CLIMATOLOGIA DE NAVARRA

RICH DAIRY PRODUCTS

Dr. D.N. Prasad¹ and Dr. S.K.Tomar²

Head¹and Sr.Scientist² Dairy Microbiology Division

NDRI, Karnal 1.0 INTRODUCTION

Electron Microscopy (EM) is being increasingly used to study the microstructure of individual components in milk products and modifications these entities undergo either alone or by interactions with each other or with additional ingredients during manufacturing processes. Such studies can be used for food structuring, texture-structure conclusions and quality evaluation (Aguilera and Stanley, 1999).

The EM techniques render a markedly higher magnification at a considerable better resolution than light microscopy. Instead of light, a beam of electrons generated from an incandescent tungsten or lanthanum hexaboride electrode is employed to magnify the image of the sample. There are two major EM modes- Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM). Magnetic lenses are used to focus the electron beam in both kinds of microscope. The specimen is placed into the path of electron beam in the TEM but in the SEM, it is placed at the end of focussed electron beam path. The image is produced in the form of a shadow on a fluorescent screen in TEM. In SEM, reflected and secondary electrons are processed by an electron detector to form a quasi three dimensional image on a monitor screen. To avoid the absorption of electrons by air, the whole operation is carried out in vacuum. An anode with an orifice in its centre is positively charged and those in the centre pass accelerated through the orifice toward the specimen. Accelerating voltage of 3 to 20 kV has been used to do SEM and 60 to 80 kV has been used in TEM of Dairy foods.

2.0 PREPARATION FOR ELECTRON MICROSCOPY 2.1 Fixation and Dehydration

As a pre-requisite to the observation of a sample with electron microscope, it is necessary to dehydrate the specimen and to fix (preserve intact) the structure in their natural orientation. The fixation and dehydration process must be carried out carefully in stages to avoid distortion of the image. Common fixatives used for this purpose are OsO4

(glutaraldehyde, formaldehyde and acrylic aldehyde) and permanganates (Potassium and barium permanganates). Other specialised compounds used for this purpose include uranyl acetate, chromium, mercury salts and phosphotungstic acid etc. Dehydration of dairy products can be accomplished by air-drying, freeze-drying and critical point-drying (Prasad, 1998).

2.2 Encapsulation

This technique is used to prepare highly viscous product like fat rich dairy product for both TEM and SEM. In this technique the specimen is concentrated in agar or other gel capsules and such sealed capsules are handled as larger solid samples.

A special encapsulation technique as devised by Veliky and Kalab(1990) are in vogue for heat-sensitive products such as cream and butter. A special apparatus is used for this purpose. A double-needle assembly consists of a central needle 1mm in diameter concentrically located in a wider needle.. The assembly is connected to two 5-ml syringes with piston to allow the food sample flow through the inner needle and a 3% sodium alginate solution is injected from another syringe to coat the food sample. Food sample and sodium alginate solution are extruded simultaneously into a 0.05 M calcium chloride solution, pH 6.5, where sodium alginate immediately forms a gel and immobilizes the food sample. The 100 to 200 mm long columns of encapsulated food so prepared may be cut into shorter segments and transferred in to a fixative for subsequent processing of sample for EM.

3.0 TRANSMISSION ELECTRON MICROSCOPY

The TEM can be performed using various techniques as discussed in the following sections.

3.1 Conventional Technique

The conventional method consists of embedding the specimen in a resin cutting thin sections(15 to 90 nm thick) with the help of an ultramicrotome, staining the structures within the sections(using heavy metal salts e.g. osmium, lead and uranium) and placing the sections in the path of the electron beam.

