Feature

Building Better Cheeses

Chemists unite in the never-ending search for the perfect taste.

Professor Charmian J. O'Connor

New Zealand researchers are working on ways of biochemically manipulating the flavour of dairy products such as cheese.

In countries where food is abundant, palatability is a major factor in determining food choice and therefore nutrient intakes. Fat is a significant determinant of palatability, and dairy foods owe their attractiveness to the very special sensory characteristics associated with milk fats. They contribute to our sensory perceptions in two ways: as a source of flavour and aromatic compounds, and as an influence on texture.

The quantities of the various main constituents of milk vary considerably between cows of different breeds and between individuals within a herd. A typical composition would be 86% water and 14% solids, with the latter being subdivided into 5.0% fat, 3.4% protein, 4.8% lactose and 0.8% minerals.

If milk is left to stand, a layer of cream (milk fat) will form on the surface. Cream consists of a large quantity of spherical fat globules surrounded by a "skin" (or membrane) consisting of proteins and phospholipids. The diameters of the fat globules range from 0.1-20 mm with an average size of 3-4 mm, and there are 3,000-4,000 million fat globules in a millilitre of whole milk.

In principle, the size of these globules has a significant effect on the yield of dairy products. The larger the globules, the easier they should be to separate from the skim milk and the lower should be the percentage of fat remaining in buttermilk after churning in the process of butter-making. However, larger globules are more easily broken and more free fat is then released into the system, creating subsequent problems.

Milk fat is a mixture of different fatty acid esters, called triglycerides, in which the backbone of glycerol is esterified by a range of short-, medium- or long-chain fatty acids, some of which may be unsaturated. Among the most abundant fatty acids generally present are myristic, palmitic, stearic and oleic. The first three of these are solids at room temperature, but oleic acid and the short-chain butyric acid and caproic acid, which are usually present in smaller amounts, are all liquids at room temperature.

Fat containing a high quantity of esterified high-melting acids, such as palmitic acid, will be hard; conversely, fat with a high content of esterified low-melting oleic acid makes soft butter. It is the release of free fatty acids induced by lipolysis (the breakdown of fat into glycerol and free fatty acids) which gives fat a rancid taste and smell. Butyric and caproic acids are particularly intense smelling.

Lipolysis is caused by the action of lipase enzymes and is encouraged by high storage temperatures. Enzymes are a group of proteins produced by living organisms. Those in milk come either from the cow's udder or from bacteria. The former (original enzymes) are normal constituents of milk and include:

• peroxidase, which transfers oxygen from hydrogen peroxide to other readily oxidisable substances
• catalase, which splits hydrogen peroxide into water and free oxygen
• phosphatase, which splits phosphoric acid esters into phosphoric acid and the corresponding alcohol
• lipase

The quantity of lipase in milk is believed to increase towards the end of the lactation cycle. Lipase is largely inactivated by pasteurisation. However, the many micro-organisms present may also produce the enzyme lactase, which is resistant to heat and which converts the milk-sugar lactose into two simple sugars (glucose and galactose). The microorganisms may also produce lactic acid.

Generally, lipolysis cannot occur unless the fat globules have been damaged so that the membranes are broken and the fat itself is exposed. Only then can the lipase attack and break down the fat molecules. In normal dairying routine, the fat globules can be easily damaged by things such as pumping, stirring and sloshing.

Cheese

The definition of "cheese", as formulated by WHO/FAO, is the fresh or ripened product obtained after coagulation and whey separation of milk, cream or partly skimmed milk, buttermilk or a mixture of these products.

Cheese contains protein, fat, water and salts in varying amounts, depending on type. Flavour is collectively referred to as the detection of taste and aroma from food. Taste buds in the mouth or tongue are stimulated by trace elements such as zinc and other transition metal ions. The addition of salt to food encourages the taste buds to become quite discriminatory in terms of a wide range of tastes and aromas.

Cheesemaking involves a number of main steps which are common to all types of cheese. Milk used for cheesemaking must be of good quality and free from antibiotics which would inhibit the starter culture. Thus milk from diseased animals and also colostrum milk must not be delivered to cheese factories.

Cheese is usually made from pasteurised milk with a relatively low bacteria count and it is therefore necessary to supply suitable bacteria in the starter culture and allow these to develop for a certain period of time, called the ripening time, before rennetting or coagulation. This is the fundamental process in cheesemaking and is usually accomplished by the addition of rennet.

