isomalto/ maltopolysaccharides bsc thesis biotechnology + code

BSc Thesis Biotechnology 044BCT

BSc Thesis Biotechnology + Code



Physical Properties of

IsoMalto/ MaltoPolysaccharides







Tom Otten

Tom Otten

Tom Otten

Tom Otten

Biobased Chemistry and Technology




February – Augustus 2016



Name course : BSc Thesis Biotechnology Number : YBT-80324 Study load : 24 ects Date : 15-08-2016 Student : Tom Otten Registration number : 940820632050 Study programme : BBT Report number : 044BCT Supervisor(s) : Piet van der Zaal and Piet Buwalda Examiners : Elinor Scott and Piet van der Zaal Group : Biobased Chemistry and Technology Address : Bornse Weilanden 9 6708WG Wageningen The Netherlands

Name course : BSc Thesis Biotechnology Number : YBT-80324 Study load : 24 ects Date : 15-08-2016 Student : Tom Otten Registration number : 940820632050 Study programme : BBT Report number : XXXBCT (you received this at the start of your thesis by email

from office BCT)

Supervisor(s) : Piet van der Zaal and Piet Buwalda Examiners : Elinor Scott and Piet van der Zaal

Table of Contents

Abbreviations ........................................................................................................................................... 1

Abstract .................................................................................................................................................... 2

Introduction .............................................................................................................................................. 3

Starch ................................................................................................................................................... 3

Modification & Application .................................................................................................................... 4

Rheology .............................................................................................................................................. 4

Thermal properties ............................................................................................................................... 5

α-4,6-Glucanotransferase .................................................................................................................... 5

IsoMalto/MaltoPolysaccharides ........................................................................................................... 6

Dextran ................................................................................................................................................. 6

Aim ....................................................................................................................................................... 7

Materials .................................................................................................................................................. 8

Methods ................................................................................................................................................... 8

Rheology .............................................................................................................................................. 8

Sample preparation .......................................................................................................................... 8

Strain sweep ..................................................................................................................................... 8

Thermal analysis .................................................................................................................................. 9

Sample preparation .......................................................................................................................... 9

Dry weight......................................................................................................................................... 9

TGA-MS............................................................................................................................................ 9

DSC .................................................................................................................................................. 9

Results & Discussion ............................................................................................................................. 10

Viscosity ............................................................................................................................................. 10

Shear behaviour ................................................................................................................................. 12

TGA-MS ............................................................................................................................................. 14

Dry weight .......................................................................................................................................... 15

DSC analysis...................................................................................................................................... 16

Conclusions ........................................................................................................................................... 19

Recommendations ................................................................................................................................. 20

DSC .................................................................................................................................................... 20

Rheology ............................................................................................................................................ 20

References ............................................................................................................................................ 21

Appendix ................................................................................................................................................ 22

DSC graphs ........................................................................................................................................ 22

1

Abbreviations

GtfB α-4,6-Glucanotransferase

IMMP IsoMalto/MaltoPolysaccharide

DP Degree of Polymerization

DE Debranching Enzyme

DSC Differential Scanning Calorimetry

TGA Thermal Gravimetric Analysis

Tg Glass transition temperature

To Onset temperature of gelatinization

Mw Molecular Weight

2

Abstract

An enzyme, named α-4,6-glucanotransferase (GtfB), was isolated from Lactobacillus reuteri. This

enzyme can be used to modify starch to form isomalto/maltopolysaccharides (IMMPs)(Leemhuis,

Dobruchowska et al. 2014). Previous research has focused on the enzyme function and the molecular

structure of enzyme products. Yet little is known about chemical and physical properties of IMMPs

besides their increased solubility and decreased digestibility. However when searching for applications

it is useful to know how the material will behave during processing or functions in the final product.

The IMMPs were compared with their native polysaccharide in several experiments. Using a

rheometer the viscosity was determined. The native polysaccharides gelled at low concentrations and

had shear thinning behaviour. Unlike the native samples, IMMPs in general have a low viscosity and

act as a Newtonian fluid. Using differential scanning calorimetry (DSC) and thermal gravimetric

analysis (TGA) the thermal properties were analysed. With TGA the onset temperature for thermal

degradation was determined.

IMMPs started to degrade at slightly lower temperatures than the native samples. The glass transition

temperature was investigated using DSC. The samples were put in standard aluminium measurement

crucibles, and subsequently sealed with a lid. For the DSC test samples were heated from 30°C to

240°C while measuring the heat flow (in Watts). As the glass transition causes a chance in a materials

specific heat, the transition is also seen as a sudden change in heat flow. However during the

measurement the weight of the samples decreased, most likely due to leaking crucibles. The weight

loss also made the measurements somewhat unreliable. From the difference in viscosity, shear

behaviour and degradation temperature it was concluded that IMMPs have less interactions either

because of the more flexible 1,6-linkage or smaller fragment size, most importantly they do not form

double helices through hydrogen bonding.

3

Introduction

Starch

Starch is a glucose, α-D-glucopyranose, polysaccharide naturally found in plants as a way of storing

energy. Most starches consist of between 20-30% amylose and 80-70% amylopectin and have a wide

range in molecular weight(Pérez and Bertoft 2010). Amylose and amylopectin consist of glucose units

linked from the 1st

to the 4th

carbon atom, see Figure 1. Additionally amylopectin has branching points

where a second chain is linked to the 6th

carbon atom.

Starch is stored in granules. Each plant has its own distinct starch granules, this is due to differences

in enzymes used for the production of these granules, the amylose/amylopectin ratio and molecular

weight (Mw). Starch granules consist of both amorphous and semi-crystalline rings, see Figure 2.