3.2 Special Technique

The EM investigation of fat rich products offers a number of difficulties. Such studies are hampered by the solubility of the fat in dehydrating agents and embedding media leading to destabilization of the fat globules and unpredictable extraction of fat. As a result, conclusions drawn from such electron -micrographs are dubious. For these reasons, fat rich dairy products are studied employing following special techniques (Kalab, 1981):

3.2.1 Negative Staining

This is relatively a simple procedure used for TEM. The specimen is in the form of submicroscopical particles semitransparent to the electron beam. Addition of phosphotungstic acid (PTA), sodium phosphotugstate, or ammonium molybdate solutions to the specimen makes the medium electron-dense but spares the particles. The thin layer of specimen so prepared is dried and finally placed into the microscope. The electron beam passes only through the semi-transparent structures under study and is absorbed by the surrounding stain of heavy metal. The structures appear light against a dark background in the micrographs.

3.2.2 Metal Shadowing

Metal shadowing is a suitable technique for studying suspensions. In this technique, the specimen is fixed and dried on a translucent film. The dried film is subsequently

shadowed with platinum or a platinum and palladium alloy. During TEM study, as the electron beam passes through the shadowed area and exposes photographic material, the shadow appears dark on the negative whereas areas with platinum deposit produce light image. This image depends on the topography of the specimen's surface.

3.2.3 Freeze Fracturing and Freeze-etching

Though laborious, these techniques enable to examine the specimen without altering it chemically (fixation) or physically (dehydration, embedding, drying).The specimen is allowed to freeze rapidly followed by freeze-fracturing at a temperature below -110^o C. The fracture plane is subsequently replicated with platinum and carbon either immediately or after certain period of freeze etching, during which a thin layer of ice in the specimen sublimes off and reveals underlying structures. The specimen is thawed; replica is separated from the specimen, and examined in the microscope.

4.0 SCANNING ELECTRON MICROSCOPY

In this system, a focussed electron beam is employed to examine the specimen. Some of these electrons get reflected while others are able to generate secondary electron from the gold coating. These secondary electrons are used to form an enlarged image of the specimen surface. In order to neutralize the negatively charged incident electrons, the specimen should be electrically conductive. This is accomplished by coating of specimen generally with gold with the help of an ion sputter coater. Gold-palladium alloy, platinum and iridium are other heavy metal used for this purpose..

5.0 MICROSTRUCTURE OF FAT RICH DAIRY AND RELATED PRODUCTS

5.1 Fat Spreads

Fat, an integral and indispensable part of our diet is consumed in large amounts as margarine and butter and is used for baking and frying and as spreads. 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 depend on the source of the fat blend and the processing conditions (Heertze & Leunis, 1997).Common fat spreads used in daily life are Shortenings, Margarine, and Butter .

Shortenings are frequently used in bakery applications. They are composed of liquid oil and fat crystals only unlike margarine and butter which contain about 16% water, in addition. While oil forms a liquid phase, fat crystals attain the form of a platelike three -dimensional crystalline network, with crystal bridges.

The microstructure of margarine is characterized by presence of water-droplet in the backdrop of a fat crystal network. Water droplets of a few micrometers in diameter are formed during intensive mixing of fat and water phases during processing. Crystals orient at the water droplet surface and thus stabilize droplet. Fat forms familiar network composed of plate-like crystal aggregates. In products (creaming-, cake- and puff pastry), the nature of the fat crystalline network differs with respect to the size, the shape, and the aggregation of the fat crystals (Heertze, 1993).

Butter offers a distinctly different microstructure exhibiting a discontinuous structure of fat globules and a crystalline fat matrix. The fat globules remain intact through the churning process. Amount of globules and the inter-globular fat phase varies with ripening procedure of the cream and other processing conditions.

5.2 Whipped Cream

A comparison of whipping of homogenized and non-homogenized cream reveals various features. The homogenized cream is characterized by smaller size of fat globules and homogenization clusters. The air bubbles decrease in proportion as the time of whipping increases and are much smaller in homogenized than in non-homogenized cream. During whipping, µlatter disrupt fat globule membrane resulting into agglomeration of fat-globules.

Further whipping results in disappearance of the air-bubbles and in the formation of butter granules similar to those found during churning (Schmidt & van Hooydonk, 1980).

