Cheese:    Development of structure

Presented By N.E.M Business Solutions                                           
Written By Miloslav Kalab

Cheeses are the most common dairy products. Milk from various animals, particularly cattle, buffaloes, goats, and sheep is used to make cheese. The basic procedure is very simple and is based on the spontaneous processes which, thousands years ago, probably led to the development of cheese. The description of cheese making procedures may be found on the Internet. Sometimes even photographs of various cheese varieties may be viewed. There are also instructions available, how to make processed cheese at school.. Various books, including one entitled Cheese - Chemistry, physics and microbiology, may be found in libraries. Another book, Cheese and Fermented Milk Foods by F. Kosikowski (F. V. Kosikowski & Associates, Brooktondale, NY, USA, 2nd ed. published in 1982) has been used by generations of dairy technologists for its thorough description of the manufacture of very many cheese varieties. Some journal papers dealing with the development of structure in cheese are richly illustrated with micrographs.

The following links lead to individual topics on this page:

Coagulation of milk, Cottage cheese, Unripened cheeses, Cream cheese, Low-fat cheeses, Curd granule junctions,
Can I see curd granule junctions? Processed cheese

Coagulation of milk

SEM of coagulated nonhomogenized milk To make cheese, milk is curdled using an agent called rennet present in the tissue of the calf stomach. Rennet (chymosin) is a proteolytic enzyme and its role is to destabilize casein micelles and make them to coagulate.. Similar enzymes are also found in plants, microorganisms, and digestive tract tissues of other animals including chickens. They break down k-casein present on the surfaces of casein micelles in milk. Deprived of the protective action of k-casein, casein micelles form a gel. When examined by electron microscopy, they form a thin matrix consisting of their clusters and short chains, encapsulating fat globules. Void spaces in the matrix are filled with the liquid milk serum called whey which is a solution of lactose, minerals, and vitamins, and a suspension of whey proteins. The subsequent steps in the cheese manufacture are aimed at separating the curd from the whey and ripening it.

Freshly coagulated milk is cut or broken into smaller particles using wire knives, stirrers or other tools and the cut or broken milk gel is slowly heated and stirred. Casein micelle clusters gradually shrink as they expel the whey and the protein matrix becomes compacted.

Another milk component - fat globules - stays with the proteins in the curd. A small number of fat globules, which were exposed during curd cutting, are washed away with the whey. The micrograph at left shows an early stage of milk coagulation. Fixation in a glutaraldehyde solution preserved the solid constituents allowing water to be removed to show the microstructure of the milk coagulum..

TEM of coagulated nonhomogenized milk TEM of coagulated homogenized milk The removal of the whey makes the casein matrix (shown blue to black) more compact and also brings the fat globules (yellow) closer together. The curd shown in the micrograph was made from fresh full-fat milk. The fat globules are shown as separate entities in the curd - they did not interact with the casein micelles in the fresh milk and they may be characterized as nonreacting inclusions. in the curd. This is true of fat globules unaffected by homogenization.

Homogenization of milk is a process during which large fat globules are disintegrated into considerably smaller particles. Their total surface is up to 6-fold larger than was the total surface of the original fat globules. Since the original fat globule membranes were fragmented by homogenization, there is a large area of unprotected exposed fat surface. Consequently, the small fat particles react immediately with any available proteins in the medium until all bare fat is again well covered. Even entire casein micelles are used for this purpose. Homogenized milk produces a different kind of curd. It is firmer than the curd made from nonhomogenized milk provided that all other parameters have been left unchanged (enzyme, total solids, pH). This phenomenon was known even before the use of electron microscopy, but now the reason for the difference is clear: The minute fat globules with casein micelles anchored on their surfaces have become part of the protein matrix. Fat is no more an inert inclusion but has become a structural constituent. And this example is one of many which show the benefits of electron microscopy in elucidating interactions among food constituents and the development of food microstructure.