Amylose forms the amorphous regions whereas amylopectin forms the semi-crystalline areas. It has

been shown that glucose chains with a degree of polymerization(DP) of at least ten can form double

helices with each other(Gidley and Bulpin 1987). Most amylose and amylopectin chains exceed this

DP and will be able to form double helices. The α-1,6 branch points in amylopectin have been shown

to have high rotational freedom this allows them to collapse back on the main chain and form a double

helix(Gidley and Bulpin 1987, O'Sullivan and Perez 1999). This results in the formation of dense

clusters of amylopectin, which explains the more compact semi-crystalline areas.

Figure 2 (a) starch granule (b) semi-crystalline and amorphous regions (c) Close-up of semi-crystalline amylopectin region (d) close-up of double helices in amylopectin

Figure 1 Schematic representation of Amylose and Amylopectin

4

Modification & Application

Starch and derivatives have been used as emulsifier, viscosifier, stabilizer, cryoprotectant, gelling

agent and texturizer (Jobling 2004, Tharanathan 2005). Starch has also proved useful in many

industrial applications among which, glue, lubricant and ink binder in the printing industry (Brimhall

1944). Bioplastics are another prominent example of starch application, as bioplastics are becoming

more and more relevant as the fossil oil required for the production of conventional plastics will

eventually run out. Starch based plastics are also biodegradable which is great from ecological point of

view but makes them less suited for wet applications (Gaspar, Benk et al. 2005, Nafchi, Moradpour et

al. 2013). Another drawback of starch based plastics is their low flexibility or crispiness. This can be

overcome by using plasticizers low Mw particles that intervene in the secondary structure.

Rheology

When native starch is heated in excess water and subsequently cooled it will form a gel. This is an

irreversible two-step process consisting of gelatinization and retro gradation. This process is

characterized by the gelatinization temperature; this temperature varies slightly depending on the

amylose/amylopectin ratio. At this temperature the mobility of the amylose and amylopectin chains is

so high they no longer form the double helices that give the starch granules their rigidness. Because of

the unwinding of the helices hydration and diffusion of the starch granules occurs thereby bringing the

amylose in solution, see Figure 3. At this point all starch components will be in an amorphous state

however upon cooling they will return to the preferred double helix structure. The amylose chains then

associate over time to form a flexible network of amylose, amylopectin, water and starch granule

remnants (Hoover 1995).

Figure 3 The mechanism of starch gelatinization and retro gradation.

5

Thermal properties

When heated at low water content, 0-0.3 gwater/gstarch, the observed behaviour is quite different. Under

these conditions starch will not gelatinize but undergo a thermal transition called the glass transition

(Tg). Similar to gelatinization the mobility of the backbone increases and the chains start sliding past

each other. However as the water content is too low to dissolve the starch it goes from being a brittle

material to a flexible or rubbery one. Studies linked this to loss of viscosity, gain of stickiness and

collapse of the structure (Roos and Karel 1991, Laaksonen, Roos et al. 2001, Chuang, Panyoyai et al.

2016). The Tg of starch is hugely dependent on smaller molecules, plasticisers. These plasticisers

form hydrogen bonds with starch. Therefore starch can form less double helices. A very common

plasticizer is water. The relation between water content and Tg is shown in Figure 4. Increase in

mobility can result in the alignment and crystallisation of the polymer. However this rarely happens in

starch as the chain length and components are to inhomogeneous. Oligosaccharides (3-10 linked

units) might “melt” and form syrup before undergoing degradation to CO2 and H2O. Due to its size

starch usually doesn’t melt but starts degrading while still solid, this starts 320°C (Liu, Wang et al.

2013).

Figure 4 Water content and Glass transition plotted versus the water activity during desiccation.

α-4,6-Glucanotransferase

α-4,6-Glucanotransferase (GtfB) is an enzyme that targets glucose oligo- and polysaccharides with a

minimum DP of four. These saccharides, like starch, are normally composed of glucose units linked

from their 1st to 4

th carbon atom and forms products with links from the 1

st to the 6

th carbon atom; see

Figure 1. GtfB has several functionalities, it can hydrolyse (cleaving α-1,4 bonds), transfer (cleaving α-

1,4 and synthesizing α-1,6 and α-1,4 glycosidic linkages) and polymerize (α1,6 linked elongation of α-

1,4 oligosaccharides). This results in a wide product range, additionally the α-1,6 : α-1,4 ratio

increased with higher DP. Lastly the branching points in amylopectin seem to inhibit enzyme function

(Kralj, Grijpstra et al. 2011, Dobruchowska, Gerwig et al. 2012).

6

IsoMalto/MaltoPolysaccharides

The main product of starch modification with GtfB was named IsoMalto/MaltoPolysaccharide (IMMP) in

reference to the mix of modified and unmodified regions in the polysaccharide. In theory amylose can

be almost completely modified. Amylopectin is expected to have lower conversion as GtfB is inhibited

by the branching points thus only forming short α-1,6 regions at the non-reducing ends. Due to the

different functions of GtfB products are not limited to IMMPs for example oligosaccharides and

glucopyranoses may be formed as a by-product of the enzymatic conversion. IMMPs have been

shown to have potential as dietary fibre, this could make them useful food additives (Leemhuis,

Dobruchowska et al. 2014).

Figure 5 Schematic representation of IMMP and Dextran

Dextran

Dextran, a very similar group of polysaccharides consisting of an α-1,6 backbone with occasional

branching at the 3rd

or 4th

carbon atom have been studied more extensively. Therefore dextran has

been used as a model saccharide under the general assumption that IMMPs will behave as an

intermediate between starch and dextran. Most dextrans are easily soluble in water, with the exception

of highly branched dextrans. Interactions in solution were also minimal as most dextran solutions have

non-Newtonian behaviour(Das and Goyal 2014). They also have great thermal stability as they start

degrading around 300°C, which makes them valuable baking additives(Das and Goyal 2014,

Tingirikari, Kothari et al. 2014). Dextran has already been used as emulsifier, viscosifier, stabilizer,

cryoprotectant, gelling agent, texturing agent, water-binding agent, bio-flocculant and has been shown

to be a prebiotic (Kothari, Das et al. 2014).

