Protein aggregation and hardening of nutritional bars as f(moisture)

Ted Labuza Peng Zhou & Amy TranUniversity of Minnesota

This research was supported by USDA, DMI and the Davisco Co.

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Typical Protein Bars

Nutrition Facts• 150 - 300 calories• 25 - 40g protein• 10 - 30g CHO• 0 - 5g fat

• Bodybuilders• Special protein

blend

Protein bar market

Est. $930 million in sales in 2006 (Mintel)35% sales growth from 2001-2006

Initial success followed by maturation

U.S. sales of nutrition and energy bars, 2001-06

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Example protein nutritional bar

Moisture ~ 15%(WB) 18%(DB)aw ~ 0.55

ProteinUp to 40% of total weight

Protein sourcesWhey protein (WPI/WPC/WPH)Caseinate/caseinMilk protein (MPI/MPC) Soy protein (SPI/SPC)Gelatin or hydrolysates

Sugar/sugar alcoholsGlycerol (glycerine)Maltitol and maltitol syrupOligofructosemaltodextrinXylitolSorbitol

Problems with protein bars

TasteMaillard ReactionTexture – bar hardeningLoss of nutritional value

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Whey protein stability in nutritional barProblem - hardening of nutritional bar during storage

Bar model test formula35% WPI25% Corn syrup25% HFCS10% Peanut butter5% glycerol

Moisture: ~15%aw: ~0.55Q10 ~ 3.6 Note initial jump presumed due to redistribution of water& humectants into protein particles as aw equilibration occurs but continuess at slower rate in long storage

0

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Storage (days)

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Possible mechanisms for hardening

1. Moisture & humectant redistribution into protein

particles.

2. Protein-protein interactions aggregation

3. Humectant effects on mobility, local viscosity/glass

transition, and protein ineraction

4. Maillard reaction effects on color & texture

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Why does hardening occur?1. Initial redistribution of water/humectants

Commercial bar model (2) stored at 35 degree C

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Stage A: Redistribution of water/humectants in matrix and particles after mixing

EVIDENCE : slight reduction in water activity over 1st 5 days

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2. Protein-protein interaction

Driving forces

Covalent bonding

Disulfide bonds

Non-covalent interactions

H-bonding, hydrophobic interactions, electrostatic interactions

% of wheyproteins SH S-S

β-Lactoglobulin ~50 1 2

α-Lactalbumin ~20 0 4

BSA ~10 1 17

α-Lactalbumin BSAβ-Lactoglobulin

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Example BSA % solubility loss @ 37°Liu et al Biotech Bioeng 37:177 1991

% s

olub

ility

loss

in 2

4 hr

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Example rbST stability @ 47°CHageman et al J Ag. Food Chem 40:342 1992

g water per g dry solids

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Figure (A).The sorption isotherm at 50°C (m0 is 4.6 g water/100 g protein)

Figure (B). Aggregation of insulin after 24 hr at 50°C as f (%RH)

Insulin aggregation as a function ofwater sorption of the protein

Costantino and others 1994, Pharm Res 11(1): 21

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Simplified bar model system

Bar simplified model system (WB): WPI and buffer system

BioPRO whey protein isolate from Davisco

Protein, 97.4% on dry basis

Lactose, < 1% of dry basis (minimal Maillard reaction)

Fat, 0.3% of dry basis ( lipid oxidation minimal)

Phosphate buffer (10 mM, pH 7)

WPI/buffer: 3/2 (WPI, 60% of the total weight)

0.67:1 water to protein ratio

Sodium Azide (0.05% of the total weight)

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Extent of whey protein aggregation as f(t,T)

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0 20 40 60 80 100 120Storage time (days)

Aggregation Rate

Q10 aggregation ~ 3.3 from 23 to 45 °C or 11 fold factor

So 1 months at 45 °C = 11 months at 23 °C

thus ~ 2.5 weeks at 45 °C = ~ 6 months at room temperature

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Aggregate particle formation