Each individual parameter used during the cheese manufacture has some effect on the cheese produced. This is probably one of the reasons for the great variety of cheeses. On the other hand, it is a great challenge for the cheese producer to keep sensory attributes of a particular product constant in a world where, for example, the original rennet has to be replaced with a similar product from a different source because there is a global shortage of calf stomachs. Enzymes with a high proteolytic activity may be efficient in quickly curdling the milk but they may also disintegrate a large proportion of the milk proteins which would consequently be lost for the cheese production. High proteolytic activity may also lead to a weakened casein matrix and alter the characteristic consistency of the cheese. Similar constrains are encountered quite frequently. However, new technologies and new ingredients offer new directions in cheese manufacture. Examples will be presented as this home page is further developed..

Rennet is added to milk along with a lactic bacteria culture. The role of the bacteria is to assist curdling by decreasing pH of the milk. This is achieved by converting lactose into lactic acid. After the whey had been removed and the curd salted and pressed, the next stage - ripening - takes places at a lower temperature for several weeks or months. This is the time when bacteria slowly degrade the milk proteins and produce substances which give the cheese its characteristic structure (carbon dioxide eyes in Swiss-type cheeses) and flavour (e.g., a low concentration of propionic acid),. The great variety of cheeses is made possible by the combinations of many varieties of specific bacteria. However, some cheeses are made with moulds (fungi}such as the Penicillium species [P. camemberti, P. roqueforti, etc.] rather than bacteria. A small group of cheeses (Paneer, Queso Blanco, White cheese) is made by coagulating milk while it is hot, with an acid, such as lactic acid. Such cheese are not ripened.

Pressing and ripening

TEM of cheddared curd - cross section TEM of cheddared curd - parallel section The increase in the density of the curd matrix as a result of whey removal and pressing has been followed in various cheeses by TEM. The micrograph at left shows a thin section of cheddared curd in a cross section. The casein micelle clusters are now more compacted (dark areas) than in the preceding micrographs. The fat globules are in contact with each other, having retained their fat globule membranes. An occasional bacterium (brown) may also be found. Cheddaring is a process, during which slabs of the warm curd are piled up in the cheese vat, subjecting the curd to a slow flow. It aligns the proteins and fat globules into a 'fibrous' structure reminiscent of a baked chicken breast. A section cut parallel with the 'fibres' shows the internal organization of the curd (micrograph at right). Similar kinds of structuring ('stretching') may be found in Italian-style Mozzarella cheese and, in particular, in 'string cheeses'. Additional images will be presented when this home is upgraded.

Cottage cheese

SEM of Cottage cheese Cottage cheese represents an early stage product in the cheese manufacture. Curdled milk, cut into cubes, is heated and gently stirred. The cubes shrink and expel whey, as has already been explained. The whey is drained off and the curd is cooled so that its grains are prevented from 'matting', i.e., fusing with each other. The microstructure of Cottage cheese, as seen by SEM at left, is still porous - consisting of distinct casein micelle clusters. Three bacteria (greenish) can be seen as if attached by filaments to the protein network. These filaments developed during sample preparation from a thin layer of a polysaccharide mucus (bacterial capsules) which surrounded the bacteria in the fresh curd. Highly hydrated polysaccharides are produced by some lactic acid bacteria. They are beneficial in our diet as a source of dietary fibre. In addition, the high viscosity of these polysaccharides modifies the mouthfeel of the food products such as Cottage cheese or yogurt. A high water-holding capacity of the bacterial polysaccharides is another beneficial property.

Unripened cheeses

Some cheeses (Indian Paneer cheese, South American Queso Blanco cheese, American White cheese, North American Ricotta cheese) are made by coagulating hot milk with an acid and separating the curd from the whey.

