablet disintegrants

CMC, MC, HEC, HEMC, HPMCCosmetics (creams, lotions, shampoos), pharmaceuticals (ointments, gels, tablets, coated tablets)

friction reduction, water retention, enhanced ignition processes

MC, HPC, HPMCEngineering (extrusion, electrode construction, ceramic sintering)

anti-redeposition power, wetting ability, suspending and emulsifying agents

CMC, HEMC, HPMCDetergents

water retention, flow characteristics, surface activity

CMC, CMSEC, HEC, HPC, HPMCDrilling industry , mining (drilling fluids)

protective colloid, surface activityHEC, HPC, HPMCPolymerization

adhesive and film-forming properties, thickening, soil release)

CMC, MC, HPMC, CMSECTextile industry (sizes, textile printing dyes)

agents for binding and suspending, sizing aids and stabilizers

CMC, HEC, HEMC, HPMCPaper manufacture

stability of suspension, thickening, film formation, wetting

CMC, HEC, HEMC,HPMC, HEMCMCPaints

water retention capacity, stability under load, adhesive strength

MC, HEMC, HPMC, CMC, HEMCMCConstruction materials (plasters, filler, pastes)

FunctionCellulose derivative*Application


Examples of Biopolymers in Solution

Xanthan:even distributionvisual impressionnatural pourabilityavoiding “sliminess“good “mouthfeeling”

HydroxyethylStarch:hypoallergenicenhanced osmotic pressurecontrolled diffusivity

Blood plasma volume expander

“Orbitz” Soft Drink

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Polysaccharides as Food Additives

Starch

GummiArabicum

Pektin

Alginates

Galactomannanes

Fermentation polymers

Celluloseether

Polymer Application

in Bakery Products and other Cereal Based Goods, Thickener for Soups and Sauces, Jams and Dessert Gels, etc.

Potatoe starchYellow maize starch Waxy rice starchWheat starch

Thickener for Ice Cream, Elasticity Enhancer in CandyE 414

Gelling agent for Gels, Preserved Food, Desserts,Thickener, Protective Colloid for Emulsions

E 440

Thyxotropic agent, Coating agent,Thickener in Ice Cream, Jams and Dessert GelsGelation agent in ready-to-use Preparations;Stabilizer for Ice Cream, Puddings, Dairy Products, Frozen Food and

Instant Meals, Emulsions und Suspensions as indigestible Starchsubstitute in Low-Energy-Food

Algin E 401Carrageenane(ι−,κ−, λ-) E 407Agar E 406

in Cheese for enhanced Water Retention, in Ice Cream for reducedSugar Crystalisation, in Salad Dressing for Emulsion Stability,

in Frozen Foods and Water Containing Instant Meals against Synärese,in Softdrinks for superior Mouthfeeling, as indigestible Starch Substitute in Low-energy-food

Guar E 412Locust BeanGum E 410

Stabilizer for Milk and Milk Products, Dressings and Canned Food, Thyxotropic Thickener in Ketchup und Sauces

Xanthan E 415

Emulsifier, Stabilizer and Thickener for Ice Cream, Milk and Fruit Juices, Soups and Sauces,

Binding Agents, Protecive Colloid

NaCMC E 466MCE 461HPC E 463HPMC E 464


Biopolymers in “Natural “ Products

Are generally not considered to be dangerous because of longtime experience

Do not have to be explicitly mentioned on an ingredient list

Are they therefore boring and not worth investigation?

4


Natural Product Beer

“Deutsches Reinheitsgebot aus dem Jahr 1516”

(German purity law of 1516)

beer may only consist of four ingredients:

o Watero Barley Malto Hopo Yeast


Problems caused by Biopolymers during the Beer Brewing Process

• Improper malting and/or mashing results in high viscosity that lowers the extract yield and affects wortrun-off

• During storage periods a gelatinous precipitate is sometimes formed which

blocks filter media in the final filtrationforms “Fisheyes” in the final productthe gel consists to 99% of (1,3)(1,4)-β-D-Glucan