The Romans were the first Europeans to describe cheesemaking in detail. To coagulate the milk proteins, an enzyme preparation from goat, lamb or even hare stomachs was mixed with sheep's or goat's milk (cow's milk was not produced on a large scale before the 13th century). After the whey had been drained from the coagulated milk, the curds that remained were salted and stored for consumption later in the year. Early cheese makers either placed strips of the kid, lamb or calf stomach directly into warm milk or prepared a crude rennet extract by soaking the strips in salt water.

World production of rennet now exceeds 25 million litres per year. Much of this is obtained from the stomachs of unweaned calves, which are slaughtered for veal production and not specifically to obtain the enzyme, or from adult cows.

In New Zealand, the NZ Rennet Company in Eltham produces the enzyme for distribution throughout the cheese-making factories, although some cheeses are now also made by using the fungal or bacterial substitutes for rennet which were developed to allow the supply of enzymes to keep pace with cheese production.

As the public's demands for a wide variety of cheeses have increased, so has the use of fat-degrading enzymes -- lipases --  which are added to promote the formation of piquant flavours as the cheese ripens. Many traditional Italian cheeses, such as parmesan, romano, provolone and mozzarella, benefit from such additions to augment the activity of the naturally occurring lipolytic microbes.

The most effective lipases for use in dairy foods are the pregastric (or lingual) lipases. These are secreted from the pharyngeal and epiglottal regions of the tongues of all mammals during suckling and swallowing. Traditionally, it was the extract from kid goats which was used in the manufacture of strongly flavoured cheeses, and which produces the characteristic peppery flavour we recognise in Italian-style cheeses.

Perhaps surprisingly, the taste of the cheeses which were produced using these extracts from lamb and calf is considered to be slightly different from the familiar tastes of the old-style cheeses which had been produced by use of the kid extract. Whereas the piquancy generated by the kid goat enzymes was "quickly cleared from the palate", that from the lamb extract caused a strong "picorino" flavour which "lingered after tasting" and the flavour from the calf extracts was "buttery and slightly peppery".

Originally, the kid enzyme was sourced from the United States, but its importation was banned in the early 1990s to protect our environment from contamination by scrapie and other endemic diseases. The Rennet Company therefore developed a method for extracting similar extracts from the tongues of calves and lambs which are slaughtered in large numbers for meat consumption.

Local Lingual Research

New Zealand is in a unique position to take advantage of market opportunities to sell lipases from disease-free stock, but in order to do so and sell the enzyme extracts on the competitive international market, it is necessary to have well documented chemical data on their reactivity and potential for use as flavour enhancers. It may also be necessary to "mix and match" these catalysts to satisfy the desires of discerning buyers.

The research group in the Chemistry Department at the University of Auckland which I lead is currently undertaking extensive exploratory work to find answers to some of these problems. In our early studies we have identified distinct differences between the ability of the extracts from calf, lamb, goat and kid to generate butyric acid from tributyrin, a lipid made up entirely of butyric acid groups attached to the glycerol backbone. However, the adult goat and kid goat extracts produce almost identical free fatty acid profiles when they are allowed to react with anhydrous milk fat.

More recently we have started to extend our work using model substrates into a study of the reactivity of the enzymes against fresh cream, in order to mimic more exactly the conditions likely to be encountered in a dairy factory. We now find that although the lipids in the milk fat globules seem to be readily hydrolysed by the lingual extracts, the phospholipids in the membrane appear to remain intact. The literature suggests that the lingual extracts do not disturb the structure of the milk fat globule membrane but penetrate through the membrane to hydrolyse the core triglycerides.

This behaviour contrasts with that seen with, say, pancreatic lipase, or bile salt stimulated lipase (an enzyme present in human, but not bovine milk). We intend to study this aspect more fully in the future and also to investigate the ability of the enzymes to react in a variety of micellar and liposomal media which will act as models for the milk emulsion.

It is hoped that this work may have considerable benefit to New Zealand and its export industry. At present the tips of lamb tongues are canned and exported as a delicacy. Perhaps future benefits will arise from an extract from the tongue roots which are currently regarded as waste product. From these tongue roots may arise a completely new range of piquant cheeses.

Professor O'Connor holds a personal chair in Chemistry at the University of Auckland.