Food Biotechnology Dr. Kamal E. M. Elkahlout Applications of Biotechnology to Food Products 3

1. Food BiotechnologyDr. Kamal E. M. ElkahloutApplications of Biotechnology to Food Products 3 Production of Fermented Foods (Fermented Foods Made from Milk)

2. Composition of Milk • Milk is the fluid from the mammary glands of animals which is meant for feeding the young of mammals. • It is a complex liquid consisting of several hundred components of which the most important are proteins, lactose, fat, minerals, enzymes, and vitamins in which emulsified fat globules and casein micelles are present. • Its composition varies from breed to breed, as shown in Table 19.1. • Proteins: Milk proteins are divided into two: caseins and whey proteins. • Caseins consist of carbohydrate, phosphorus, and protein ( glyco-phspho-protein) and make up 85% of the total milk proteins.

4. Casein exists in milk as the calcium salt, ie, as calcium caseinate in globules (micelles) ranging from 40-300 mμ in diameter. • Casein exists in four types designated, , and and depending on their electric charges. • The proportion of the various types in milk depends on the breed of the cow producing the milk. • The letter ‘s’ after s - caseins indicates its sensitivity to precipitation by calcium. • Whey proteins consist of different components which are normally stable to acid, but very sensitive to heat. • Lactoglobulin forms about 66% of the total whey proteins, followed by -Lactabumin (22%). • The immune globulins from about 10% of the total, and contribute towards the immunity derived by the young from the consumption of colostrum.

5. Lactose: The main carbohydrate in milk is lactose, which is found only in milk. • It is a dissacharide of glucose and galactose and has a low sweetening ability, as well as low solubility in water. • Fat: Fat consists of one molecule of glycerol and three of fatty acids. • Over 60 different acids are known in butter, many of them, being of low molecular weight of about 10 carbon atoms or less, and include saturated and unsaturated fatty acids. • Enzymes: Enzymes found in milk include proteases, carbohydrases, esterases, oxidases/reductases. • Minerals: Milk is a major source of calcium; other minerals in milk are phosphorous, magnesium, sodium, potassium, as well as sulphate and chloride ions.

6. When fat is removed from milk such as during butter making, the remnant is skim milk. • On the other hand, when casein is removed such as during cheese manufacture, the remnant is known as whey. • Whey is high in lactose and its disposal sometimes poses some problem as not all microorganisms can break down whey. It is however used in the production of yeasts to be used as food or fodder. • Cheese • Cheese is a highly proteinaceous food made from the milk of some herbivores. • Cheese is believed to have originated in the warm climates of the Middle East some thousands of years ago, and is said to have evolved when milk placed in goat stomach was found to have curdled.

7. The scientific study and manipulation of milk for cheese manufacture is however just over a hundred years old. • Most cheese in the temperate countries of the world such as Western Europe and the USA is made from cow’s milk, the composition of which varies according to the breed of the cattle, the stage of lactation, the adequacy of its nutrition, the age of the cow, and the presence or absence of disease in the breasts (udders), known as mastitis. • In some subtropical countries milk from sheep, goats, the lama, yak, or ass is also used. • Sheep milk is used specifically for the production of certain special cheese types in some parts of Europe (e.g. Roquefort in France, and Brinsen in Hungary).

8. Milk from the water buffalo may be used in India and other countries, while milk from the reindeer and the mare may be used in northern parts of Scandinavia and in Russia, respectively. • Cheese made from the milk of goat and sheep has a much stronger flavor than that made from cow’s milk. • This is because the fat in goat and sheep milk contain much lower amounts of the lower fatty acids, caproic, capryllic, and capric acids. • These acids confer a sharp taste (similar to that of Roquefort cheese) to cheese made from these mammals. • Future discussion of cheese in this chapter will however refer to that made from cow’s milk.

9. About a thousand types of cheese have been described depending on the properties and treatment of the milk, the method of production, conditions such as temperature, and the properties of the coagulum, and the local preferences. • Stages in the manufacture of cheese • The manufacture of all the types of cheeses include all or some of the following processes: • (a) Standardization of milk • The quality of the milk has a decided effect on the nature of cheese. • Cheese made from skim milk is hard and leathery; the more fat a cheese contains the smoother its feel to the palate.

