Heertje studied the microstructure of food and detergent products for
25 years at the Unilever Research Laboratory in Vlaardingen, the
Netherlands, lately as senior scientist. In the foods area his main
interests were proteins and protein gelation, carbohydrates, dairy
products, emulsions, and bakery and fat
products. In this area, he published about over 20 scientific papers
in the open literature and lectured at many conferences and institutions
throughout the world. He was the former editor of the journal Food
Structure for Europe and is now retired. In the last part of his
scientific career he explored, in particular, the use of liquid
crystalline phases in the structuring of food products. From his wide
gallery of micrographs, he is contributing images of fat crystals and fat
spreads, like margarine and butter. His three contributions to the Guest
Food Microscopist sections were earlier available at the
distans.lth.se:2080/ server but they have now been included in this
Guest Food Microscopists 2 section.
is an indispensable part of our nutrition. It is a main energy source for
the body. It is consumed in large amounts as margarines and butter, and is
used for baking and frying and as spreads. On eating those types of
products, we probably do not realize that we are eating crystals
(Figure 1). But indeed in fat spreads the fat molecules of
high-melting fats are crystallized in a regular arrangement into solid
crystals. The type and the size of the crystals and their mode of
interaction vary depending on the source of the fat blend and the
aggregate of needle-shaped and plate-like crystals, deposited on a
substrate, in a margarine observed by TEM after replication.
Shortenings, frequently used in bakery applications,
are composed of liquid oil and fat crystals only, whereas margarine and
butter contain about 16% water in addition. Solid/liquid ratios of the fat
phase in those products may vary widely, and are typically within the
limits of 0.2 to 0.35 in margarine and butter at room temperature. Those
products derive their consistency in particular from the organization of
the fat crystals. An example of the product structure of a shortening is
presented in Figure 2. The fat crystals form a plate-like
three-dimensional crystalline network, with crystal bridges. The plates
are in fact aggregates of single crystals as shown in Figure 1.
|Figure 2. Fat crystalline
network in a shortening observed by Cryo-Scanning EM. Careful
inspection of the original micrograph reveals that the plates
visible in this structure are in fact aggregates of single crystals.
The open spaces in the structure indicate the areas where oil had
originally been present before it was removed during the preparation
of the sample for electron microscopy; oil removal was necessary to
show the crystals.. |
|Figure 3. Fat crystalline
network (yellow) and a water droplet structure (blue) in margarine
observed by Cryo- Scanning EM. The shell around the water droplet
has a crystalline structure. Bar: 10 µm. For open spaces see the
caption at Figure 2. |
Margarine also derives its
from a fat crystal network. The most striking difference, on comparing
these products, is the presence of water droplets in margarine (Figure
3). Water droplets of a few micrometers in diameter are formed during
intensive mixing of fat and water phases during processing. Crystals
orientate at the water droplet surface and stabilize the water droplet.
The continuous fat matrix again shows an interconnected network structure
composed of plate-like crystal aggregates. In margarine products for
various applications (creaming-, cake- and puff pastry), the nature of the
fat crystalline network will vary with respect to the size, the shape, and
the aggregation of the fat crystals.
a completely different microstructure: it shows a discontinuous structure
of fat globules and a crystalline fat matrix (Figure 4). Butter is
made by churning dairy cream. The fat globules observed have remained
intact through the churning process. Different amounts of globules and the
interglobular fat phase may be observed depending on the ripening
procedure of the cream and other processing conditions.
|Figure 4. Fat structure in
butter showing a discontinuous arrangement of fat globules (yellow
arrow) and fat crystals (green arrow) observed by Cryo-Scanning EM.
Bar: 10 µm. For open spaces see the caption at Figure 2.
Many people prefer the taste and the mouthfeel of butter over margarine
and not without reason there is a
product on the market named: I Can't Believe It's Not
Butter. Apparently, the differences in structure between margarine
and butter are reflected in their functional properties. But, at the same
time, establishing the relationship between structure and function offers
the possibility to manipulate the structure in such a manner that desired
functional properties can be obtained. This is, in fact, one of the main
objectives for doing microstructural research.