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(1,3)(1,4)-β-D-GlucanLinear Polysaccharide consisting of β-Glucose monomers30% (1,3)-glycosidiclinkage70% (1,4)-glycosidiclinkageStatistical distribution of the linkagesOnly isolated (1,3)-glycosidic linkages

⇒Hot water soluble


Sources of (1,3)(1,4)-β-D-Glucan• (1,3)(1,4)-β-D-Glucan

is a structure-polysaccharide in the cell walls of:

BarleyOatLichenan (Island Moss)Yeast

Schildchen Mehlkörper (Endosperm)

Blattkeim

Wurzelkeim

Aleuron- oder Kleberschicht

Spelze

Fruchtschale und Samen

Root Seed

Leaf Seed

Endosperm

Spelt

Alueron

Fruit Hull

Shield

• 70% (1,3)(1,4)-β-D-Glucan• 20% Arabinoxylan• 6% Protein• 2% Cellulose

Barley:

The Endosperm of Barley consists of:

6


The Beer Brewing Process

(1,3)(1,4)-β-D-glucan is enzymatically freed from the cell wall during soaking, germination and kiln drying processDuring the malting and mashing process the glucan is enzymatically degraded

Barley Malt Wort Beer

Water Hop Draff

1. Soaking2. Germination3. Kiln Drying

1. Grinding2. Mashing3. Refining

4. Wort Boiling

1. Fermenting3. Storing3. Filtering4. Bottling

YeastWater


Isolation of β-Glucan• From beer: • precipitation from chilled

solutions• Forced gelation under shear

• From Draff: • coupled enzymatic and chemical

extraction

• From Barley Oat or Lichenan:

Gerste

Rohextrakt( -Glucan, Arabinoxylan, Proteine, Maltodextrine)β

β-Glucan

Mahlen

Kochen in 70 % EtOH

Zentrifugieren

Fällen in 50 % EtOH

Fällen mit 30 % (NH ) SO4 2 4

ProteineMaltodextrine

Arabinoxylan

Grinding

Barley

Boiling in 70 % EtOH

Centrifugation

Raw Extract

Precipitation in 50 % EtOH

Precipitation with 30 % (NH4)2SO4

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Polymer AnalysisCode Origin M w M n M w/M n [η ] kH

(103 g/m ol) (103 g/m ol) (cm 3/g) (g/m l)

Barley G lucan SEC/M ALLS/DRI Viscosim etry GG375 Chem ically isolated from Barley 374 236 1,6 509 0,855 GG300 Megazym e 298 206 1,5 458 0,832 GG275 Megazym e, St.* 274 179 1,5 444 0,762 GG200 Megazym e, St.* 202 140 1,4 333 0,772 GG165 Megazym e 166 86 1,9 255 0,621 GG140 Megazym e, St.* 140 90 1,6 256 0,726 GG100 Megazym e 103 74 1,4 211 0,632 GG70 Ultrasonically degraded GG100 72 47 1,5 172 0,637 GG50 Ultrasonically degraded GG165 43 33 1,3 115 0,565 GGBeergel Isolated from sheared Beer 56 36 1,6 n.b. --- GGFrost Isolated from frozen Beer 23 17 1,4 n.b. --- Oat G lucan HG220 Megazym e 218 152 1,43 335 0,747 HG40 U ltrasonically degraded HG220 40 32 1,25 n.b. --- Lichenan LN55 Megazym e 55 32 1.72 n.b. --- Glucan Code Glucose Xylose Other Sugars

Acid Hydrolysis and Borat-Complex-Anion Exchange-Chromatographie com. available barley glucane (Megazyme )

GG300 ... GG50 97,5 2,5 0*

Glucan isolated from barley GG375 95,9 3,8 0,3* Glucan from beer GGFrost 96,2 3,8 0* Glucan from beer gel GGBiergel 96,3 0 3,7* Oatglucan HG 220 99,7 0 0,3* Lichenan LN55 97,2 0 2,8*

Protein < 1,9% (KJELDAHL andCHN-Analysis)


Definition of a GelCompositional Definition:

Colloidal System with at least 2 phases one of which forms a 3-dimensional network that acts like an elastic solid

Structural Definition:Highly ordered mesophase structures; covalent polymer networks; physical networks with ordered domains; disordered rodlike structures

