InsideFood Symposium, 9-12 April 2013, Leuven, Belgium 1 | P a g e

Multi-length scale structural imaging of freeze-dried carrots and their rehydration behaviour.

Gerard van Dalena, Adrian Voda

a, Arno Duijster

b, Lucas van Vliet

b, Frank Vergeldt

d, Ruud van der Sman

c, Henk

Van Asd, John van Duynhoven

a,d

a Unilever Research & Development, Olivier van Noortlaan 120, 3133AT Vlaardingen, the Netherlands

b Quantitative Imaging Group, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands

c Food Process Engineering, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands

d Laboratory of Biophysics and Wageningen NMR Centre, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The

Netherlands

ABSTRACT

Complementary imaging techniques (µCT, SEM, MRI), NMR and quantitative image analysis

methods were used to obtain multi-length scale information of the porous structure of carrots in their

freeze-dried, rehydrating and rehydrated form. This work established a predictive relation between

freezing rate and freeze damage. Water imbibition rates could be explained from the porous structure

induced during freeze-drying. In the rehydrated state, remaining structural integrity and anisotropy

could be correlated with the textural quality.

1 Introduction

Dried fruits and vegetables that are currently available on the market are a poor compromise between

convenience (rehydration kinetics) and sensorial quality. This is a major bottleneck for consumers to “Make the

Healthy Choice the Easy Choice” and it also negatively impacts market growth. As an example of compromised

quality, Fig. 1 shows (A) the visual appearance and (B) the rehydration kinetics for carrots that underwent

different pre-treatments (blanching and freezing at different temperatures) before being freeze-dried. Non-

blanched carrots clearly show a decrease in rehydration mass with decreasing freezing temperature. This was

not observed after blanching. Blanched samples rehydrate substantial faster than non-blanched ones, except the

ones at -28oC (Fig. 1B).

Fig. 1 Effect of blanching and freezing temperature on visual appearance and rehydration behaviour of freeze dried carrot

cylinders (5 min. rehydration at 90oC, I=dry, II=rehydrated).

Currently rational optimization of drying processes is impeded by lack of insight which structural features

determine rehydration kinetics (convenience) and texture (sensorial quality) upon rehydration. We first studied

the impact of thermal pre-treatment conditions on the structure of the cortical tissue of winter carrots by a suite

of complementary imaging techniques (X-ray microtomography (µCT)1, Scanning Electron Microscopy (SEM)

and micro Magnetic Resonance Imaging (MRI)2) in combination with Nuclear Magnetic Resonance (NMR)

relaxometry and diffusometry. Dedicated 3D image analysis methods were developed to obtain quantitative

information about the microstructure in the dried state in order to establish predictive models for structural

damage incurred by thermal pre-treatment. Next, the relation between these different freeze-damaged structures

and rehydration behaviour as visualized by real-time in-situ MRI and NMR was studied. In the rehydrated state

the relation between distribution of water, its anisotropic diffusion behaviour and texture was assessed.

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2 Material and Methods

2.1 Carrot pre-treatments

Cylindrical samples with a diameter of about 6 and 10 mm and

a length of about 10mm were cut from winter carrots. Different

thermal pre-treatments were applied to the samples before freeze-

drying under vacuum (0.4 mbar) from -30oC up to 25

oC during about

27 hours4. The first pre-treatment was blanching, which was

performed for one minute in boiling water. Non-blanched samples

were also selected for the next step. Secondly, samples were frozen at

four different temperatures: -28oC (freezer), -80

oC (dry ice, CO2), -

150oC (N2 gas) and -196

oC (liquid N2).

2.2 µCT

The internal porous structures of the dry carrot samples were visualized using a desktop X-ray micro-

tomography system (SkyScan 1172, Belgium, http://www.skyscan.be) and a synchrotron system (ID19

beamline at the European Synchrotron Radiation Facility, ESRF in Grenoble). With the SkyScan system stacks

of 2480 horizontal cross sections (4000 x 4000 pixels) with a pixel size of 4.0 µm were produced and with the

synchrotron system stacks of 1000 horizontal cross sections (2048*2048 pixels) with a pixel size of 0.56µm.

Details about the procedure can be found in a paper about µCT imaging of freeze-dried vegetables3.

