3D imaging of freeze-dried vegetables using X-ray microtomography

G. van Dalen1, M. Koster1, J. Nijsse1, E. Boller2, J. van Duynhoven1

1 Unilever R & D, Olivier van Noortlaan 120, NL-3133AT Vlaardingen,

2 European Synchrotron Radiation Facility, Grenoble Cedex, France

Introduction Dried vegetables are important ingredients in many convenience food products like instant soups, sauces, ready-to-eat meals and snacks. They are easy to prepare-by just adding hot water. Rehydration should be fast. Changes in macro – and microstructure are very important factors for the rehydration properties and textural quality of these dried materials. Products available on the market are a compromise between convenience and sensorial/nutritional quality. With the currently available drying technologies, dried fruits and vegetables cannot be rehydrated to their original fresh-like, volume, shape and texture. Vegetables are generally dried convectively with heated air. A disadvantage is the substantial degradation in quality attributes, such as appearance (shrunken, shrivelled, darkened), nutrients and flavour and the low rate of rehydration. Higher quality products can be obtained using much more expensive freeze drying methods. Freeze drying allows for quick rehydration of the product. However the very porous structure leads to a loss in texture and increase in friability. Most developments in drying of vegetables have been engineering-driven. In typical engineering approaches, emerging new drying technologies have been optimized to obtain rehydration properties and texture that match as much as possible the original properties. So far no systematic approach that considers the underlying micro structural events during the fresh-dried-rehydrated processing chain has been applied. A major barrier to embark on such an approach has been the lack of adequate measurement technologies that would enable decision making based on sound micro structural data. Hence we have deployed X-ray microtomography (µCT) [1] to investigate the micro structural impact of thermal pre-treatments and freeze drying [2,3]. 3D image analysis methods were developed to obtain quantitative information about the porous microstructure. This information will further be used to develop mathematical models for simulation of the moisture transport within the product. These models will focus on the control of ice crystal growth, structure and speed of

rehydration. Finally these models will be validated using MRI [4]. In this paper, the usability of µCT will be demonstrated on different types of freeze-dried broccoli, carrots, bell peppers and mushrooms. Images obtained with a laboratory μCT instrument are compared to those obtained using high resolution phase contrast Synchrotron Radiation (SR) μCT [5] and Scanning Electron Microscopy (SEM). For SR-μCT a new 3D volume reconstruction was used (Paganin‘s single distance phase retrieval) [6]. Freeze drying is based on the dehydration by sublimation of a frozen product. It results in a very open structure with large cavities promoting fast rehydration. These cavities are not the plant cells but are created by the ice crystals. The size of these

cavities is therefore mainly influenced by ice crystallisation behaviour or the cooling rate[7]. With air drying the structure shrinks and collapses during drying, driven by the capillary force created by air-water interfaces[8]. A very dense structure is created which will rehydrate very slowly.

Method Samples Cylindrical samples with a diameter of 6-10 mm were cut from bell peppers, mushrooms and winter carrots (Figure 3). For broccoli pieces of the stem below the flower canopy were used, having diameters of about 3mm (Figure 4).

Figure 3: Preparation of cylinders from a winter carrot.

Figure 4: Sampling of broccoli with outline of selected stem in red.

Different thermal pretreatments were applied to the samples before freeze-drying under vacuum (0.4 mbar) from -30oC up to 25oC during about 27 hours[4]. The first pretreatment 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), -80oC (dry ice, CO2), -150oC (N2 gas) and -196oC (liquid N2). µCT (SkyScan1172) A SkyScan 1172 desktop X-ray micro-tomography system (Belgium, http://www.skyscan.be) was used with power setting of 60kV and 167μA. μCT produced two-dimensional images of projections of a sample. For samples with a diameter of about 6mm and length of 10mm, a stack of 5200 flat cross sections (4000 x 4000 pixels) was obtained after tomographic reconstruction of images (4000 x 2096 pixels) acquired under different rotations over 180 degrees with a step size of 0.20 degrees (frame averaging = 2). The acquisition time for one projection was 1178 ms (exposure) resulting in a total acquisition and read-out time per scan of about 80 min (993 projection images/scan). A pixel size of 2.0 µm was selected. The samples were scanned using 3 scans, connected in the vertical direction to increase the axial field-of-view (oversized scan) and subsequently merged together during the