10. The fat/protein ratio is often adjusted through fat addition in order to yield a cheese of consistent quality. • In the US, pasteurization (High Temperature Short Time) or (Long Temperature Short Time) must be given to milk to be in certain types of cheeses, such as cottage or cream cheese. • For others the milk need not be pasteurized but must be stored at for at least 60 days at 2°C. • If however the ‘starter’ is slow acting or souring is delayed, food-poisoning staphylococci could develop and produce toxins in the cheese. • Sub-pasteurization temperatures are often the legal compromise. • Pasteurization gives a better control over the processes of cheese production. • However, the organisms present in raw milk are important during the ripening processes.

11. The milk may also be homogenized by forcing it at high speed through small orifices to reduce the milk fat globules for use in producing soft cheeses. • (b) Inoculation of pure cultures of lactic acid bacteria as starter cultures. • In the past, lactic acid was produced by naturally occurring bacteria. • Nowadays they are inoculated artificially, by specially selected bacteria termed starters. • Indeed lactic acid formation is necessary in all kinds of cheese. • The propagation and distribution of lactic acid bacteria for use in cheese manufacture is an industry in its own right in the US. • For cheese prepared at temperatures less than 40°C strains of Lactococcuslactis are used.

12. For those prepared at higher temperatures the more thermophilicStreptococcus thermophilus, Lactobacillus bulgaricus, and Lact. helveticus are used. • Lactic acid has the following effects: • (i) It causes the coagulation of casein at pH 4.6, the isoelectric point of that protein, which is used in the manufacture of some cheeses, e.g. cottage cheese. • (ii) It provides a favorably low pH for the action of rennin the enzyme which forms the curd from casein in other types of cheeses. • (iii) The low pH eliminates proteolytic and other undesirable bacteria.

13. (iv) It causes the curd to shrink and thus promotes the drainage of whey. • (v) Metabolic products from the lactic acid bacteria such as ketones, esters and aldehydes contribute to the flavor of the cheese. • Problems of lactic acid bacteria in cheese-making • (i) Attack by bacteriophages: Bacteriophages sometimes attack the lactic acid starters and besides choosing strains that are resistant to phages, rotations (i.e., using different lactic mixtures every three or four days) helps eliminate them. • (ii) Inhibition by penicillin and other antibiotics: Lactic acid bacteria, being Gram-positive are particularly susceptible to penicillin used to treat diseased udder in mastitis if the antibiotic finds its way into the milk; other antiobiotics also have an inhibitory effect on them.

14. (iii) Undesirable strains: Some strains of lactic acid bacteria are undesirable in cheese making because they produce too much gas, undesirable flavors, or produce antibiotics against other lactic acid bacteria. • They arise by mutation. • (iv) Sterilant and detergent residues: Sterilant and detergent residues may inhibit the growth of starter bacteria. • The minimum concentration required for inhibition varies with the different anti-microbial agents and between different strains of starter bacteria. • Residues gain entry to milk at the (a) farm, (b) during transportation to the factory, and (c) the factory due to careless use of sterilants or detergents, incomplete draining or inadequate rinsing of equipment.

15. The inhibitory effects of sterilant and detergent residues are prevented by the correct and ethical use of these materials. • Proper use includes the use of the chemical at the correct concentration and adequate rinsing and draining. • Their presence is mitigated by dilution with uncontaminated milk. • (c) Adding of rennet for coagulum formation • The classical material used in the formation of the coagulum is ‘rennet’ which is derived from the fourth stomach, abomasum or vell of freshly slaughtered milk-fed calves. • Besides those of calves, the abomasum of kids (young goats), lamb or other young mammals have been used. • Rennet is produced by soaking and/or shredding air-dried vells under acid conditions with 12-20% salt.

16. Extracts from young calves contain 94% rennin and 6% pepsin and from older cows, 40% rennin and 60% pepsin. • Rennin (chymosin) is the enzyme responsible for the coagulation of the milk. • Pepsin is proteolytic and too high an amount of pepsin can result in the hydrolysis of the coagulum and a resulting low yield of cheese, and a bitter taste may result from the amino acids. • Due to the high cost of animal rennet, other sources, mostly of microbial origins, have been found (Table 19.2).