Based on similarities between IMMP, starch and dextran it is assumed that GtfB modification will

cause a loss of viscosity and shear thinning behaviour. As the glass transition temperature(Tg) is

related to the molecular mobility and the α-1,6-linkage is more flexible than the α1,4-linkage, the Tg is

likely to go down. An increase in thermal stability is also expected as this is observed in both dextran

and dextrinized starch(Tharanathan 2005, Das and Goyal 2014).

7

Aim

The molecular structure of IMMPs has been analysed using ‘H-NMR and the enzyme function has

been studied using monosaccharides. Based on previous research it is possible to theorize about

physical properties. However besides a notable increase in solubility and solution stability, the effect of

GtfB on starch’s physical properties has yet to be experimentally proven. Therefore the goal of this

research will be to determine the effect of GtfB modification on the physical properties of starch to give

insight in future applications of IMMPs. The central research question is: “What is the effect of GtfB

modification on the physical properties of starch?”. This question was subdivided into rheological and

thermal properties. The viscosity and shear behaviour of IMMPs have been selected for determination

to get a general impression of rheological properties. The glass transition and degradation temperature

have been selected as interesting indicators of thermal properties.

To answer these questions several starches and their respective IMMPs as well as a α-1,6-

oligosaccharide were compared in several experimental setups. Using a rheometer to analyse the

viscosity and shear behaviour. Thermal properties were investigated with differential scanning

calorimetry and thermal gravimetric analysis.

8

Materials

Three polysaccharides were selected to investigate the effect of GtfB modification. These were potato

starch, waxy potato starch (Eliane 100) and Etenia (Etenia 457) all acquired from Avebe. These were

all modified using GtfB a second Etenia sample was treated with both GtfB and debranching enzyme

(Promozyme D2 pullulanase). The percentage of α-1,6 bonds was analysed pre- and post-modification

with ‘H-NMR. The resulting IMMPs will be referred to as potato IMMP, waxy potato IMMP, Etenia

IMMP, and Etenia IMMP+DE with respective α-1,6 percentage of, 25.5%, 6.1%, 8.2%, and 91.2%. The

native polysaccharides already had α-1,6 bonds; potato starch(3.7%), waxy potato starch(4.1%),

Etenia(3.2%). From the difference between the native and GtfB modified polysaccharides the increase

in α-1,6 bonds was determined to be 21.5% for potato starch, 2% for waxy potato starch, 5% for

Etenia, and 88% for Etenia+DE. For the rheology experiments a buffer was made containing Sodium

Chloride (Merck KGaA 6471 Darmstad, Germany) and Citric Acid (Sigma-Aldrich). A water bath

(Julabo Shaketemp SW22) was used for rheology sample preparation. An Anton Paar rheometer MCR

302 with DG 26.7(Double Gap 26.7mm diameter) measuring cup and cylinder controlled by Rheoplus

was used for the measurements. Thermal analysis was carried out on a Mettler Toledo DSC/TGA1

using standard 40µl aluminium crucibles with lids. Also a Thermostar gas analysis system with

prismaplus QMG220 mass spectrometer (Pfeiffer vacuum) was used to measure gas concentrations in

the outlet gas. The mass spectrometer was controlled by the Quadera software (Pfeiffer Vacuum).

Methods

Rheology

Sample preparation

For the rheological measurements several sample solutions were made from IMMPs and Vita Fibre

containing 0.01; 0.02; 0.03; 0.04; 0.05 w/v (g/l) and reference solutions were made from potato starch,

waxy potato starch and Etenia at 0.005; 0.01; 0.015; 0.02; 0.025 w/v concentrations. Weight volume is

given in gram polysaccharide per ml buffer, water containing 10 mM NaCl and 10mM Citric Acid. As

water was added in excess the water content of the polysaccharides was neglected. First the highest

w/v ratios were made using a 50ml volumetric flask and 2.5g of sample (1.125g for potato, waxy potato

and Etenia). The samples were put in a water bath at 90°C for 30 minutes, to dissolve the

polysaccharides completely. These solutions were then diluted with preheated buffer, vortexed, and

allowed to cool at room temperature. Subsequently the samples stored at 9°C overnight. After ±18

hours viscous liquids were selected for measurements while Etenia IMMP 0.04 w/v and 0.05 w/v were

discarded as these had gelled.

Strain sweep

To determine shear behaviour and viscosity an Anton Paar Modular Compact Rheometer 302 with a

DG 26.7(Double Gap 26.7mm diameter) measuring cup and cylinder was used. To load the sample,

3.8ml, in the measuring cup a 5ml pipet was used. The pipet tips were cut off to broaden the inlet and

minimize shear. A strain sweep was performed, using a shear rate range of 1 to 300s-1

in 3 cycles of 2

minutes (upward, downward and upward again). Using Microsoft Excell the sweeps were averaged

and the shear force was plotted against the shear rate while viscosity was plotted against shear rate

(1/s) and concentration (% w/v).

9

Thermal analysis

Sample preparation

Samples were put in a desiccator with zeolite and a light vacuum was applied. Dry weight was

determined before and after desiccation.