Changes in micro-structure (scanning electron microscopy)

1µm 1µm

�Storage at 45 °C for 3 months(Particle diameter 50~100 nm)

Fresh control

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Changes in the conformation of whey protein molecules by DSC

0.05

0.1

0.15

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0.25

0.3

20 40 60 80 100

Temperature (Degree C)

Control

45 C, 100 days

α-Lactalbumin and BSA

β-Lactoglobulin

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Confirmation by FTIR for WPI

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Mechanisms of protein aggregation

Solubility of aggregates in various solutions

Solutions Aggregate solubility(%)

Buffer (10 mM, pH 7) 4.4 ± 0.6Buffer with 0.1% SDS 7.2 ± 0.5Buffer with 6 M guanidine HCl 10.9 ± 0.7Buffer with 8 M urea 11.6 ± 1.7Buffer with 10 mM dithiotreitol 92.2 ± 0.9Buffer with SDS and dithiotreitol 97.1 ±1.7

Non-covalent

Covalent(Disulfide bond)

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Changes in the texture of whey protein bar model system

Texture measurementTA-XT2 texture analyzerPlunger: 3mm diameterCompression speed: 1mm/sDeformation strain: 50%Hardness is recorded as the maximum force during the compression

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Aggregation and Hardness of whey system vs time

Formation of aggregates Hardening in texture

1

10

100

1 10 100Storage time (days)

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Suggested primary mechanism for texture change in whey bars due to disulfide bond interactions

Fresh bar base

Formation of separate aggregates between tiny particles

Formation of aggregate network

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3. Potential Influence of sugars/polyols (humectants) on hardening in whey bar systems

Bar systems need an aw of < 0.7 to prevent microbial growth otherwise antimicrobial agents required

Accomplished by replacing water with sugars or sugar alcohols (polyols) as plasticizers in whey system

Lowers aw and will influence:Local viscosity of liquid phase which controls mobility & thus reaction rates (find maxima in aw 0.6 to 0.8 range)

Tg of system which affects molecular mobility and texture

Protein conformation

Maillard reaction (browning) if humectant has reducing groups (HFCS)

Crystallization (graining) if use sucrose to control Maillard

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0 °C

Te

Tg

Glassy state

rubberystate

Crystal melt lineSolution

freezing lineice

super sat solution

State Diagram

-140°C

and

boiling line

vapor

BAT = 23°C

0 0.2 0.4 0.6 0.8 1Water acti

Reaction rate & mobility as f(T-Tg)

Added humectant

Modified from Roos and Karel Food Tech 45(12): 66 1991

Tm

Tg dry

A= soft 15%wb

B= soft @ 8% wb

0 % solids 85% 100 %1 water activity 0.8 0.7 0.6 0.5 0.3 0

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Effects of sugar/polyols on lowering of aw

0.0

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0.4

0.6

0.8

1.0

0 20 40 60 80 100[Polyol/(Polyol+H2O)]%

Critical micro level

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Effects on texture after 1 week @ 45°C

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Effects of sugar/polyols on whey protein aggregation after 1 week @ 45°C

PG causes significant amounts of protein aggregationOther sugar/polyols delay the protein aggregation

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4. Maillard reaction in the whey bar

Driving forces Whey proteins are rich in lysine (>10 g/100 g protein) The presence of reducing sugars such as fructose/glucoseThe aw of most nutritional bars is between 0.5 ~ 0.7, in the reactive range for Maillard reaction

0 0.2 0.4 0.6 0.8 1Water activity

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Maillard browning problem

Storage at 45 °C for 7 Days

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Non enzymatic browning in protein bars

Model system: Initial water activity: 0.7825 days to 50% lysine loss (FDNB method) at 35°C

18.53g/100 g solidWater

20Microcrystalline

cellulose

20Apiezon B oil

30Whey protein

20Glycerol

10Glucose

0.3K-sorbate

%Ingredient

Schnickels, Warmbier, Labuza: Effect of Protein Substitution on Nonenzymatic Browning in an Intermediate Moisture Food System, Journal of Agricultural and Food Chemistry, 1976

