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Feature

A Close-Up Look at Food

Food researchers really like to see what they're eating!

Tony McKenna

Looking closely at food, using a variety of sophisticated techniques now available, gives us a better understanding of how food is formed, behaves and even tastes. As a New Zealand Dairy Research Institute scientist, I've been studying food microstructure techniques at the AFRC Institute of Food Research in England, freezing a range of foods for examination under electron microscopes.

The New Zealand dairy industry produces a wide range of dairy ingredients which are used in numerous foods including baked goods, confectionery, dairy foams, cultured milks, cheese products and meats. A proper understanding of the behaviour of a food requires an understanding of the structural arrangement of the food components within the food product.

Food microstructure, the examination of food by light (resolution to 0.2 mm) and electron (resolution to 0.2 nm) microscopy techniques, allows the morphology, distribution and interaction of components within food to be determined. The range of microstructural techniques available for examining the structure of foodstuffs has expanded considerably over the last few decades, increasing the type of food able to be examined.

Cryo-preparation Techniques

The preparation of a sample prior to examination in the microscope can be a painstaking process, but it is critical for the production of micrographs which accurately represent the original food. The high vacuum inside the electron microscope causes instability of any volatile components such as fat and water. Therefore these components need to be immobilized or removed prior before examination. The volatile components can be chemically fixed by the use of gluteraldehyde, formaldehyde and osmium tetroxide followed by dehydration, embedding in resin and thin sectioning. However, it's a laborious process which may lead to distortion of the original structure.

The limitations of these chemical techniques resulted in the development of quick-freezing techniques for immobilization of the food components. Two of these techniques, one used to prepare samples for scanning electron microscopy (SEM) and the other for transmission electron microscopy (TEM), are being developed for food microstructure research at the NZDRI.

A new cryogenic preparation unit lets us freeze food samples which would normally be deformed in the vacuum and under the electron beam, using liquid nitrogen at a temperature of -196C. The samples are inserted into the cryogenic unit, fractured to expose a fresh surface, sputter coated with gold, transferred to the cold stage on the SEM and viewed. This technique has been used to study a wide range of food products including low-fat spreads, whipping cream and ice-cream.

The spatial distribution of the three phases of ice cream can be very effectively studied by cryo-SEM. Fat globules can be seen in both the aqueous phase and adsorbed to the interface which makes up the internal surface of an air bubble. A featureless glass-like surface is the third phase component, a fractured ice crystal. This technique allows the assessment of the size distribution of air bubbles, fat globules and ice crystals (all major texture determinants) after manufacture and during storage.

Rather large specimen areas can be studied by cryo-SEM allowing detailed topographical interrelationships of the component parts to be investigated. For example, the laminations in puff pastry can be clearly visualized. However, cryo-SEM (resolution 5-10 nm) can not achieve the resolution of transmission electron microscopy (resolution 0.2-0.5 nm).

Freeze-Etching Food

The process of freeze-fracture replication, commonly referred to as freeze-etching, involves producing a replica from a fracture plane made across a frozen specimen. It is the replica which is viewed under the microscope, not the actual product itself. The process involves rapidly cooling the food sample in either liquid nitrogen, propane or freon and introducing the sample into a freeze-fracture apparatus. There it is fractured and the fracture face is shadowed with platinum to create selective electron contrast with a carbon layer used to produce a continuous, exact replica. Once thawed, the food sample is removed and the replica can then be viewed in a conventional TEM.

Freeze-fracture replication has been extensively used to study milk and milk products, observing the shape of proteins, protein gels, fat crystals in a range of fat products, milk powders, cream, coffee cream, ice-cream and confectionery. In-process examinations can be carried out by simply sampling at the relevant stages during a manufacturing process and rapidly freezing by plunging the sample into the cryogen and then transporting the frozen samples to the freeze-fracture apparatus for replica preparation. Samples may be stored in the cryogen for long periods of time thus enabling transport of the samples over long distances.

The structural differences between UHT cream made from fresh and imitation, homogenized cream were determined by freeze-fracture replication. The fat globules in the recombined cream were smaller and ellipsoid (caused by the formation of large fat crystals within the fat globule), whereas those in the fresh cream were larger and spherical. Casein micelles were present in both creams as were fat crystals (free fat) in the aqueous phase of the recombined cream.

In liquid samples, such as milks and creams, the technique used for freezing will influence the freezing rate. If this rate is too slow, then artifacts resulting from the presence of ice-crystals will influence the structure of the sample and hence the replica. Glycerol may be added to liquid samples to depress the freezing point and thus allow simple plunge-freezing in liquid nitrogen without ice-crystal formation. For foams such as whipped cream, glycerol would destroy the structure, and so a faster freezing technique must be found.

Looking with Lasers

A relatively new light microscopy technique, confocal laser scanning microscopy (CLSM), is set to revolutionise the field. This technique uses a laser to illuminate and scan points in a very thin plane of the sample. Only the information from this very thin plane (down to 0.5mm) passes through the confocal pinhole (a small aperture) to the detector. This leads to increased resolution, the possibility of identifying up to four separate food components by specific fluorochrome labelling, deep non-invasive penetration of the sample (up to 100mm) and the ability to obtain sequential optically thin sections through a food. These sequential sections may be computer reassembled to form three-dimensional images.

The preparation of salad dressings, low-fat spreads, butters, creams, and ice-creams for conventional light microscopy is difficult. Confocal microscopy is ideally suited to the study of such emulsions as the lipid and/or aqueous phase can be stained with an appropriate fluorescent dye. Staining a solid sample such as cheese can be achieved by placing a few crystals of a fluorescent dye on the surface of the foodstuff and allowing it to diffuse into the sample. In studying processed cheese, the cheese was stained with crystals of Fast Green FCF for aqueous phase staining to show up the continuous protein phase of the cheese and the dispersed oil phase.

Crystalline material can be imaged by reflectance to determine the size and position of crystals in materials such as milk powder and processed cheese. Milk powders, some containing crystalline lactose, have been examined by reflection. The different food components in chocolate and other confectionery products can be studied by staining the fat and examining the crystalline material by reflection.

Currently there are three confocal microscopes in New Zealand, one each at Otago and Auckland Medical Schools and the third at Victoria University. The NZDRI have assisted Massey University in the purchase of a confocal microscope which will be installed in the University's plant biology department in August 1995.

Studying the microstructure of foodstuffs can result in valuable information about the arrangement of component phases. In conjunction with other analyses, it can bring a greater understanding to the effects of compositional and processing alterations on the functional properties of a wide range of food products.

The New Zealand dairy industry has a growing presence in the world marketplace and with it comes the opportunity to supply new products, ingredients and processes. To exploit these opportunities a rapid development response is required, which must be based on a good understanding of the relevant food system. There are a great number of ways to measure the performance characteristics of a food. Often a close look at the structure can help us to understand why a food has such performance characteristics.

Tony McKenna is a food structure scientist with the food science section of NZDRI.