Core-and-shell structure The development of microstructure in Paneer, Queso Blanco, and White cheeses has been described in Food Structure. These cheeses have several features in common: the milk is first heated to at least 85C and then is coagulated using an acid such as citric, lactic, acetic, or hydrochloric acid (or an acid precursor such as glucono-d-lactone) to a final pH value of 5.5. This means that the curd is not too acidic. The coagulated milk is then cooled and the whey is separated. The microstructure of the casein particles has a characteristic 'core-and-shell' structure (micrograph at left). (The whey contains very little whey proteins since they coagulated due to the heating and became part of the curd).

How the microstructure develops has not yet been fully explained but it is known that three essential conditions must be met: The milk must be coagulated at a temperature higher than 85C so that whey proteins may interact with the k-casein; Whey proteins and the milk salt system must be present in the milk; The final pH value must be 5.5 0.1.

Cream cheese

As the name indicates, Cream cheese is made from pure cream or from mixtures of cream and milk. It has a rich, mildly acidic flavour and a smooth buttery consistency.

In the traditional system of manufacturing, the cream mixture is pasteurized, homogenized, and coagulated using a lactic bacterial culture. The curd is then heated to 52-63C, drained, and hot-packed or cold-packed. This kind of manufacturing procedure yields whey which has to be disposed off.

In a newly formulated method of 'whey-less' manufacturing, the cream-and-milk mixture has the total solids composition of the cheese. The mixture is also pasteurized, homogenized, and incubated with a lactic bacterial culture at ~30C. Then the solidified mixture is homogenized again and packed without cooling. Products which have not been made by the traditional procedure may not be called 'cream cheese' and terms such as 'cream cheese food' or 'cream cheese spread' are used.

Another new procedure has been suggested by Dr. H. W. Modler. It consists of producing curd, mixing it with high-fat (58%) cultured cream, and homogenizing the mixture. Whey is produced but whey proteins are retained in the curd depending on how high the milk is heated prior to coagulation. Heated milk (up to 98C) is coagulated using a 2.5% citric acid solution until pH of 5.3-5.5 is reached and the curd is drained. A mixture of the curd and high-fat cream is heated at ~70C, homogenized, and the cream cheese spread is hot-packed.

Cream cheese TEM
Microstructure of traditional Cream cheese Fat globule clusters (dark yellow) are covered with protein (dark blue) in the aqueous medium (light yellow).
Cream cheese TEM
Microstructure of newly formulated Cream cheese Large fat particles (brown) are not closely associated with protein (dark blue) in the aqueous medium (yellow)
Cream cheese TEM
Microstructure of Cream cheese made from high-fat cream and acid-coagulated hot milk Fat globules (brown) and the curd (which shows the core-and-shell ultrastructure of casein particles - dark blue) are the major ingredients dispersed in the aqueous phase (yellow).
Microstructure Structural differences between the cheeses are best observed using TEM of thin sections. This technique makes it possible to examine the interior of the cheese particles whereas SEM would show their surfaces.

In the traditional Cream cheese, electron microscopy reveals a very high fat content in the form of minute fat globules. Their surfaces are covered with protein particles. The protein frequently covers fat globule clusters rather than each individual fat globule.

The newly formulated Cream cheese spread structure is different. This is noticeable at the first glance: the fat is present in the form of relatively large fat particles which are not associated with protein. Protein is relatively evenly distributed through the body of the spread in the form of small clusters attached to the fat particles at random.

Cream cheese spread made by homogenizing high-fat cream with fresh curd differs from the products mentioned above and clearly reflects the manufacturing procedure. Most of the fat is in form of small globules in clusters and the protein is in the form of relatively large particles. The structure of the curd also reveals its origin - acid-induced coagulation of hot milk to pH 5.5 shows the core-and-shell ultrastructure of the casein particles. Although the curd undergoes homogenization with cream, the 'protein shells' are clearly noticeable (figure at right).