Previous study – lysine loss

Color measurement

Minolta Chromameter Model CR-200

L values: 0 (black) to 100 (white)Previous study: 100% WPI bar 83 → 50 in 30 days at 35ºC

Small scale study – color changes

HFCS/CS bars stored at 35 C

0 1 2 3 4 5 Weeks of storage time

Storage study – HFCS/CS bars at 35 °C

Remaining reactive lysine in protein bars stored at 35 C vs time

y = 78.1e-0.3531x

R2 = 0.8785

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storage time (weeks)

% re

mai

ning

reac

tive

lysi

ne

Series1Expon. (Series1)

t1/2 = 10.5 days

Remaining reactive lysine of protein bars stored at 45 C vs time

y = 91.581e-0.2545x

R2 = 0.9543

10

100

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Storage time (days)

% re

mai

ning

reac

tive

lysi

ne

Maltitol bar

HFCS/CS bar

Expon. (HFCS/CS bar)t1/2 = 2.25 days Q10 =4.7

1 day AT 45 °C= 1 mo. @ 23 °C

Storage study – bars at 45 C

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NEB effects on protein quality @ aw = 0.65

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NEB effects on protein quality

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HOW to make and keep a soft bar and of high nutritional quality ?

Potential solutions:

1. Add reducing reagents and thiol-blocking reagents if allowed

2. Add whey protein hydrolysates (Lowering the Tg of bar system)

3. Control the types and ratio of humectants

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1 . Reducing or thiol blocking reagents

Try to slow down protein aggregation and hardeningReducing reagents: Cysteine, glutathioneThiol blocking reagents: N-ethylmaleimide

Model ID Protein (6g) Buffers (4g) FunctionB WPI 10 mM phosphate buffer ControlC-L WPI 0.06 M L-cysteine ReducingC-H WPI 0.45 M L-cysteine ReducingG-L WPI 0.06 M L-glutathione ReducingG-H WPI 0.45 M L-glutathione ReducingE-L WPI 0.06 M N-ethylmaleimide Thiol blockingE-H WPI 0.45 M N-ethylmaleimide Thiol blocking

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Storage of whey model WB at 45 °C

30 day equivalent to 1 year at 23 C

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2. Addition of protein hydrolysatesH1= 5.2% hydrolyzed H2= 8.8% H3= 14.9%

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Water activity

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Hydrolysate effectiveness is not related to greater water holding capacity

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Whey protein hydrolysates (Davisco)

Bar model formula (25% replacement of WPI with whey protein hydrolysates)

40% total protein

30% corn syrup

20% HFCS

10% glycerol

~ 15% water

aw = 0.65

Degree of hydrolysis 5.2% 8.8% 14.9%

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Hardness development at 45 °C Day 7 vs Day 0

WCFG model system with 40% proteins ~7 day at 45C equivalent to 3 months at 23 °C

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Hydrolysate substitution lowers Tg so softer based on T-Tg

WPI: y = -47.051Ln(x) + 205.86R2 = 0.9643

H1: y = -54.61Ln(x) + 199.22R2 = 0.9948

H2: y = -65.562Ln(x) + 212.89R2 = 0.9975

H3: y = -64.534Ln(x) + 200.85R2 = 0.996

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1 10 100Water content (g H2O / 100 g solid)

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Solutions reviewed

Add either reducing agents or thiol blockersThe reducing agents do NOT work for the model systemCannot find a food grade thiol blocking agent yet

Partly replace WPI with 25% whey protein hydrolysateMakes a softer bar, partially slowing down the hardening

Control type and ratio of humectantsNo propylene glycolEliminate HFCS, use sucrose instead for sweetness or artificialUse glycerol in combo with sorbitol, maltitol, and xylitol

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Questions and/or comments?