Dr. Heertje's scientific publications on foods:
- HEERTJE I., BOSKAMP M.J., KLEEF F. van, GORTEMAKER F.H. 1981. The
microstructure of processed cheese. Neth. Milk Dairy J. 35,
- HEERTJE I., VISSER J., SMITS P. 1985. Structure formation in acid
milk gels. Food Microstruct. 4, 267-277.
- HEERTJE I., KLEEF F. van.1986. Observations on the microstructure
and rheology of ovalbumin gels. Food Microstruct. 5, 91-98.
- VISSER J., MINIHAN A., SMITS P., TJAN S.B., HEERTJE I. 1986. Effects
of pH and temperature on the milk salt system. Neth. Milk Dairy J.
- HEERTJE I., LEUNIS M., ZEYL W.J.M. van, BERENDS E. 1987. Product
morphology of fatty products. Food Microstruct. 6, 1-8.
- HEERTJE I., VLIST P van der, BLONK J.C.G., HENDRICKX H.A.C.M.,
BRAKENHOF G.J. 1987. Confocal scanning laser microscopy in food
research: some observations. Food Microstruct. 6, 115-120.
- HEERTJE I., EENDENBURG J. van, CORNELISSEN J.M., JURIAANSE A.C.
1988. The effect of processing on some microstructural characteristics
of fat spreads. Food Microstruct. 7, 189-193.
- JURIAANSE A.C., HEERTJE I. 1988. Microstructure of shortenings,
margarine and butter - a review. Food Microstruct. 7, 181-188.
- HEERTJE I., NEDERLOF J., HENDRICKX H.A.C.M., LUCASSEN-REYNDERS E.H.
1990. The observation of the displacement of emulsifiers by confocal
scanning laser microscopy. Food Struct. 9, 305-316.
- HEERTJE I., HENDRICKX H.A.C.M., KNOOPS A.J., ROIJERS E.C., TURKSMA
H. 1991. Use of mesomorphic phases in food products. European
patent: 0 558 523 B1
- HEERTJE I., 1993. Microstructural studies in fat research. Food
Struct. 12, 77-94.
- HEERTJE I. 1993. Structure and function of food products: a review.
Food Struct. 12, 343-364
- HEERTJE I., PÂQUES M. 1995. Advances in electron microscopy. In:
New physico-chemical techniques for the characterization of complex
food systems (Dickinson E., ed). Blackie Academic, London, New York.
- HEERTJE I., AALST H. van, BLONK J.C.G., DON A., NEDERLOF J.,
LUCASSEN-REYNDERS E.H. 1996. Observations on emulsifiers at the
interface between oil and water by confocal scanning light microscopy.
Food Sci. Technol. 29, 217-226.
- HEERTJE I., LEUNIS M. 1997. Measurement of shape and size of fat
crystals by electron microscopy. Food Sci. Technol. 30, 141-146.
- HEERTJE I., ROIJERS E.C., HENDRICKX H.A.C.M. 1998. Liquid
crystalline phases in the structuring of food products. Food Sci.
Technol. 31, 387-396.
- HEERTJE I. 1998. Fat crystals, emulsifiers and liquid crystals. From
structure to functionality. Polish J. Food Nutr. Sci. 7/48, 7-18
- HEERTJE I. 1997. Vet zonder vet. Natuur en Techniek, 65 (2),
© I. Heertje 1998
|Figure 1. Structure of a 40% fat
spread, containing 60% water observed by SEM. Water droplets (w)
in a continuous fat matrix. Protein particles (e) in the water
In the elaborate range of
food products, fat spreads play an important part. In the earlier days,
shortenings (100% fat), and margarines and butter (80% fat) set the scene.
These products derive their consistency mainly from fat crystals and the
way those crystals interact.
|Figure 2. Structure of a 25% fat
spread with 4% protein and 1% carbohydrates in the aqueous phase,
observed by CSLM.|
Top: The fat, in between the droplets, was
made visible by the fluorescer: Nile blue.
protein, in particular at the interface of the droplets, was made
visible by the fluorescer: fluorescein
To decrease the proportion of fat in the diet of consumers in the
Western world, spreads with 60, 40, 20 and even 0% fat have appeared
on the market(1).
(2)With the decreasing
fat content, the composition and the structuring of the aqueous phase
becomes gradually more important for obtaining a good fat spread.