2.3 SEM

A piece of dried carrot was cut into two halves in such a way that a cross-section was obtained. A very

thin slice was cut off from the surface with a razor blade to obtain a high quality cross-sectional surface of the

remaining piece of dry tissue. This surface was sputter coated with platinum for better SEM imaging contrast.

The Pt coated sample was inserted into a Jeol 6490 LA Scanning Electron Microscope and both the peripheral

and central areas were imaged at magnifications ranging from 10x to 10.000x.

2.4 MRI, NMR relaxometry and diffusometry

Rehydration to study rehydrated carrots was done at 95 C. The rehydration process itself was studied by

MRI and TD-NMR at room temperature. After rehydration, the samples were gently rolled on an absorbent

paper tissue to remove the water dripping over the surface, and wrapped in cling-film to prevent moisture loss

during measurements. Time-domain NMR relaxation experiments were carried out at room temperature (22 C)

on a Maran Ultra spectrometer (Resonance Instruments, Oxford, UK) operating at proton frequency of 30 MHz.

Magnetic resonance imaging (MRI) experiments (Fig. 10) were performed on a 0.7 T MRI spectrometer

operated by a Bruker Avance spectrometer. Details are reported in reference 4 and 9.

2.5 Image analysis

The pore and wall size distribution was measured from the 3D µCT images using a morphological sieve

method5. In this approach a scale-space (granulometry) is build using differences between closings with

spherical structuring elements of increasing diameter. Details will be reported in a separate paper6.

3 Results

3.1 Structure-processing relationship: impact of thermal-pretreatment on carrot structure

The effect of the freezing temperature on the internal porous structure of freeze dried carrot cylinders

with a diameter of 10mm (2.1) is shown in Fig. 3. The µCT images clearly show a decrease in pore size with

decreasing the freezing temperature. Slow freezing facilitates the formation of large ice crystals resulting in

elongated cavities (pores) up to 2 mm with broken walls. Cell sizes in a fresh carrot tissue range from 10 to

100µm. Many of these plant cells are compressed between large ice crystals resulting in pore walls consisting of

several layers of dehydrated cells up to a thickness of about 10 µm (Fig. 4). The microstructure was better

preserved at lower freezing temperatures. However freezing via liquid nitrogen (-196oC) is prone to freeze

cracking. The 3D porous microstructures of the freeze dried carrot cylinders frozen at -28 and -150 C are

visualised in Fig. 5. Freezing at -150 C resulted in a better cellular integrity and retention of the size and shape

Fig. 2 Cross-section of a carrot with

sampling location and μCT image after FD

(numbers indicate different tissue regions).

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of the freeze dried carrot pieces. SEM images of the same freeze dried carrot samples are shown in Fig. 4. These

images show the same effect of the freezing temperature on the microstructure as shown by μCT. The SEM

images reveal the porous architecture at a much higher resolution giving detailed information about the pore

wall morphology. However it will not show the microstructure in 3D, hampering quantitative image analysis. To

tackle this, samples have to be sectioned to reveal the internal microstructure. This destructive nature makes it

unsuitable for multimodal imaging (e.g. to correlate the microstructure with the spatial moisture ingress imaged

with MRI).

Fig. 3 Horizontal cross sections of μCT images of freeze dried carrots frozen at -28, -80, -150 and -196 C (image size:

12.1mm*12.1mm, pixel size = 4.0µm).

Fig. 4 SEM images of freeze dried carrots frozen at -28, -80, -150 and -196 C. High magnification SEM images (right)

showing the microstructure of the pore walls consisting of several layers of compressed cells (-28oC non-blanched).

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Fig. 5 3D surface rendering of μCT images of freeze dried blanched carrot cylindrical samples frozen at -28 and -150 C

showing the total (left) and clipped sample volume (right).

The results of the pore size distributions analysed by granulometry computation are shown in Fig. 6. The

granulometries of the samples with fine structures rise at small diameters, while the curves of samples with large

voids rise at larger diameters. A characteristic value for every curve is derived as the diameter where the

cumulative granulometric curve is 0.50, i.e. where half of the total void space contains voids smaller than this

characteristic diameter.

Fig. 6 Granulometry (left) of eight different freeze drying procedures (four different

temperatures and two pre-treatments) with characteristic pore diameters (right) and

corresponding estimations of the standard deviation (carrot diameter = 10mm).