Dry weight

Dry weight determination was done through TGA (on a Mettler Toledo TGA/DSC1). 5±1 mg of

sample, placed in a standard 40µl Aluminium measurement crucible without lid, was used for the TGA

method. The sample was heated from room temperature to 300°C at a rate of 10°C/min, weight

normally stabilized around 150°C and the dry weight was determined at 200°C. The dry weight

percentage was defined as:

𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡% = (𝑒𝑛𝑑 𝑤𝑒𝑖𝑔ℎ𝑡/𝑠𝑡𝑎𝑟𝑡 𝑤𝑒𝑖𝑔ℎ𝑡) ∗ 100%

TGA-MS

To determine the degradation temperature of the samples the Mettler Toledo TGA/DSC 1 gas outlet

was coupled to a mass spectrometer. About 10±0.5 mg of sample was put in a 40µl aluminium crucible

without lid, then loaded into the TGA and heated from 30°C to 300°C. During this weight measurement

the outlet gas was analysed for O2, and CO2. Data was acquired through the Quadera program and

analysed using Microsoft Excel.

DSC Thermal analysis was carried out using a Mettler Toledo TGA/DSC 1. 5±0.5 mg of sample was pre

weighed and hermetically sealed in a standard 40µl aluminium crucible; sample contained around 0.09

w/w gwater/gpolysaccharide. A scan range of 30°C to 240°C with a heating rate of 10°C/min was used. Data

was acquired through the STARe program and analysed using Microsoft Excel.

10

Results & Discussion

Viscosity

Before doing the strain sweep, suitable concentrations had to be found. Originally this meant finding

the gelation point of each sample so the gel viscosity could be determined. During the first trials none

of the IMMPs gelled even at concentrations of 0.3 w/v (g/l). The native polysaccharides started gelling

at concentrations below 0.03 w/v. So instead two sample ranges were made. To keep the native

polysaccharide samples below the gelling point, a concentration range from 0.005 w/v to 0.025 w/v

was used. However the effect of IMMPs and Vita Fibre on the viscosity was noticeably smaller so to

have a larger concentration range these were prepared at 0.01 w/v to 0.05 w/v.

The strain sweep was carried out and the resulting viscosity data was plotted versus the shear rate.

The results for all the 0.02 w/v measurement are shown below in Figure 6. In Figure 7 a close up of the

lower values is shown. There are clear distinctions between Native potato, Waxy potato and their

respective IMMPs. The native polysaccharides decrease in viscosity at higher shear rates whereas the

IMMPs are largely unaffected. Also the viscosity at 300 s-1

is almost ten times higher for the native

samples. The results of Etenia suggest a mistake was made during labelling, as opposing to results for

potato and waxy potato, GtfB would have increased the viscosity. The viscosity at 300s-1

was used to

compare the different sample concentrations, see Figure 8. This graph shows three types of curves;

exponential increase, linear increase and flat lines. These three curve types suggest three different

kinds of interaction taking place. Vita Fibre and Etenia IMMP+DE both show almost flat lines so the

fragments are probably too small to have any interaction. Higher DP samples probably do have some

random entanglement or other size interaction resulting in the linear increase of viscosity at higher

concentrations. A secondary effect like double helix formation can explain the exponential increase.

Again the Etenia results contradict the potato and waxy potato results. Additionally after the sample

preparation it was already noted that Etenia IMMP had gelled at 0.04 w/v and 0.05 w/v. While native

Etenia showed barely any increase in viscosity. As Etenia IMMP did not gel at concentrations below

0.3 w/v during the first trails it seems likely the Etenia samples got swapped before dilution. So in the

conclusions section it will be assumed that the Etenia IMMP displayed in this section is actually Etenia

and vice versa.

Figure 6 Viscosity (Pa*s) plotted versus the shear rate (1/s). Both the native and IMMP samples are at 0.02 w/v (g/l).

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 50 100 150 200 250 300

Vis

cosi

ty (

Pa*

s)

Shear rate (1/s)

viscosity at 0.02 w/v (g/l) Potato Waxy potato

Vita Fibre Etenia

Potato IMMP Waxy Potato IMMP

Etenia IMMP Etenia IMMP+DE

Demi Water

11

Figure 7 Viscosity (Pa*s) plotted versus the shear rate (1/s). Both the native and IMMP samples are at 0.02 w/v (g/l).

Figure 8 Viscosity at 300 s-1

plotted versus the concentration in weight/volume for all the polysaccharides. For the three IMMP samples the α1,6 percentage is also displayed.

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

0 50 100 150 200 250 300

Vis

cosi

ty (

Pa*

s)

Shear rate (1/s)

viscosity at 0.02 w/v (g/l)

Vita Fibre Etenia Potato IMMP

Waxy Potato IMMP Etenia IMMP+DE Demi Water

25.2% α1,6

6.1 %α1,6

91.2% α1,6

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.00 0.01 0.02 0.03 0.04 0.05

Vis

cosi

ty (

Pa*

s)

w/v (g/l)

Viscosity as result of sample concentration

PotatoWaxy PotatoEteniaVita FibrePotato IMMPWaxy potato IMMPEtenia IMMPEtenia IMMP+DE

12

Shear behaviour

The shear force data obtained from the strain sweep was also plotted. This was done to observe the

shear behaviour. When plotted like this Newtonian fluids give linear curves; the slope of these curves

is dependent on the molecule size. While non-Newtonian fluids will deviate up or down from a straight

line as a result of secondary interactions.

The shear force of the 0.02 w/v samples has been plotted in Figure 9. Potato, waxy potato and Etenia

IMMP all show a clear non-linear response while the other samples seem linear. However the close up

of these samples, Figure 10, shows all samples have non-linear behaviour. Even the blank sample,

demi water, shows a slight downward deviation. This shows there is a small measurement error. At

higher concentrations this measurement error becomes relatively smaller so the 0.05 w/v results are

shown in Figure 11 The curves at 0.05 w/v are all linear indicating Newtonian behaviour in IMMPs and

Vita Fibre. As with the viscosity results it is assumed Etenia and Etenia IMMP got swapped before

dilution. So in the conclusions section it will be assumed that the Etenia IMMP displayed in this section

is actually Etenia and vice versa.