Low fat spreads (20% fat) have a structure comparable to that of
margarine. In general, these products are water-in-oil emulsions, in which
a continuous fat matrix surrounds the dispersed water droplets (Figures
1 and 2).
Therefore, those products
behave in many respects (mouthfeel, spreadability) similar to classical
fat spreads. With even lower amounts of fat (<20%) it becomes
impossible to keep the fat in the continuous phase. When water becomes the
main component of the food system, we are faced with the problem to
structure it while giving the product fat-like properties.
|Figure 3. Fine stranded
polysaccharide gel network of curdlan (Courtesy T. Harada)
observed by TEM.|
|Figure 4. Globular particles in the
formation of a particle gel of ovalbumin observed by
Various strategies to achieve this have been developed. Large amounts
of water can be enclosed in fine stranded networks of polymer chains, with
many entanglements and junction zones.
Carbohydrates (carrageenan, pectin, curdlan (Figure 3) and
proteins (gelatin) may show this behaviour. In other cases, in particular
for proteins, coarse particle gels are formed by aggregation of
macromolecular assemblies in globular form (e.g., yogurt)
However, in the design of very low and zero fat spreads, these
strategies fail to imitate the properties of margarine. This was
accomplished by using liquid
crystalline or mesomorphic phases.
|Figure 5. Aggregation behavior
of emulsifier molecules|
(a) Head group larger than the
tail: micelle formation in water
molecule: formation of parallel bilayers
(c) Head group
smaller than the tail: formation of inverted micelles,
||Figure 6. A
schematic drawing of a lamellar phase. The emulsifier
molecules are arranged in parallel sheets in such way that the
hydrophilic head groups of the molecules are in contact with
water and that the hydrophobic chains are in contact with each
other and excluded from
Such phases can be formed from emulsifiers such as monoglycerides
(monoacylglycerols) and phospholipids. The molecules of these compounds
are characterized by a hydrophilic water-attracting head and a hydrophobic
water-rejecting tail and display a specific aggregation behavior that is
determined by their geometry (Figure 5). The situation of the
greatest interest in the structuring of food products is the one involving
more or less cylindrically shaped molecules (Figure 5b). In that
case, bilayer structures are formed, which may enclose water layers, which
give rise to a so-called lamellar phase, a three-dimensional structure of
alternating bilayers and water layers (Figure 6). The lamellar
phase can be obtained by heating, e.g. monoglycerides, in the presence of
water to above a certain transition temperature and can, under certain
conditions, take up all the available water into a continuous swollen
lamellar phase (Figure 7).
When this phase is cooled down below its transition temperature, the
lamellar phase structure is retained and a highly viscous phase, the
so-called a-gel phase is formed. By
crystallisation this phase in turn may be transformed into an even stiffer
gel type, the coagel.
|Figure 7. Monoglycerides (0.05g/g)
and water heated above the transition temperature (60°C), observed
by LM. Swollen streaky lamellar texture and some (partly
swollen) multilamellar vesicles (arrows).
It appears to be composed of a
network of monoglyceride crystals enclosing water (Figure 8). Both
gel types have much to offer in the structuring of food products. a-Gel can be applied in softer products and is optimal
in aerated products because of its strong foaming capacity in contrast to
the coagel. Coagel has been applied in the development of a "fat" free
margarine for the North-American market. This product contains 3% of
monoglyceride in a coagel state. The product shows a remarkably fatty
impression. This may well be related to the striking resemblance of the
network of monoglyceride crystals (Figure 8) with the network of
triglyceride crystals in a shortening or an 80% fat spread such as
margarine. Apparently, the enclosed liquid, i.e., oil in the case of
high fat products and water in a "fat" free margarine, is of minor
importance for the perceived fatty impression. The structure of the
network and the function of the product appear to be clearly related.
Undoubtedly, the further exploration of the relation between structure and
function will open up new roads in the design of new food products.
|Figure 8. Network structure of
monoglyceride (0.05g/g) crystals in a coagel observed by SEM. In
the open space between the crystals water was originally
KROG, N., BORUP A.P. 1973. Swelling behaviour of lamellar phases of
saturated monoglycerides in aqueous systems. Journal of the Science
of Food and Agriculture 24, 691-701.