Fig. 7 Ice crystal size against

freezing rate. Solid line: regression

on crystal size versus freezing rate.

Grey: literature data7.

In Fig. 7 the average pore sizes determined by granulometry were compared with the scaling laws for ice

crystal growth and experimental data of ice crystal sizes for comparable (bio) materials7. The freezing rate is

estimated via Plank’s equation7. It can be observed that all data collapse to a single curve, and our data fall on

this common line - except for the blanched carrot frozen at -28 C. The mean pore size of the slow frozen

blanched sample is twice that of the non-blanched sample. We expect that at slow freezing (-28 C) the elastic

properties of the carrot might play a role in the moisture transport, and thus the ice crystal growth. Linear

regression shows that the ice crystal size (λ), scales with the freezing rate (Ṫ) by a power law: λ = Ṫ-0.25

. Thus a

predictive relation has been established between freezing rate and freezing damage.

The pores in the freeze dried carrots are not uniformly distributed. This can be attributed to the different

tissue types within the fresh cylindrical carrot samples used for freeze drying. These fresh samples include parts

of the central stele and peripheral cortex, containing different types of cells having different size, shape and

orientation with different cell wall thickness and strength. Four different regions can be distinguished as shown

in Fig. 2. During freezing the growth of an ice crystal ruptures, pushes and compresses cells. This process is

influenced by the strength of the cell walls8. Pores and cavities are left after sublimating the ice crystals from the

carrot matrix. The ice crystals will grow in the cell direction creating elongated pores which are much larger

than the original cells at slow freezing.

Carrot cylinders with a diameter of 6mm were used to study the impact of thermal pre-treatment on the

microstructure of the freeze dried carrots at higher resolution. By using smaller carrot samples a more uniform

part of the carrot could be sampled, excluding the central part (parenchyma cells). Blanched and non-blanched

carrots cylinders were freeze dried at -28 C and -150 C. Images obtained using the ESRF SR-µCT system are

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shown in Fig. 8. The pores are much smaller than observed for the 10mm carrots and the influence of the

freezing temperature is less pronounced. This could be caused by the different cell type and by the smaller

diameter of the sample. The freezing rate depends on the dimensions and shape of the sample, particularly

thickness. Blanching before freeze drying at -150 C resulted in smaller pores which are more homogeneously

distributed (Fig. 9). During blanching membrane disruption will result in a sugar homogenization over the

tissue. Therefore, a blanched carrot can better accommodate intracellular ice crystal growth and will thus suffer

less structural damage. This is not necessarily valid in the case of slow cooling where freezing occurs

extracellular and blanching may hardly have any effect. Blanching resulted in a smaller average wall size (Fig.

9).

Fig. 8 Horizontal (top) and vertical (bottom) cross sections of ESRF SR-µCT

images of carrots (diameter = 6mm) freeze dried at -28C and -150C, with and

without blanching pre-treatment (image width = 1.15mm, 1 pixel = 0.56µm).

Fig. 9 Pore and wall size distribution

of FD carrots analysed from the ESRF

SR-µCT images shown in Fig. 8.

3.2 Structure-functionality relationship: freeze dried structure and rehydration behaviour

In order to verify models for water imbibitions, MRI methods were implemented (Fig. 10) for the real-

time assessment of the spatial water mobility. Although MRI time series provide both temporal and spatial

information on rehydration processes, special care should be taken to avoid erroneous quantification due to

susceptibility artefacts9, even at field strengths as low as 0.72 T. T2 relaxometry complemented the MRI

measurements and enable more reliable quantification. The rehydration studies with MRI and TD-NMR were

done at room temperature.

Fig. 10 Longitudinal centre slice from 3D (top) and 2D (bottom) MRI Turbo Spin Echo (0.7T) time series recorded during

rehydration at room temperature of FD blanched (B) and non-blanched (NB) carrot pieces. Pixel size = 0.22mm. FOV =

14×7×7 mm3 (NB) and 14×7×1 mm3 (B), effective echo time = 5.3ms (NB) and 4.6 ms (B), experiment Time = 100s (NB)

and 6s (B).

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Non-blanched carrot pieces frozen at −28 °C rehydrate faster compared to those frozen at −150 °C due to

differences pore size. For blanched samples there is an extra mechanism that overwhelms these differences in

pore size and strongly enhances water ingress. Freeze-drying leads to a better connectivity of the pore structure.