Figure 9 Shear Force plotted versus Shear Rate for demi water, the reference saccharides and the IMMPs. Sugar solution contain 0.02 w/v sample.

0

1

2

3

4

5

6

0 50 100 150 200 250 300

She

ar f

orc

e (

Pa)

Shear Rate (1/s)

Shear force at 0.02 w/v

Potato Waxy Potato

Etenia Vita Fibre


Etenia IMMP Etenia IMMP + Pullulanase

Demi Water

13

Figure 10 Shear Force plotted versus Shear Rate for demi water, the reference saccharides and the IMMPs. Sugar solution contain 0.02 w/v sample.

Figure 11 Shear Force plotted versus Shear Rate for Vita fibre, and demi water. Sugar solutions contain 0.05 w/v sugar. The linearity confirms that these solutions maintain Newtonian properties.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 50 100 150 200 250 300

She

ar f

orc

e (

Pa)

Shear Rate (1/s)

Shear force at 0.02 w/v Etenia Vita Fibre


Etenia IMMP + Pullulanase Demi Water

0

0.5

1

1.5

2

2.5

3

0 50 100 150 200 250 300

She

ar F

orc

e (

Pa)

Shear Rate (1/s)

Shear force at 0.05 w/v

Vita Fibre

Potato IMMP

Waxy Potato IMMP

Etenia IMMP + Pullulanase

Demi Water

14

TGA-MS

To attribute different weight losses to different thermal events a mass spectrometer (MS) was coupled

to the TGA/DSC1 machine’s gas outlet. With the MS the offset gas could be analysed. The O2 and

CO2 concentrations together with the weight percentage were plotted versus the temperature. As this

test was mostly meant to show the difference between the two major weight losses only native potato

starch, Figure 12, and Potato IMMP, Figure 13, were measured.

The first weight loss is only accompanied by an increase in O2 concentration. This indicates that the

weight loss is due to evaporation. During the second weight loss CO2 is formed therefore the sample

must be degrading. The onset of CO2 production has been used to determine the degradation

temperature. For potato starch this was found to be 288°C and 272°C for potato IMMP. So potato

IMMP starts degrading slightly sooner. This possibly indicates an increase in reductive ends and

therefore more but also smaller fragments.

Figure 12 TGA-MS analysis results for potato starch. O2 and CO2 are plotted as a percentage of the outlet gas and the weight as percentage of the starting weight.

Figure 13 TGA-MS analysis results for potato starch IMMP. O2 and CO2 are plotted as a percentage of the outlet gas and the weight as percentages of the starting weight.

288

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0

20

40

60

80

100

120

30 80 130 180 230 280

Gas

co

nce

ntr

aio

n (

%)

Sam

ple

we

igh

t (%

)

Temperature (°C)

Potato Starch TGA-MS

Weight percentage O2 CO2

272

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0

20

40

60

80

100

120

30 80 130 180 230 280

Gas

co

nce

ntr

atio

n (

%)

Sam

ple

we

igh

t (%

)

Temperature (°C)

Potato IMMP TGA-MS

Weight percentage O2 CO2

15

Dry weight

As the water content can have a large influence on the glass transition temperature the DSC samples

were desiccated. To determine the dry weight TGA was used. Samples were put in open cups and

heated from 25 °C to 300°C. The weight was plotted as percentage of the initial weight, see Figure 14.

As shown during the TGA-MS the first weight loss is due to evaporation and the second was caused

by burning. Therefore the stable weight between these weight losses is dried sample. For all samples

weight was most stable between 150 °C and 250 °C. Therefore the dry weight percentage for all

samples was determined at 200 °C, see Table 1. After desiccation there was an average 1% increase

in dry weight percentage. More importantly the standard deviation became five times smaller. Also the

second weight loss in IMMPs starts at lower temperatures than the native samples. This suggests a

drop in degradation temperature, in accordance to the TGA-MS results.

Figure 14 Post-desiccation dry weight determination TGA curves, weight is displayed as a percentage of the initial weight with a temperature range of 25 to 300°C.

Table 1 Dry weight percentages determined for each sample pre- and post-desiccation. The average dry weights and standard deviation are also shown. Vita fibre was not desiccated thus it is displayed last.

Pre-desiccation (%w/w) Post-desiccation (%w/w)

Potato 84.63 90.16 Waxy potato 85.32 90.37 Etenia 89.54 91.47 GtfB Potato IMMP 93.09 91.61 GtfB Waxy potato IMMP 92.42 91.74 GtfB Etenia IMMP 93.42 92.01 GtfB Etenia IMMP+DE 94.23 92.09

Average 90.38 91.35

Standard devation 3.68 0.72

Vita Fiber 95.76 N.A.

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300 350

We

igh

t p

erc

en

tage

(%

w/w

)

Temperature (°C)

Dry weight determination curves Potato

Waxy Potato

Etenia

Potato IMMP

Waxy Potato IMMP

Etenia IMMP

Etenia IMMP+DE

16

DSC analysis

To try and find the glass transition temperature a DSC was run and the heat flow was plotted against

the temperature. A sudden change in heat flow would indicate a shift in specific heat which could have

resulted from passing the glass transition temperature. Before the final measurements trials were done

to determine optimal heating rates and sample weights. Heating rates from 5 till 50°C/minute were

checked. At rates above 20°C/minute the sample temperature started to fall behind on the cell

temperature so the heat flow to the sample seemed to have a limit. Similarly high amounts of sample

also caused this thermal lag. As seen with the TGA-MS the polysaccharides will start burning at

temperatures above 250°C. Finally to keep the moisture content equal during the measurement,

crucibles with lid were used. Taking all these variables into account a final DSC measurement was set

up; using 5±0.5mg sample in a closed 40 µl aluminium crucible with a heating rate of 10°C/minute over

a scan range of 30°C to 240°C. This measurement was done in duplo and each duplo was measured

three times cooling at room temperature in between runs.