reconstruction. For tomographic reconstruction the following settings were used: no smoothing, ring artefact correction = 16 and beam hardening correction = 60%. SR-µCT Samples were imaged using the ID19 beamline at the ESRF (European Radiation Synchrotron Facility) in Grenoble. A set of horizontal cross sections was obtained after tomographical reconstruction of 1002 projection images (radiographs) acquired using different rotations over 180 degrees. An exposure time of 0.2 s was used for all projection images. The sample-detector distance was 10 mm. As detector, a FReLoN 2048*2048 pixels camera was used with 10x and 2x objectives. The image pixel sizes of 0.56 and 2.8 µm correspond to field of views of 1.15*1.15 mm2 and 2.8*2.8 mm2. This multi-scale approach is illustrated in Figure 5. An energy of 17.6 keV was chosen in function of sample absorption. The samples were inserted in plastic cylindrical sample holders.

Figure 5: μCT images of a freeze dried carrot obtained using a Synchrotron system at magnifications of 2x and 10x resulting in pixel sizes of 2.8µm and 0.56µm and field of views of 5.7mm and 1.15mm respectively.

Images obtained using the SkyScan 1172 μCT and the ESRF SR-μCT system are compared in Figure 8 and Figure 9. The images of the horizontal cross sections were obtained at exactly the same location. The low resolution images can be used for the determination of the pore size distribution of the tissue and for the size and shape of the pieces. High resolution SR-μCT is needed to obtain information about the pore wall thickness and connectivity between pores. The contrast obtained in traditional laboratory or desktop X-ray tube based µCT instruments, is due to the intrinsic differences in linear attenuation coefficients between the sample constituents (related to atomic number and density). An improved contrast can be obtained for materials with low or nearly the same attenuation coefficient by using phase contrast SR µCT. Images show enhanced contrast at edges and interfaces. However quantitative analysis of these images is more difficult than for images showing only absorption contrast. The features of interest in phase contrast images cannot be segmented using classical thresholding methods. In a previous study[9] holotomography was used to retrieve the different phases. The method is however more complex, requiring 3D images obtained at different distances between sample and detector, alignment of these different positions and a more sophisticated reconstruction procedure. In this study a new reconstruction method, Paganin, was used to retrieve the different phases (Figure 6). This method requires images at only a single distance. The single-distance non-iterative phase retrieval algorithm described by David Paganin [6] et al. has been implemented in the ESRF reconstruction software using GPUs[10]. The resulting images can be segmented using local thresholding (Figure 7).

Figure 6: Standard (left) versus Paganin (right) 3D volume reconstruction for Synchrotron μCT images of a freeze dried carrot (top) and mushroom (bottom). Image width = 0.25mm*0.25mm. Pixel size = 0.56 µm.

Figure 7: Binary images obtained after thresholding of standard (left) and Paganin (right) reconstructed Synchrotron μCT images of a freeze dried carrot (top) and mushroom (bottom) shown in Figure 6.

Figure 8: μCT images of a freeze dried carrot (-28oC, blanched) obtained using the SkyScan 1172 and the ESRF Synchrotron system at magnifications of 2x and 10x. Image analysis and visualisation

For image processing and analysis the image analysis toolbox DIPlib (version 2.3)

from the Delft University of Technology (NL, http://www.ph.tn.tudelft.nl/DIPlib/) running under MATlab (vers. 2009a) from MathWorks was used. For 3D visualisation and transformations in 3D space the Avizo Fire software package (version 7.1) from the Visualization Sciences Group was used.

Figure 9: μCT images of a freeze dried mushroom (-28oC, blanched) obtained using the SkyScan 1172 and the ESRF Synchrotron system at magnifications of 2x and 10x.

The μCT images in the result section are shown after inversion of the contrast (grey level ranging from 0 (black) to 255 (white) corresponding to high to low absorption) which is more common in food research.

Results Broccoli The influence of the freezing rate on the microstructure of small pieces of freeze dried broccoli stems is illustrated in Figure 10. Freezing was done in a freezer at -20oC (slow), in a blast air freezer at -35oC (fast) and by submerging in liquid nitrogen at -196 oC (very fast).

Figure 10: μCT images of freeze dried blanched Broccoli obtained using slow (left), fast (middle) and ultra fast (right) freezing. Top: horizontal cross sections (3.9mm*3.9mm), middle: surface rendering and bottom clipped surface rendering (clipped pore walls in red). Box size = 3.2mm*3.2mm*4.0mm. Pixel size = 4.2µm.