NMR relaxometry showed that rehydration is followed by slow, diffusion-driven leaching of solutes out of the

carrot tissue9.

3.3 Structure-functionality relationship: rehydrated carrot structure and texture

Micro-scale morphology in rehydrated freeze-dried specimens was assessed by means of NMR

transversal relaxation and diffusometry. Three relaxation components could be identified for all samples. The

short (20ms) and intermediate (100-180 ms) T2‘s correspond to the non exchangeable protons of the

macromolecules (cellulose, carotenoids, sugars and pectin) and to the water present in the swollen cell walls.

The main (long T2) component (400 up to 600 ms) corresponds to the water, filling the rehydrated pores, and it

is shown in Fig. 11a for all carrot samples. It can be observed that the variation of relaxation times with the

freezing temperature occurs on a relative narrow interval. Assuming the surface relaxivity of the solid carrot

matrix is independent of the freezing rate, one would expect that the NMR relaxation rate would scale inverse

proportional with the pore size, which is confirmed in the experimental data shown in Fig. 11b (see

granulometry in Fig. 6).

Cell wall network tortuosity was determined by assessment of self-diffusion data as a function of

diffusion time (Fig. 11b). It can be observed that the tortuosity of slow frozen samples at -28°C is very close to

unity, which indicates that water molecules can diffuse through the porous structure with hardly any

obstructions. This confirms that native (membrane separated) compartments have been destroyed after freeze-

drying and that their functionality is not recovered after rehydration. The disruption of membranes makes the

rehydrated system to behave more like a sponge characterized by network tortuosity.

Fig. 11 Micro- and macroscopic features of rehydrated cortical tissue of carrots freeze-dried at -196, -150, -80 and -28 C,

with and without blanching pre-treatment: (A) proton transverse relaxation time, T2, and (B): network tortuosity (from water

self diffusion by NMR).

The water distribution in rehydrated carrots was visualized by MRI (Fig. 12A). The signal intensity in the

images depends on the water amount and on the carrot tissue density per pixel. As a first observation, all

samples show partial rehydration after 15 min, indicated by spots in the image with low signal intensity. A

difference in the structure between the blanched and non-blanched -28oC samples can be identified; smaller non-

hydrated cavities are present in the blanched samples. The dried microstructure revealed by μCT can be

correlated with the water distribution in the rehydrated structure. Thereby, it is clear that pre-treatments have an

impact on both the dried and rehydrated states.

Fig. 12 MRI images (left) of rehydrated (15 min. 95oC) cortical tissue of carrots freeze-dried at -28 and -196 C, with and

without blanching pre-treatment. Diffusional anisotropy (ratio between NMR self-diffusion constants at Δ=1s along

longitudinal and transversal direction) and texture results (right).

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Structural anisotropy was assessed by measuring water self-diffusion coefficients along longitudinal and

transversal directions (axial is defined as aligned with the static magnetic field and along the carrot root). The

ratio between diffusivities along the two directions, estimated at long diffusion times (>1 s), gives an indication

about the anisotropic nature of the system. The anatomy of fresh carrots is known as bi-dimensional anisotropic,

due to cells elongated along the direction of the growth. It was found out that anisotropy to a certain extent

remains after thermal treatments and drying. Samples frozen at lower temperatures show more anisotropy, thus

confirming that faster freezing preserves cell wall morphology (Fig. 12B), and consequently resulting in a better

texture (higher compression force).

4 Conclusions and perspectives

A combination of complementary imaging techniques followed by image analysis resulted in multi-

length scale information of the porous structure of carrots in their freeze-dried state, during rehydrating and in

rehydrated form. A predictive relation between freezing temperature and freeze damage as reflected in pore

sizes in freeze-dried carrots was established. Water imbibition rate as observed by real-time MRI and NMR

could be understood from the porous structure induced during freeze-drying. In the rehydrated state, the

remaining structural integrity and anisotropy related to textural quality.

The quantitative structural information obtained from µCT can be used as input for modelling the

rehydration of freeze-dried carrots10

. Model verification can be performed by real-time assessment of water

imbibition as provided by MRI in combination with image analysis.

Acknowledgements

Martin Koster for µCT imaging and Jaap Nijsse and Caroline Remijn for SEM imaging.

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