Figure 15 shows the results for the first duplo measurement of potato starch. It also shows one of the

problems with this measurement the weight drops by almost 0.6 mg only to regain some of the weight

during measurements. This is observed in all measurements and probably means the seal on the

crucibles wasn’t strong enough to withstand the vapour pressure or was leaky from the start. As all

samples were essentially dry before 150°C it would be impossible to find a Tg. However the general

trend could still be assessed to this end the first run of all the samples was plotted in Figure 16 and the

second run in Figure 17. The other graphs can be found in the appendix.

During the first run the IMMPs and to a lesser extend potato and Etenia had a peak around 80°C. From

125°C onward the native samples and IMMPs follow similar trends although the IMMPs are shifted to

the right. The Etenia IMMP+DE measurement is especially interesting as this was the only one where

the weight didn’t start dropping at the beginning of the measurement. Instead the weight remained

constant till just above 140°C when the weight and as a result the heat flow drastically dropped. In the

second run the samples become a lot more similar, the peaks at 80°C are a lot smaller. The initial

slump in heat flow due to water evaporation is still present. However beyond 150°C the curves do get

a bit more pronounced with more little peaks. Sadly, these curves are the best results and no Tg was

determined.

Although all variables were minimized there were still two problems. The STARe software module for

Tg determination was not present and most importantly for determination of thermal events in complex

materials a modulated DSC is needed. This was a piece of hardware we simply did not have at our

disposal.

17

Figure 15 DSC curves for the 1st, 2

nd and 3

rd run of potato starch. The solid lines represent the heat flow, the

corresponding weight values are plotted using dashed lines.

Figure 16 DSC curves for all samples on the first run. Closed crucibles containing 5±0.5 mg heated at 10°C/minute from 30°C to 240°C.

4.5

4.6

4.7

4.8

4.9

5

5.1

5.2

5.3

5.4

5.5

-2

-1.8

-1.6

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0 25 50 75 100 125 150 175 200 225 250

Sam

ple

we

igh

t (m

g)

He

at F

low

(W

/g)

Temperature (°C)

Potato DSC Heat flow and weight

1st run 2nd run 3rd run

1st run weight 2nd run weight 3rd run weight

-2

-1.8

-1.6

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0 25 50 75 100 125 150 175 200 225 250

He

at F

low

(W

/g)

Temperature (°C)

first DSC run

Potato Waxy potato

Etenia Vita Fibre



18

Figure 17 DSC curves for all samples on the second run. Closed crucibles containing 5±0.5 mg heated at 10°C/minute from 30°C to 240°C.

-2

-1.8

-1.6

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0 25 50 75 100 125 150 175 200 225 250

He

at F

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/g)

Temperature (°C)

Second DSC run

Potato Waxy potato

Etenia Vita Fibre



19

Conclusions

The rheology experiments showed that upon modification with GtfB shear thinning behaviour is lost

and the viscosity decreases. There is also a slight correlation between the GtfB conversion percentage

and loss of viscosity between potato IMMP, waxy potato IMMP and Etenia IMMP+DE. Both of these

findings imply that IMMPs do not form double helices and thus has more interaction with water. This is

further supported by their increased solubility. However it can also be explained by a decrease in

degree of polymerization or molecule size. This is especially the case with Etenia IMMP+DE which

behaves very similar to Vita Fibre.

The TGA-MS shows that the first and second weight loss can be attributed to respectively evaporation

and burning. While evaporation remains unaffected, the enzyme treatment did result in a lower

degradation temperature. This could indicate that there are more reducing ends and thus more chain

fragments in the IMMP sample.

The dry weight percentages show that the desiccation was successful at equilibrating the samples.

Also with a dry weight percentage of almost 9% the Tg should have been between 100°C and 150°C,

way below the degradation temperature.

Reducing the spread in water contents alongside pre-weighing samples and using a proper heating

rate did improve reproducibility and reduced noise during DSC measurements. Sadly, the standard

aluminium crucible did not withstand the pressure of the water vapour. So they burst and water content

was not maintained during the DSC measurements. The samples were essentially dried during the

measurement, this might have increased the Tg to a temperature above the temperature range used

for the DSC. In addition the heat flow curves, despite best efforts, were too noisy to determine the Tg.

This noise is probably caused by the inhomogeneity of the samples which causes overlapping of

thermal events.

The decrease in viscosity shows IMMPs interact less on an intermolecular level. The Newtonian

behaviour and viscosity vs. concentration data show that the mode of interaction is altered. These

findings support the idea of α-1,6-linked glucose chains being more flexible and not forming double

helices. However this can also be attributed to smaller molecule size as implied by the lower

degradation temperature observed for IMMPs.

20

Recommendations

DSC

The determination of the glass transition temperature (Tg) of starch turned out to be more complicated

than originally believed. During the DSC measurement it is important to have a constant water content.

The standard aluminium crucibles could not maintain water content as the pressure got to high. This

can be solved by using high pressure crucibles for future measurements. These have a gold plated

copper seal and are closed by screwing the lid on, resulting in a allowed maximum pressure of 15

MPa. The medium pressure crucibles are save up to 2 MPa this might also be enough. However the

Kel-F (polytrifluorochloroethylene, PCTFE from 3M) O-ring used to seal these crucibles melts at

220°C (Mettler-Toledo GmbH 2000). So the DSC method should be altered to have a lower end

temperature.