Phenomenological Definition:Semi-soft, firm material made from two components (small solid concentration) that has a frequency independent storage modulus G’ and a loss modulus that is at least one decade lower

10-1 100 101 10210-1

100

101

102

103

104

105

G'

|η*|

G''

G''

G'|η*|

|η* |

/ [P

a·s]

ω / [rad·s-1]

10-2

10-1

100

101

102

103

104 Barley Glucan GG165Molar Mass: 165.000 g/mol6% (w/w) in H2O

Blue: freshly prepared SolutionRed: after 72h

G'

G'' /

[Pa]

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Time dependent Gelation of β−Glucan

• The gelation of β-glucan is thermo reversible (Tc = 349K - 355K)

• The solution stays in the sol state for an induction period before the onset of gelation

• Problem: How to find– Gelpoint– Speed of

Gelation

0 10 20 30 40 50 60100

101

102

103

104

|η* | /

[Pa·

s]

t / [h]

100

101

102

103

104

|η*|

G''

G'

G',

G''

/ [P

a]


tanδ = 1 as the Gelpoint?

• At tan δ = 1 (G’=G’’) the elastic properties become dominant over the viscous properties

• But the crossover of G’ and G’’ is not independent of the frequency

0 10 20 30 40 50 60

10033.3

103.3

1 0.33

0.01

0.1

1

10

100

tan δ

t (min) ω (rad/s)

40 min

29 min

Barley GlucanMolar Mass: 103.000 g/mol10% (w/w) in H2O

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Stress Relaxation Test • Shear Modulus in the linear

viscoelastic regime as measured for the onset of build up of a solid structure

• Good method to detect the buildup of a global network

• But every data point is a single experiment:

– Large amount of samples needed

– Time

0 20 40 60 80 1000

2

4

6

8

10

12

G(t)

(kP

a) a

t t =

45

s

t (min)

Gel Point

Barley Glucan GG100Molar Mass:100.000 g/mol10% (w/w) in H2O( ) ( )tG t

τγ

=


The Elasticity Increment• The Elasticity Increment

IE (the maximum slope of the storage modulus G’ ) is a measure of the gelation speed

• It also gives a gelation point

• IE is independent of the Frequency

• IE includes the induction period, the shape of the curves is the same for a certain polymer solvent system

0 10 20 30 40 50100

101

102

103

104

105

IE= 0,05 h-1

IE = 0,91 h-1

IE = 5,28 h-1 50.000 g/mol 103.000 g/mol 298.000 g/mol

10% (w/w) in H2O

G' /

[Pa]

t / [h]

maxE t

GlogI

∂

′∂=

10


Speed of Gelation • Elasticity Increment IE:

increasing Concentration

increasing Molar Mass

increasing Polydispersity

4 5 6 7 8 9 1010-2

10-1

100

101

MW= 43.000 g/mol, MW/Mn = 1,25 MW= 72.000 g/mol, MW/Mn = 1,49 MW= 166.000 g/mol, MW/Mn = 1,93 MW= 103.000 g/mol, MW/Mn = 1,39 MW= 298.000 g/mol, MW/Mn = 1,45

I E / [h

-1]

c / [% w/w] ⇒ The spontaneous Gelation is determined by the low molar mass molcules

⇒ No spontaneous Gelation below a critical concentration


Shear induced Gelation• Barley glucan (molar mass

298.000 g/mol) shows an induction period > 4h at a concentration of 10% (w/w)

• This induction time can be shortened by applying a shear rate of 2s-1 for 2 min and a rest period of 10 min (1 cycle)

• After 3 cycles (44 min) a global network has formed

0 2 4 60

40

80

120

100 %

73 %

35 %

26 %

Creep recovery

after 3. Cycle

after 2. Cycle

Creep test fresh

after 1. Cycle

γ / [%

]

t / [min]

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Shear induced Gelation in dilute Solutions

• A dilute solution of barley glucan (Mw = 165,000 g/mol, c = 0.025%), sheared for 10 min with an Ultraturrax (10.000 rpm) shows a precipitation of a gel-like sediment that contains 95% of the former dissolved glucan after 96 h.