5.3 Ice cream

The EM studies of ice cream mix depict it as an emulsion comprising of tiny fat droplets dispersed in the water phase, each surrounded by a membrane of proteins and emulsifiers. The sugars get dissolved in the water phase. During cooling, milk fat partially solidifies so that each droplet consists of solid fat crystals cemented together by liquid fat. Ice crystals and air bubbles are two additional phases which come into existence during whipping and freezing of mix. They are dispersed in the concentrated unfrozen mix. The water contributed by milk or cream in the mix freezes in to ice. As a result, the dissolved sugar get increasingly concentrated in the unfrozen phase as more ice forms.Thus microstructure of ice cream comprises four distinct phase, ice crystals, air bubbles, fat droplets and the unfrozen phase. The process of freezing and aeration of the mix causes the emulsion to undergo a process called partial coalescence. During this process, fat droplets form clusters and aggregates of fat that surround and stabilize the air bubbles as it happens in whipped cream.

5.4 Butter Milk

Fat is present in dispersed state in cream and fat globules measuring 0.5 to 10 µm in diameter are encased in membranes composed of lipoproteins which stabilizes them in the milk and inhibits their aggregation. Most of the fat globule membranes are disrupted during churning leading to aggregation of globules in to butter. Most of the membranes fragments are released into the butter milk while others are retained in the butter. Consequent upon removal of the butterfat, though the composition of butter milk made from sweet cream is similar to that of skimmilk with respect to protein and carbohydrates yet it contains additionally excessive membranous material and slightly higher lipid content.

Due to lower price, there is a temptation to blend small amount of butter milk into skim milk; chemical detection of butter milk may prove to be difficult due to identical composition. The EM studies can be used to detect differences in the morphology of butter milk and skim milk particles in blends and also of reconstituted products.

Apparently, spray-dried skimkmilk and butter milk appear similar under SEM. Both exist in the form of spheres or clusters of spheres widely ranging in dimensions. A closer look, however, offer some striking dissimilarities. In spray-dried skim milk, majority of spheres are severely wrinkled and occasionally displaying the apple like structure. On the

other hand, spray dried butter milk is characterized by less deep wrinkled spheres and absence of collapsed structures frequently found in spray-dried skim milk. The former has been found more porous, a feature related to the fat content. Another possibility of adulteration of blending fluid butter milk with skim milk and spray dry the mixture could not be detected by SEM. This could be ascertained by observing the presence of fragments of fat globule membrane by TEM (Kalab, 1980).

6.0 CONCLUSION

Electron microscopy though a sophisticated and expensive technique is highly valuable in establishing the relationship of various attributes of finished product e.g.

composition, rheology as well as manufacturing conditions with its microstructure. The study of microstructure has ample application in quality control, product development and process control.

7.0 REFERENCES

Aguilera, J.M. and Stanley, D.W.1999. Microstructural principles of food processing and engineering.2^nd ed.Aspen Publishers,Inc, Maryland, USA.

Heertze, I.1993.Microstructural studies on fat research. Food Struct.12:77-94

Heertze, I. and Leunis, M.1997. Measurement of shape and size of fat crystals by electron microscopy. Food Sci. Technol.30:141-146.

Kalab, M.1980. Possibilities of an electron microscopic detection of butter milk made from sweet cream in adulterated skimmilk .Pages 645-652.Scanning Elect. Microscopy.1980/III, SEM Inc, AMF O' Hare, USA.

Kalab, M.1981. Electron microscopy of milk products: A review of techniques. Pages 453-472. Scanning Elect.Microscopy.1981/III, SEM Inc, AMF O' Hare, USA.

Prasad, D.N.1998.Microstructure of traditional dairy products. CAS 4^th Short Course on Advances in Traditional Dairy Products (Dec 16,1997-Jan.6,1998) NDRI, Karnal.

Schmidt,D.C and van Hooydonk, A.C.M.1980. A scanning electron microscopical investigation of the whipping of cream.Pages.653-658.Scanning Elect. Microscopy.1980/III, AMF O Hare, USA.