HEERTJE I., HENDRICKX
H.A.C.M., KNOOPS A.J., ROIJERS E.C., TURKSMA H. 1991. Use of mesomorphic
phases in food products. European patent 0 558 523 B1
I., ROIJERS E.C., HENDRICKX H.A.C.M. 1998. Liquid crystalline phases in
the structuring of food products. Food Science and Technology
© I. Heertje 1999
How do you like your
eggs in the morning?
Some not fully respected people may have
experienced that the content of raw eggs is quite different from a nice
hard-boiled or scrambled egg consumed at the breakfast table. Those of us,
who prepare their own food, know that an egg consists of 3 parts: the
shell, the eggwhite, and the yolk. Only the latter two are used as a food.
It is easy to observe how the eggwhite and the yolk solidify during
frying. The solidification actually is a complex process consisting of a
sequence of structural changes. Macroscopically, the translucent viscous
mass turns white and solid.
The hardening is due to ovalbumin, which is
the main eggwhite protein. When an aqueous solution of this protein
(20g/100g) is heated, it forms an irreversible gel. The gel formation can
be followed by measuring the shear modulus (some measure of the hardness)
as a function of temperature. It appears that at about 75°C the hardness
increases dramatically up to about 90°C. On subsequent cooling and
reheating, the hardness is not much affected. This in contrast to gelatin,
another well-known protein, which, after gel formation, dissolves again on
Proteins play an important role in many
food systems. Food technologists, who understand structure formation and
relationships between macroscopic properties microstructure and molecular
properties, may fruitfully apply this information in the development and
the processing of food products.
In this context, ovalbumin gels, prepared
by heating at 100°C under different conditions of pH were examined by
scanning and transmission electron microscopy and rheologically
characterized. The latter was performed by stretching a small sheet of the
gel and measuring the elongation as a function of the applied force. In
addition, changes in the conformation of the protein molecules were
examined. This was accomplished by nuclear magnetic resonance (NMR)
|Fig. 1. A heat-set
ovalbumin gel at pH 5. Dark area represents protein. Note an
aggregated inhomogeneous protein structure. Thin-sectioning TEM.
Bar: 0.5 µm|
|Fig. 2. A heat-set
ovalbumin gel at pH 5. Note the granular structure. SEM after
freeze-drying. Bar: 2 µm|
|Fig. 3. A heat-set
ovalbumin gel at pH 10. Dark area represents protein. Note the
homogeneous distribution of protein filaments. Thin-sectioning TEM.
Bar: 0.5 µm|
|Fig. 4. A heat set
ovalbumin gel at pH 10. SEM after freeze-drying. The cellular
structure is induced by ice crystals formed during sample
preparation. Bar: 2 µm |
Using this technique it is possible to
study the behaviour of the protein molecules at the different conditions
of pH and temperature. In particular, information is obtained on the
degree of unfolding of the protein molecules, which is important to
understand the aggregation behaviour of the protein molecules.
An ovalbumin gel prepared at pH 5, shows an
inhomogeneous distribution of strongly aggregated protein particles (Figs.
1 and 2), whereas a homogeneous distribution of fine protein strands is
observed at pH 10 (Figs. 3 and 4). This behavior is ascribed to the
molecular properties of the proteins. At the low pH, close to the
isoelectric point of the ovalbumin at pH 4.5, strong hydrogen bonds,
ionic- and strong hydrophobic interactions occur between the uncharged
protein molecules. They result in the formation of dense protein
aggregates. In contrast at high pH, under influence of electrostatic
repulsion, protein molecules unfold and are irreversibly cross-linked
mainly via covalent disulfide bonds without much further interactions. For
this reason, gels prepared at such a high pH will not be affected by
subsequently lowering the pH.
The differences are further reflected in
the mechanical and other properties of the gels. A structure, which is
composed of flexible, unfolded, but not strongly interacting protein
chains is much more extensible than the aggregated structure composed of
compact, strongly interacting protein molecules. In the latter structure,
regions of low protein concentration tend to act as weak points, resulting
in a gel, which breaks easily. This also explains why gels at pH 5 are
opaque and easily release water, whereas pH 10 gels are transparent and do
not loose water.
Combining this type of knowledge on all
levels of structural organization is of great help in the optimal use of
proteins and modified proteins in foods and food processing.
© I. Heertje 2001