• The gel formed at early gelation stages consists mainly of the high molar mass parts

20 24 28 32Eluate Volume / [ml]

SolutionGel

Barley glucan 165.000 g/mol

Eluation Spectrum of the Size-Exclusion-Chromatography (SEC) after 48 h and 96 hours:

The shear induced gelation is caused by the high molar mass molecules


Sol State of β-Glucan

10-2 10-1 100 101 102 103 10410-3

10-2

10-1

100

101

102

Mw= 224, 3,0% Mw= 166, 3,0% Mw= 166, 2,0% Mw= 166, 1,0% Mw= 166, 0,5%

Mw / 103 [g/mol] Mw= 285, 6,0% Mw= 285, 4,0% Mw= 285, 3,0% Mw= 285, 2,0% Mw= 285, 1,7% Mw= 285, 1,0% Mw= 285, 0,5% Mw= 285,0,25% Mw= 285, 0,1%

η / [

Pa⋅s

]

g / [s-1]10-2 10-1 100 101 102 103

10-2

10-1

100

101

102

103

104

G' G'' Mw / 103 [g/mol] 6% Mw= 285, 6,0% 4% Mw= 285, 4,0% 3% Mw= 285, 3,0% 2% Mw= 285, 2,0% 1% Mw= 285, 1,0%

G',

G''

/ [Pa

⋅s]

ω [rad/s]

• During the induction period, the glucan solution behaves as a typical viscoelastic fluid, no evidence of aggregates

12


Birefringence in Polymer solutions

d'n2'0λ∆π

=δ

Phase-difference

Field Vector E'0

Field Vector E'1

d'n2'0λ

=δ

δ’


Stress-Optical Rule• In the scope of the stress-

optical rule, it is found that, independent from the shear velocity:

• - the mean orientation of the segments and the first principal tensor τI take up the same angle in relation to the shear direction ( φ = χ )

• - the birefringence ∆n‘ (= n‘I -n‘II) is proportional to the first principal tensor difference ∆τ(= τI - τII )

principal tension ellipsoid

segment orientation

τI

τII

τI

τII

n'I

n'I

n'II

n'II

∆n‘ = C ⋅ ∆τ

13


Flow Birefringence of β-Glucan

10-2 10-1 100 101 102 10310-9

10-8

10-7

10-6

10-5

10-4

3,0% 2,0% 1,0% 0,5%

∆n' /

[-]

g / [s ]10-2 10-1 100 101 102 103

10-10

10-9

10-8

10-7

10-6

10-5

10-4

Mw / 103 [g/mol] Mw= 285, 6,0% Mw= 285, 4,0% Mw= 285, 3,0% Mw= 285, 2,0% Mw= 285, 1,7% Mw= 285, 1,0% Mw= 285, 0,5% Mw= 285,0,25% Mw= 285, 0,1%

∆n' /

[-]

g / [s ] • The Flow Birefringence shows deviation from the expected linear dependence on the shear rate in the Newtonian regime

Mw=166,000 g/mol


Stress Optical Coefficient

21

221 N4 +τ⋅=τ∆

φ⋅′∆⋅⋅

=τ 2sinnC2

121

100 101 102 103 104100

101

102

103

104

τ21 N1 6,0% 4,0% 3,0% 2,0%

τ 21, N

1 / [P

a]

g / [s-1]

10-1 100 101 102 1030

2x10-8

4x10-8

6x10-8

8x10-8

6,0% 4,0% 3,0% 2,0%

∆n'/∆

τ / [

Pa-1]

g / [s-1]

• The Stress-Optical Coefficient C can be calculated from the birefringence and shear experiments

• The Stress-Optical Rule is valid only for high shear rates

Mw=285,000 g/mol

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c(x)

x

c(x)

x

c(x)

x

mäßig konzentrierteNetzwerklösung

mäßig konzentriertePartikellösung

mäßig konzentrierteNetzwerklösungmit Aggregaten

∆ ∆ << c/ x 1∆ ∆ >> c/ x 1

Form Birefringence

τ⋅⋅

⋅

∂∂

+τ⋅=+Tk

Ncn

ncCnn

B

2

fiForm birefringence is only detectible

• if

• and if there is a “form”:

2n 0c

∂ ≠ ∂

c 1x

∆ ∆


Intrinsic vs. Form Birefringence

In the case of finite expandable or shear instable aggregates the form birefringence(aggregates) is overcompensated by the intrinsic birefringence (polymer segments) above a critical shear rate.