The initial water content during the DSC was 9%, this should have been enough water to keep the Tg

below degradation. However the Tg increases exponentially at lower concentrations. Therefore a

water content between 10-20% is more desirable as the change in Tg is linear between these water

concentrations. This water content can be achieved by using a salt with a water activity between 0.2

and 0.6 Aw for desiccation instead of the zeolite used for equilibration in this experiment. Starch is a

very inhomogeneous material and IMMPs might be even more inhomogeneous. This causes problems

with normal DSC as several thermal events might overlap. The solution to this problem is modulated

DSC where instead of a linear heating profile, a sinusoidal heating pattern is used. Modulated DSC

allows for simultaneous determination of heat flow and heat capacity while also increasing sensitivity.

This makes modulated DSC very well suited for Tg determination, because the Tg is observed as a

small change in heat capacity resulting in a small change in heat flow. With data on both the heat flow

and capacity it possible to distinguish between low DP fragment degradation and an actual

Tg(Instruments , Thomas 2005). DSC modulation requires an expansion on the standard STARe

software provided by Mettler Toledo, this expansion is called TOPEM. However the DSC Evaluation

module might still be needed to determine the Tg automatically.

So in short, use high pressure crucibles (Mettler Toledo part number 51140404) with a high pressure

seal (Mettler Toledo part number 51140403) on a DSC machine controlled by STARe with the TOPEM

(Mettler Toledo part number 51141971) and DSC analysis (Mettler Toledo part number 119457)

software expansions.

Rheology

The results from the rheology experiments imply less retro-gradation in IMMPs. However these

samples were only stored overnight. Therefore it is not excluded that IMMPs start retrograding or

settling after a longer storage time. To follow up on this the viscosity could be monitored over a one

month period. This can be done using the sample preparation and strain sweep methods used during

this research. During the concentration determination trials it appeared that IMMP samples react

differently to reheating. This again relates to retro-gradation which makes re-gelling impossible in

starch, see figure 3. A temperature sweep, set shear rate with variable temperature, can be used to

analyse the re-gelling properties. When performing this temperature sweep it might be interesting to

use a several step heat program, heating from room temperature to 99°C and back down to room

temperature with a resting period before going down to -30°C and back again. This way GtfB’s effects

on both the re-gelling capacity as well as the freeze-thaw stability can be investigated with one

experiment.

21

References

Brimhall, B. (1944). "Structure of pyrodextrins." Industrial & Engineering Chemistry. Chuang, L., N. Panyoyai, L. Katopo, R. Shanks and S. Kasapis (2016). "Calcium chloride effects on the glass transition of condensed systems of potato starch." Food Chemistry 199: 791-798. Das, D. and A. Goyal (2014). "Characterization and biocompatibility of glucan: a safe food additive from probiotic Lactobacillus plantarum DM5." Journal of the Science of Food and Agriculture 94(4): 683-690. Dobruchowska, J. M., G. J. Gerwig, S. Kralj, P. Grijpstra, H. Leemhuis, L. Dijkhuizen and J. P. Kamerling (2012). "Structural characterization of linear isomalto-/malto-oligomer products synthesized by the novel GTFB 4,6-α-glucanotransferase enzyme from Lactobacillus reuteri 121." Glycobiology 22(4): 517-528. Gaspar, M., Z. Benk, G. Dogossy, K. Reczey and T. Czigany (2005). "Reducing water absorption in compostable starch-based plastics." Polymer Degradation and Stability 90(3): 563-569. Gidley, M. J. and P. V. Bulpin (1987). "Crystallisation of malto-oligosaccharides as models of the crystalline forms of starch: minimum chain-length requirement for the formation of double helices." Carbohydrate Research 161(2): 291-300. Hoover, R. (1995). "Starch retrogradation." Food reviews international. Instruments, T. "THERMAL ANALYSIS REVIEW MODULATED DSCTM THEORY." Retrieved 8-8-2016, 2016, from http://www.eng.uc.edu/~beaucag/Classes/Characterization/ModulatedDSC_TAinst.pdf.

Jobling, S. (2004). "Improving starch for food and industrial applications." Current Opinion in Plant Biology 7(2): 210-218. Kothari, D., D. Das, S. Patel and A. Goyal (2014). "Polysaccharides." Polysaccharides: 1-16. Kralj, S., P. Grijpstra and S. S. van Leeuwen (2011). "4, 6-α-Glucanotransferase, a novel enzyme that structurally and functionally provides an evolutionary link between glycoside hydrolase enzyme families 13 and 70." Applied and …. Laaksonen, T. J., Y. H. Roos and T. P. Labuza (2001). "Comparisons of the use of desiccators with or without vacuum for water sorption and glass transition studies." International Journal of …. Leemhuis, H., J. M. Dobruchowska, M. Ebbelaar, F. Faber, P. L. Buwalda, M. J. E. C. van der Maarel, J. P. Kamerling and L. Dijkhuizen (2014). "Isomalto/Malto-Polysaccharide, A Novel Soluble Dietary Fiber Made Via Enzymatic Conversion of Starch." Journal of Agricultural and Food Chemistry 62(49): 12034-12044. Liu, X., Y. Wang, L. Yu, Z. Tong, L. Chen and H. Liu (2013). "Thermal degradation and stability of starch under different processing conditions." Starch‐Stärke.

Mettler-Toledo GmbH, A. (2000). Crucibles Overview DSC and TGA/SDTA. Nafchi, M. A., M. Moradpour and M. Saeidi (2013). "Thermoplastic starches: Properties, challenges, and prospects." Starch‐ ….