Slow freezing facilitates the formation of large ice crystals resulting in elongated cavities (pores) up to 1.5 mm with broken walls. Cell sizes in a fresh broccoli stem 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 20 µm (Figure 12). Fast freezing yields many small ice crystals resulting in a homogeneous distribution of pores up to 0.2 mm. Ultra fast freezing facilities the formation of even smaller ice crystals within the plant cells.

However, freezing via liquid nitrogen easily induces tension cracks in the tissue during freezing, and shrinkage during freeze drying. SEM images of the same freeze dried broccoli samples are shown in Figure 11 and Figure 12. 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, making quantitative image analysis more difficult and 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).

Figure 11: SEM images of freeze dried blanched Broccoli obtained using slow (left), fast (middle) and ultra fast (right) freezing.

Figure 12: SEM images of freeze dried blanched Broccoli obtained using slow freezing showing the microstructure of the pore walls consisting of several layers of compressed cells.

Prior to freeze drying the broccoli samples were blanched (cooking 3min at 100oC) to inactivate enzymes which can cause off-flavours. Blanching will have an effect on the microstructure which will be investigated in the next sections for carrots, bell peppers and mushrooms.

Carrots Carrot (type winter carrot) cylinders with a diameter and length of about 10mm were

blanched (1min at 100oC) and frozen at -28, -80, -150 and -196 C., after which they were freeze dried. The effect of the freezing temperature on the internal porous structure is shown in Figure 13. The images obtained using the SkyScan 1172 show clearly a decrease in pore size with decreasing freezing temperature. The microstructure will be 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 Figure 15. Freezing at -150 C resulted in a better cellular integrity and retention of the size and shape of the freeze dried carrot pieces.

Figure 13: Vertical (top) and horizontal (bottom) cross sections of μCT images of freeze dried

blanched carrots frozen at -28, -80, -150 and -196 C. Image size: 9.2mm*9.2mm (top) and 12.1mm*12.1mm (bottom). Pixel size = 4.0µm.

The impact of the freezing temperature on the porous microstructure is also visible in the SEM images of the freeze dried carrot samples shown in Figure 14.

Figure 14: SEM images of freeze dried blanched carrots frozen at -28, -80, -150 and -196 C.

Figure 15: 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 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, orientation with different cell wall thickness and strength. Four different regions can be distinguished as shown in Figure 16. During freezing the growth of an ice crystal ruptures, pushes and compresses cells. This process is influenced by the strength of the cell wallsError! Bookmark not defined.. 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 at slow freezing much larger than the original cells. The average length of cells within region 1 of a fresh carrot is around 0.1mm compared to a maximum pore length of 3mm after freezing at -28oC.

Figure 16: A photograph of a cross section through a winter carrot (left) with schematic representation (middle) indicating 4 distinct regions (1-pith mainly containing parenchyma cells, 2-parenchyma cells and xylem vessels, 3-outer cells of the vascular tissue and phloem vessels, and 4-cortex tissue). The disc represents the sampling spot of the 10mm cylindrical sample and corresponds to the μCT image of a freeze dried carrot (-28

oC) shown on the right

.

Carrot cylinders with a diameter of 6mm were used to study the impact of thermal pretreatment on the microstructure of the freeze dried carrots. 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 SkyScan 1172 µCT and the ESRF SR-µCT system are shown in Figure 17. Sub-volumes of the SR-µCT images are visualized in 3D in Figure 18. 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. 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 effectError! Bookmark not defined.. The influence of different blanching pre-treatment methods on the microstructure and texture of carrots has been widely studied[11,12].

Figure 17: Vertical (A) and horizontal (B&C) cross sections of μCT images of carrots freeze

dried at -28C and -150C, with and without blanching pre-treatment (A&B: SkyScan 1172, 1 pixel = 2.0µm; C: ESRF SR-µCT, 1 pixel = 0.56µm).

Figure 18: 3D surface rendering of SR-µCT images (Figure 17) of freeze dried carrots (box size: 0.50mm*0.50mm*0.25mm).

Bell peppers Bell peppers (sweet peppers) are important vegetables in dried savoury products. The tender skin of fresh bell pepper covers a crisp, fragile flesh that is easily bruised, cracked or crushed. Fresh cylindrical samples were cut perpendicular to the skin including the total tissue from outer to inner skin. Blanched and non-blanched bell

pepper samples were freeze dried after freezing at -28 C and -150 C. Images obtained using the SkyScan 1172 µCT are shown in Figure 19 and Figure 20. The influence of blanching pre-treatment is clearly visible. Without blanching very dense areas are observed which are caused by cell collapse. Blanching prevents shrinkage during freeze drying.