Information on Cream cheese products presented in this section is based on my earlier experimental work described in several papers co-authored by A. G. Sargant, D. A. Froehlich, and H. W. Modler in Food Structure and in Milchwissenschaft 40(4):193-196 (1985) (Milk gel structure. XV. Electron microscopy of whey protein-based Cream cheese spread).


Low-fat cheeses

An example of low-fat cheese where a fat substitute based on protein (light green-coloured globular aggregates of micro- particulated protein) was used to replace a small portion of milkfat. Fat globules originally present in the cheese were extracted from the sample while it was prepared for scanning electron microscopy. Initially they occupied the spaces which now appear empty in the protein body of the cheese.
Electron microscopy has also been used in studies of low-fat or fat-free cheeses, where fat has been replaced with one of the so-called fat replacers. They may be based on proteins, polysaccharides, or even on indigestible fats and oils. Their action is based on the fact that our tongue receives stronger signals about the dimensions of the particles than about their chemical nature. Particles 1 to 3 µm in diameter are perceived as fat. In the micrograph at left, a protein-based fat replacer has been incorporated in cheese. It affects, because of its protein nature, only sensory attributes and instrumental measurements of the cheese. Protein- and polysaccharide-based fat replacers do not melt and their use is limited to specific situations, for example frozen desserts, salad dressings, and some other applications.

The low-fat cheese sample shown at left was freeze-fractured. This procedure makes it possible to fracture (break) even minute particles and study their internal structure by scanning electron microscopy.

Additional scientific papers are being processed for this page. Images of fat substitutes used in cheese, the structure of cheeses made without the aid of microorganisms, Cottage cheese and other cheeses such as Mozzarella will be gradually presented at this site. Processed cheese is another important dairy product in which interesting discoveries have been made using electron microscopy.

Brick cheese vs. Cheddar cheese

Two different structures compared

Differences between cheeses can not only be tasted but also seen, because manufacturing processes impart special features on the cheese microstructure.

To make cheese, curdled milk is cut using steel wire knives or the milk gel is broken into small particles using a propeller stirrer. Subsequent heating shrinks and compacts the particles, as whey is drained off. The curd particles are pressed together and they fuse to make a uniform body of cheese.

The sites of contact, where adjoining curd particles meet, are called curd granule junctions. Their development is schematically shown in the diagram. The gelled (nonhomogenized) milk which contains fat globules (yellow disks) is cut (red vertical line). The fat globules thus exposed are washed out from the curd (arrows). The surface of the granules heals (middle figure). Pressing of the 2 granules together (last figure) causes the superficial layers depleted of fat to fuse. The junction is shown as an area devoid of fat globules.

If homogenized milk is used where the fat globules are considerably smaller, the width of the junctions is markedly reduced.

The junctions can easily be seen by scanning electron microscopy (SEM) as compact zones. To obtain the micrograph shown, a small Brick cheese sample (1x1x10 mm) was fixed in a glutaraldehyde solution, dehydrated in ethanol, defatted in n-hexane, returned into absolute ethanol, and rapidly frozen in liquid Freon 12. The frozen sample was transferred into liquid nitrogen, where it was freeze-fractured, returned into absolute ethanol, where it thawed, and then it was critical-point dried from liquid carbon dioxide. Defatting and freeze-fracturing were the essential steps to show the junctions. Removal of fat makes the fracture plane of the sample 'rough' - full of cavities (initially occupied by fat) - in the area showing the interior of the curd granules. The protein walls separating the cavities scatter light in all directions. Light scattering causes this area to appear lighter than the compact structure of the curd granule junctions, which is mostly free of fat globules. The contrast between both structures creates the curd granule junction patterns.

The junctions are visible even to a naked eye. Brick cheese (left figure below) made from curd obtained by knife cutting shows granules relatively similar in size. Such images may be obtained even at a high school chemical laboratory and instructions on how to proceed are given below. They are also characteristic of other 'stirred-curd' cheeses such as Farmer's, Eidam, Gouda, etc.