O'Sullivan, A. C. and S. Perez (1999). "The relationship between internal chain length of amylopectin and crystallinity in starch." Biopolymers 50(4): 381-390. Pérez, S. and E. Bertoft (2010). "The molecular structures of starch components and their contribution to the architecture of starch granules: A comprehensive review." Starch‐Stärke.

Roos, Y. and M. Karel (1991). "Phase transitions of mixtures of amorphous polysaccharides and sugars." Biotechnology Progress. Tharanathan, R. N. (2005). "Starch--value addition by modification." Critical reviews in food science and nutrition 45(5): 371-384. Thomas, L. C. (2005). "Modulated DSC® Paper #1 Why Modulated DSC®? ; An Overview and Summary of Advantages and Disadvantages Relative to Traditional DSC." Retrieved 8-8-2016, from http://www.tainstruments.co.jp/application/pdf/Thermal_Library/Technical_Papers/TP006.PDF.

Tingirikari, J., D. Kothari and A. Goyal (2014). "Superior prebiotic and physicochemical properties of novel dextran from Weissella cibaria JAG8 for potential food applications." Food & function 5(9): 2324-2330.

http://www.eng.uc.edu/~beaucag/Classes/Characterization/ModulatedDSC_TAinst.pdf

http://www.tainstruments.co.jp/application/pdf/Thermal_Library/Technical_Papers/TP006.PDF

22

Appendix

DSC graphs

Figure 18 Potato DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation.

Figure 19 Waxy potato DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation.

-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250

He

at f

low

(W

/g)

Temperature (°C)

Potato starch DSC


-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250

He

at f

low

(W

/g)

Temperature (°C)

Waxy potato DSC


4.54.64.74.84.9

55.15.25.35.45.5

0 50 100 150 200 250W

eig

ht

(mg)

Temperature (°C)

4.54.64.74.84.9

55.15.25.35.45.5

0 50 100 150 200 250

We

igh

t (m

g)

Temperature (°C)

23

Figure 20 Etenia DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation.

Figure 21 Vita fibre DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation.

-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250

He

at f

low

(W

/g)

Temperature (°C)

Etenia DSC


-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250

He

at f

low

(W

/g)

Temperature (°C)

Vita fibre DSC


4.54.64.74.84.9

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0 50 100 150 200 250

We

igh

t (m

g)

Temperature (°C)

4.54.64.74.84.9

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0 50 100 150 200 250

We

igh

t (m

g)

Temperature (°C)

24

Figure 22 Potato IMMP DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation.

Figure 23 Waxy potato IMMP DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation.

-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250

He

at f

low

(W

/g)

Temperature (°C)

Potato IMMP DSC

1st run 2nd run

-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250

He

at f

low

(W

/g)

Temperature (°C)

Waxy potato IMMP DSC

1st run 2nd run

4.54.64.74.84.9

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We

igh

t (m

g)

Temperature (°C)

4.54.64.74.84.9

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We

igh

t (m

g)

Temperature (°C)

25

Figure 24 Etenia IMMP DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation.

Figure 25 Etenia IMMP+DE DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation.

-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250H

eat

flo

w (

W/g

) Temperature (°C)

Etenia IMMP

1st run 2nd run

-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250

He

at f

low

(W

/g)

Temperature (°C)

Etenia IMMP+DE


4.54.64.74.84.9

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0 50 100 150 200 250

We

igh

t (m

g)

Temperature (°C)

4.54.64.74.84.9

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0 50 100 150 200 250

We

igh

t (m

g)

Temperature (°C)

26

Figure 26 potato duplo DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation.

Figure 27 Waxy potato duplo DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation. While inserting this sample for the third time the sample robot somehow removed the lid.

-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250H

eat

flo

w (

W/g

) Temperature (°C)

Potato


-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250

He

at f

low

(W

/g)

Temperature (°C)

Waxy Potato


4.54.64.74.84.9

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We

igh

t (m

g)

Temperature (°C)

4.54.64.74.84.9

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0 50 100 150 200 250

We

igh

t (m

g)

Temperature (°C)

27

Figure 28 Potato IMMP duplo DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation. The sample toppled over during the third measurement hence the dramatic weight decrease at 100°C.

°C)

Figure 29 Waxy potato IMMP duplo DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation.

-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250

He

at f

low

(W

/g)

Temperature (°C)

Potato IMMP


-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250

He

at f

low

(W

/g)

Temperature (°C)

Waxy Potato IMMP


4.54.64.74.84.9

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igh

t (m

g)

Temperature (°C)

4.54.64.74.84.9

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0 50 100 150 200 250

We

igh

t (m

g)

Temperature (°C)

28

Figure 30 Etenia IMMP duplo DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation.

Figure 31 Etenia IMMP+DE duplo DSC curve using a closed aluminium crucible with 5 mg of sample at a heating rate of 10°C/minute with scan range 30-240°C. Measurement was repeated three times, sample was cooled at room temperature in between measurements. The insert displays the corresponding weight curves over the same temperature range. Only the weights from 4.5mg-5.5mg are shown as these fall within the maximum sample weight deviation.

-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250

He

at f

low

(W

/g)

Temperature (°C)

Etenia IMMP


-2.5

-2

-1.5

-1

-0.5

0

0 50 100 150 200 250

He

at f

low

(W

/g)

Temperature (°C)

Etenia IMMP+DE 1st run 2nd run 3rd run

4.54.64.74.84.9

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0 50 100 150 200 250

We

igh

t (m

g)

Temperature (°C)

4.54.64.74.84.9

55.15.25.35.45.5

0 50 100 150 200 250

We

igh

t (m

g)

Temperature (°C)

isomalto/ maltopolysaccharides bsc thesis biotechnology + code

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