Figure 19: Vertical (A) and horizontal (B) cross sections of μCT images of bell peppers freeze

dried at -28C and -150C, with and without blanching pre-treatment (SkyScan 1172, 1 pixel = 2.0µm).

Figure 20: 3D surface rendering of μCT images of cylindrical samples of bell peppers freeze

dried at -150C showing the total (left) and clipped sample volume (right).

Freezing of blanched bell peppers at -150 C gives a better preservation of the microstructure (smaller pores). The size, shape and orientation of the pores correspond to those of the cells in the original fresh material. Fresh bell pepper samples include different types of cells with an increasing size from outer to inner epidermis (from skin to the wall of the seed cavity), ranging from 20 to 600µm[13]. The cells (20-30 µm) in the outer epidermis are arranged in rows of 5 to 7, parallel to the skin with very thick walls (13-15 μm). The central parachyma cells (50-600 µm) are arranged perpendicular to the skin. SR-µCT images of a sub-volume of comparable parts of tissue of blanched bell

pepper freeze dried at -28 C and -150 C are visualized in Figure 21. The pore walls

of the -28C freeze dried sample are often thicker than the -150C freeze dried sample, probably due to collapse of cells during freezing.

Figure 21: Horizontal cross sections (top) of SR-µCT images of bell peppers samples freeze

dried at -28C and -150C with 3D surface rendering of the cellular tissue (bottom). Image size: 0.50mm*0.50mm, box size: 0.50mm*0.50mm*0.25mm, 1 pixel = 0.56µm.

Mushrooms Mushrooms are popular in dried foods for adding taste and flavour. Mushrooms are not plants like carrots or bell peppers, but are fungi. They contain long tubular cells (diameter ~10µm) with large intercellular spaces. The cell walls of mushrooms are composed of beta glucan and not cellulose as in the walls of plant cells. Mushrooms are soft, fragile and very sensitive to temperature. Therefore selection of the right drying method is very important[14,15]. Blanched and non-blanched mushroom

(Agaricus bisporus) cylinders were freeze dried at -28 C and -150 C. Images obtained using the SkyScan 1172 µCT and the ESRF SR-µCT system are shown in Figure 22. Sub-volumes of the SR-µCT images are visualized in 3D in Figure 23.The original tubular cells of the fresh mushrooms are collapsed and flattened by the ice crystals resulting in a fine porous structure. At -150oC smaller pores are formed. Blanching resulted in a more homogeneous porous structure.

Figure 22: Vertical (A) and horizontal (B, C & D) cross sections of μCT images of mushrooms

freeze dried at -28C and -150C, with and without blanching pre-treatment (A&B: SkyScan 1172, 1 pixel = 2.0µm; C&D: ESRF SR-µCT, 1 pixel = 0.56µm).

Figure 23: 3D surface rendering of SR-µCT images (Figure 22) of freeze dried mushrooms (box size: 0.25mm*0.25mm*0.125mm).

Conclusion µCT imaging of pieces of freeze dried broccoli, carrots, bell peppers and mushrooms showed a porous microstructure. The pores correspond to the spaces originally occupied by the ice crystals. Slow freezing allows ice crystals to grow outside plant cells, causing damage by cell collapse and rupture. Fast freezing determines ice crystals to grow inside cells with very little cell separation and much less damage. The size and orientation of the ice crystals are affected by the size and directionality of the plant cells and the strength of their cell walls. These effects depend on the type of vegetable, the size and shape of the pieces and the processing and pre-treatment conditions. Blanching pre-treatment resulted in a more homogeneous porous structure in case of fast freezing. For bell peppers it prevented shrinkage and deformation of the freeze dried pieces during freeze drying. This study demonstrates the capability of µCT to visualise the 3D internal porous microstructure and external morphology of pieces of freeze dried vegetables. The microstructure covers length scales from sub-micron through sub-millimeter and can be observed with laboratory µCT and SR-μCT. High resolution SR-μCT is needed to obtain information about the pore wall thickness and connectivity between pores. SR-μCT is further used to study the water sorption in the porous matrix[16]. The µCT imaging methods presented in this paper will be used in combination with complementary imaging techniques (SEM and µMRI)) and image analysis methods to obtain multi-length scale information of the porous structure of freeze dried fruits and vegetables which serves as input for modelling of rehydration behaviour[4].

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