10-2 10-1 100 101 102 10310-10

10-9

10-8

10-7

10-6

10-5

∆n'i (Intrinsic Part)

∆n'f (Form Part)

∆n' /

[-]

g / [s ]

10-2 10-1 100 101 102 10310-10

10-9

10-8

10-7

10-6

10-5

∆n'i (Intrinsic Part)

∆n'f (Form Part)

∆n' /

[-]

g / [s ]

10-2 10-1 100 101 102 10305

1015202530354045

(Intrinsic φi Part)

φf (Form Part)

φ / [

°]

g / [s-1]10-2 10-1 100 101 102 10305

1015202530354045

(Intrinsic φi Part)

(Form Part) φf

φ / [

°]

g / [s-1]

Only the Orientation of the dominating part of the birefringence can be seen, creating typical tilted s-curves

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Orientation of the Birefringence

10-6 10-510-4 10-3 10-210-1 100 101 10205

1015202530354045

6,0% 4,0% 3,0% 2,0% 1,7% 1,0% 0,5% 0,2% 0,1%φ

/ [°]

g/gcrit / [-]

Barley GlucanMolar Mass:285.000 g/mol( )[ ]

ϕλ⋅γ

+

λ⋅γ+⋅

γ⋅λ=φ

bn2

b0

bn

b0

0

1

11arctan21

• Orientation of the intrinsic flow birefringence in the vincinity of the stress optical rule, dependinging on empirical parameters:

0γ ⋅λ


Reduced Birefringence

0.01 0.1 1 10 100 100010-9

10-8

10-7

10-6

10-5

10-4

6,0% 4,0% 3,0% 2,0% 1,7% 1,0% 0,5% 0,2% 0,1%

∆n'/η

0 / [P

a-1⋅s

-1]

g / [s-1]

( )[ ] ( )bn4

b02

0bn2

b00 11C2n

⋅⋅

ϕλ⋅γ

+⋅λ⋅γ+λ⋅γ+⋅γ⋅η⋅⋅=′∆

Barley GlucanMolar Mass:285.000 g/mol

Flow Birefringence in the vicinity of the stress optical rule, depending on empirical parameters:

• The high shear rates are dominated by the intrinsic birefringence and do not allow a detection of the aggregate behavior• Below a concentration of 1% a change in the flow behavior of the aggregates can be seen

γ⋅η⋅⋅=′∆γ<<γ

0C2nkrit

16


Dichroism of Aggregates in Solution

Loss of Intensity

Field Vector E''0 Field Vector E''1


Form Dichroism• Dichroism is only caused by

form partsOnly deformation behavior of Aggregates visible, no contribution of the single polymer chain

10-2 10-1 100 101 102 10310-10

10-9

10-8

10-7

6,0% 4,0% 3,0% 2,0% 1,7% 1,0% 0,5% 0,2% 0,1%

∆n''

/ [-]

g / [s-1]

• Plateau region is formed either by:Finite deformable Aggregates orEquilibrium between Aggregate breakdown and shear force


17


Orientation of Aggregates in the flow field

• The rise of the Orientation angle to more statistic orientations at higher shear rates indicates an aggregate breakdown

10-1 100 101 102 10305

1015202530354045

6,0% 4,0% 3,0% 2,0% 1,7% 1,0% 0,5% 0,2% 0,1%

θ [°

]

g [s-1]

Statistic Orientation

Total Orientation



Plateau regions of Flow Dichroism

• The reduced Dichroism ∆n’’/c in the plateau regions represents the sum of absolute Deformability of an aggregate and its concentration in Solution

• Below a concentration of 1%, ∆n’’/c reaches a new plateau value, indicating a change in the aggregate structure