Cheddar cheese manufacture involves considerably more work. The fused curd granules (curd slabs) are piled one over the other in the cheese vat in traditional Cheddar making. Heat and the presence of fat in the curd make it to slowly flow down. This cheddaring process elongates the granules. The slabs are gradually replaced so that all are exposed to maximum flow and then the slabs are milled into finger-like pieces. These are salted and pressed together. Milling produces new cuts and thus new junctions (right figure). They are called milled curd junctions and are noticeably thicker than the curd granule junctions.

Also mechanized and automated cheddaring produces both kinds of junction. Different equipment leaves its marks in the junction patterns. 

These findings are of practical importance. For example, they show that any attempts to alter a certain cheese manufacturing procedure would easily be detected. The findings ensure that the higher price which the consumers pay for Cheddar cheese compared to Farmer's cheese is justified and that they buy a product which really is Cheddar cheese.


Readers who would like to see the junctions in their piece of cheese who would like to see the junctions in their piece of cheese and who have access to a chemical laboratory, will find that the procedure is relatively simple.

Needs:

fume hood
3 to 4 petri dishes
cheese slicer or a sharp knife
a piece of filter paper
a pair of tweezers
2 glass or metal plates (at least 10x10 cm)
weights
fine sand paper
2 to 5% glutaraldehyde solution
96% (denatured) alcohol
n-hexane or acetone


Procedure:

Obtain thin (~2 mm) slices the cheese under study, about 5x5 cm large, and immerse them in an aqueous 2 to 5% glutaraldehyde solution in separate petri dishes overnight. This treatment will fix the cheeses and make them easy to handle. It will also increase the contrast between the junctions and the curd interior. Next day, replace the glutaraldehyde solution with 96% denatured alcohol, about 3 times after 30 minute periods. Then replace ethanol with n-hexane or acetone (twice) to extract fat from the cheese. Using a pair of tweezers, place the cheese slices between two filter papers, place them, with the papers, between two glass or metal plates, place a weight on them, and let the cheese slices dry overnight. All this work must be done in a fume hood.

When the cheese slices are dry, they are light and brittle. Sand them carefully with a fine sand paper and see the junctions emerge as sanding progresses. Why is necessary to sand the slices? Because cutting had smeared cheese protein on the slices and it is necessary to remove it.

Stretched Mozzarella, cheeses with very low fat contents, and processed cheeses do not reveal curd junction patterns.

Processed cheese

Processed cheese has a relatively short history. First experiments started at the end of the last century but success was achieved only in 1912, when citric acid was introduced as a melting salt. This happened in Switzerland. A few years later, sodium phosphates were added to sodium citrate and have been used since that time.

The initial idea of processing cheese was to increase the shelf life of cheese and, more importantly, to utilize cheeses which may have had various defects. If it was possible with butter by rendering it, some people believed, it should also be possible with cheese. However, when cheese is melted without any additive, fat separates from protein and the result is terrible. The secret of 'processing' cheese is in keeping the fat in the protein matrix. Heating, however, decreases the ability of the cheese proteins to keep the fat globules in the dispersed state, which means that the emulsifying capability of the proteins has been reduced. Melting salts restore it by binding (sequestering) calcium which is present in the caseins. Melting salts with very strong calcium-binding ability (affinity for calcium) lead to the production of hard processed cheeses which contain fat in the form of very small globules. For those readers who like chemistry, the affinity increases in the following order:

It has to be emphasized that the melting salts are not emulsifiers but they restore the emulsifying ability of the milk proteins very efficiently.

Principles of cheese processing are simple: Various natural cheeses are shredded and then blended with the melting salts and other ingredients such as various kinds of milk solids such as milk powder, whey powder, coprecipitates, cream, butter or butter oil, and sometimes also previously processed cheese. Vegetables and spices may also be added and some processed cheeses may contain 'muscle food ingredients' such as ham, salami, or fish. Other additives such as preservatives, colouring and flavouring agents, binders, and salt and water complete the list of the ingredients.