18. The major effect of the milk-clotting enzymes is the conversion of casein from a colloidal to a fibrous form. • First the pH of the milk is brought down from pH 6.8 -7 to pH 5.5 by the action of lactic acid bacteria which produce lactic acid from lactose in the milk. • On addition of rennet, the active component, rennin, catalyses the hydrolyses of k-casein to release para-k-casein and k-casein macropeptide. • The latter goes into whey, while the para-k-casein remains part of casein micelles, which now bind together to form the curd following the removal of carbohydrates with the k-casein macropeptide and the exposure of binding surfaces.

19. The events up this coagulation are aided by lowered pH and by increasing temperatures up to 45°C. • Most of the bacteria, fat, and other particulate matter are entrapped in the curd. • When casein is removed the remaining liquid containing proteins, lactalbumin, globulin, and yellow-green riboflavin (vitamin B2) is whey. • The whey proteins may be precipitated by heat, but not acid or rennin and they are used in making whey cheese. • The enzymes used in cheese making are now obtained from microorganisms, mainly fungi. • (d) Shrinkage of the curd • The removal of whey and further shrinkage of the curd is greatly facilitated by heating it, cutting it into smaller pieces, applying some pressure on it and lowering the pH.

20. In many types of cheeses, such as Parmesan, Emmenthal and Gruyere, there is a stage known as ‘scalding’ in which the temperature can be as high as 56°C in the preparation. • Acid produced by the lactic starters introduce elasticity in the curd, a property desirable in the final qualities of cheese. • (e) Salting of the curd and pressing into shape • Salt is added to most cheese varieties at some stage in their manufacture. • Salt is important not only for the taste, but it also contributes to moisture and acidity control. • Most importantly however it helps limit the growth of proteolytic bacteria which are undesirable. • The curd is pressed into shape before being allowed to mature.

21. (f) Cheese ripening • The ripening or maturing of cheese is a slow joint microbiological and biochemical process which converts the brittle white curd or raw cheese to the final full-flavored cheese. • The agents responsible for the final change are enzymes in the milk, in the rennet and those from the added starter microorganisms as well as other micro-organisms which confer the special character of the cheese to it. • Among the cheese whose peculiar characteristics are dependent on particular microorganisms are the blue-veined cheese Roqueforti, Gorgonzola, Stilton, conferred by Penicilliumroquefort.

22. Swiss cheese, with its characteristic flavor and holes produced by the fermentation products and gases from Propionibacterium spp. Yeasts, micrococci, and Brevibacterium linens impart the characteristic flavor of Limburger cheese. • In soft cheese, such as Camembert, the protein is completely broken down to almost amino acids, whereas in the hard cheese, the protein remains intact.

23. Yoghurt and Fermented Milk Foods • Many types of fermented milks are produced and drunk around the world (Table 19.3). • Yoghurt is a fermented milk traditionally believed to be an invention of the Turks of Central Asia, in whose language the word yoghurt means to blend, a reference to how the milk product is made. • Although accidentally invented thousands of years ago, yoghurt has only recently gained popularity in the United States. • While yoghurt has been present for many years, it is only recently (within the last 30-40 years) that it has become popular. • This is due to many factors including the introduction of fruit and other flavorings into yoghurt, the convenience of it as a ready-made breakfast food and the image of yoghurt as a low fat healthy food.

25. In the manufacture of yoghurt, two kinds of lactic acid bacteria, Lactococcus spp. & Lactobacillus spp., are generally used with usually unpasteurized milk. • Most commonly used are Lactococcussalivarius and thermophilus, and Lactobacillus spp., such as Lacto. acidophilus, bulgaricus and bifidus. • The bacteria produce lactic acid from lactose in the milk causing the pH to drop to about 4-5 from about 7.0. • This drop in pH causes the milk to coagulate. • The lactic acid gives yoghurt its sour taste and limits the growth of spoilage bacteria. • Yoghurt is flavored usually with fruits.