10-1 100 101 102 10310-7

10-6

10-5

6,0% 4,0% 3,0% 2,0% 1,7%

∆n''/

c [m

l⋅g-1]

g [s-1]100 101 102 103

10-7

10-6

10-5

∆n

''/c

[ml⋅g

-1]

1,0% 0,5% 0,2% 0,1%

g [s-1]


18


Aggregate Size Depending on Concentration• At concentrations higher than

c*, the competition between possible aggregation points leads to smaller aggregates

• The formation of bigger aggregates below the critical concentration leads to a greater distance between the single aggregatesthe buildup of a global network between free polymer chains and aggregates is not possible below c* . It can only be induced, for example by a shear force that enhances the random contacts between the single aggregates

c < c* c > c*

T > 353K

T = 298K


Comparison of different glucansGlucan Mn

[g/mol]

Gelation Speed

IE / [h-1]

Transmission

T(sol) / T(gel)

lichenan 32 000 >50 64 barley glucan 33 000 5,3 31 oat glucan 32 000 0,86 24

20 40 60 80 100102

103

104

105

lichenan barley glucan oat glucan

65° C62 °C

73 °C

G' /

[Pa⋅

s]

T / [°C]

• The speed of the gelation shows differences between the different glucans, though their number average molar mass is nearly the same

• The transmission quotient as well as the gel softening temperature indicate the biggest cluster size for lichenan, followed by barley and oat glucan

• The cluster size coincides with the gelation speed

c = 6%

19


(1,3)-(1,4) Linkage Ratio

110 105 100 95 90 85 80 75 70 65 60 55

ppm

Haferglucan

Gerstenglucan

Lichenan

C-1

1,3- 1,4-Verknüpfung

17 18 19 20 21 22 23

1,3,5-Tri-O-acetyl-2,4,6-tri-O-methyl-D-glucitol

1,4,5-Tri-O-acetyl-2,3,6-tri-O-methyl-D-glucitol

Gerstenglucanbarley glucan

barley glucan

oat glucan

lichenan

linkage

13C-NMR Spectroscopie:

GC-Spectrum of methylated, hydrolised, reduced and acetylated barley glucan:

t / [min]

Polysaccharid Linkage Ration (1,3) : (1,4)

Methylation 13C-NMR

Lichenan - 27 : 73 Barley glucan 28:72 28 : 72 Oat glucan 29:71 29 : 70


5 10 15 20 25

Inte

nsitä

t

Zeit (min)

P = 3 6 7 8 954

Lichenan

Gerste

Hafer

(1,3)-(1,4) Linkage Distribution

t / [min]

(1 4)→(1 4)→

(1 4)→(1 3)→

Intensity

Oat

Barley

Lichenan

glucane P=3 : P=4 : P=5

Lichenan 23 : 1: 2,5 Barley glucan 2,5 : 1 : 0,12 Oat glucan 1,9 : 1 : 0,08

• specific enzymatic cutting of the (1,3)(1,4)-β-glucan chain with Lichenase (E.C. 3.2.1.73) only at (1,4) positions after a (1,3) linkage

•oligomer analysis with anion exchange chromatography shows that lichenan has the most regular distribution of cellotriose units and oat glucan the most irregular distribution

20


New Aggregation Mechanism

Old Mechanism:Association via

hydrogen bonds of long cellulose-like sequences ((1,4)-linkages)

β-(1,3)

{Cellotriose- Einheit

celluloseartigeSequenz

a) b)

cellulose-likesequence

cellotrioseunit

New Mechanism:Association via

hydrogen bonds of of regularly distributed sequences of cellotriose units


Conclusions and ways to optimizethe beer brewing process

• Addition of enzymes to degrade (1,3)(1,4)-β-D-glucanNot allowed for German beer

• Temperature control during the mashing processMostly optimized

• Avoiding high shear rates during pump-, transportation and filtration process

Slower output and therefore higher costs• Degradation of high molar mass (1,3)(1,4)-β-D-glucan with mechanical

methods to avoid shear induced aggregation• Selection and breeding of new barley with irregular distribution of

cellotriose units

biopolymers in solution - mitweb.mit.edu/nnf/people/clasen/harvard2002_presentation.pdf · –...

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