The blend is heated with constant stirring until a smooth mass is formed. In a continuous processed cheese production, the temperature is increased to 140C for a few seconds to destroy harmful bacteria (such as clostridia) if they happen to be present in some of the ingredients.

TEM micrograph of processed cheese
TEM of processed cheese. Undissolved melting salt crystals (white), fat being emulsified (yellow), and a calcium phosphate crystal (red) are all clearly noticeable. Bar: 5 µm
SEM micrograph of processed cheese
SEM of processed cheese. Cavities left in the protein matrix by undissolved melting salt crystals (blue arrow), dark globular cavities initially occupied by fat, and a calcium phosphate crystal (red) are also noticeable by SEM. Bar: 20 µm
From the structural viewpoint, many features characteristic of natural cheeses are destroyed, for example, the curd granule junction patterns and the original fat globule membranes. On the other hand, new features are formed. In most processed cheeses, undissolved melting salt crystals may still be evident. Adding the salts in crystalline form rather than in the form of an aqueous solution to the cheese blend leaves some crystals undissolved. Interactions between the melting salts and calcium in the natural cheeses lead to the formation of insoluble calcium phosphates. Emulsification of fat - that means disintegration of large fat globules into smaller droplets - is also often noticeable in processed cheese.

 

Processed cheese rework
Hot melt in processed cheese Compact electron-dense structures (purple arrow) developed in processed cheese made with 2.7% trisodium phosphate (used as the melting salt) due to excessive heating (82C for 5 hours).
Hot melt in processed cheese Two kinds of electron-dense structure developed in process cheese made with 20% rework consisting of hot melt. The hot melt was obtained by processing cheese with 2.7% sodium citrate and heating it at 82 for 5 hours (green arrow). The shredded hot melt was added to a fresh cheese blend and the mixture was processed (using 2.7% trisodium phosphate) and excessively heated. Purple arrows point to structures developed in the fresh cheese blend.

The amount of heat absorbed by processed cheese blends during processing may vary and may even be excessive at times. A blend may receive too much heat during continuous processing if, for example, packaging is delayed for some reason and the flow of the viscous processed cheese blend in the pipes is reduced. The blend eventually thickens and stops moving. Then it is called hot melt. It is removed from the pipes and is frozen for future use. Reworking or reprocessing consists of thawing and shredding the hot melt and adding a small quantity of it to a fresh blend. This is often done on purpose to modify the melting properties of processed cheese in a desired manner. Hot melt is thus a type of process cheese food that is not packaged for sale although it would meet product specifications. If it is re-used, it is called rework.

Electron microscopy revealed structural changes in the proteins of rework in the form of small (<1 µm in diameter) dark areas in the micrographs of thin sections. Darkening may be the result of compaction of the cheese proteins or alterations in their chemical structure whereby the heat-modified proteins would react more intensively with heavy metals used during fixation and staining. Tests, in which heavy metals (Os, Pb, U) were omitted from the fixatives and stains during sample preparation for electron microscopy indicated that the proteins in the dark areas were rather compacted than chemically altered. The submicrostructure of the compacted areas was found to be related to the melting salt used to make the processed cheese. Hot melt contained considerably less undissolved melting salt crystals apparently because the crystals had time to dissolve during the entended exposure to heat. Rework dispersed rapidly in the freshly processed cheese blend. This visual observation was confirmed by optical microscopy.

What do these findings mean in processed cheese production? They show that the loss of meltability is associated with structural changes in cheese proteins. They make it possible to detect if rework was used in cheese processing; even at 10% rework, there was a high concentration of the dark areas in the micrographs. Finally, these findings point to interesting thermal effects on processed cheese, which may eventually be studied in greater detail and explained.


                                       Updated: June 23, 2000.
                                        Author: M. Kalab