Estonian University of Life Sciences, Institute for Veterinary Medicine, Tartu, Estonia

Environmental impacts on and of animals Challenge of artificial environment to domestic animals: problems to be solved in relation to quality issues of local food products Milk composition Nutritional role of milk and milk products Part I. Milk composition [E-B 1] Milk is a complex biological fluid consisting practically of all chemical components (carbohydrates, proteins, lipids, minerals, and vitamins etc. (Table 1), necessary for building and functioning of living cells. The number of milk ingredients increases due to technical improvement of analysis methods. At present about 100 000 components have been identified. Chemical composition and physical properties of milk vary within wide ranges. The variability can be resulted from the following factors: 1. genetical difference between breeds and individuals; 2. physiologically specific features resulted from lactation stage, heat, age of an animal and gestation; 3. environment, especially feeding- and keeping conditions, but also climate. The main constituent of milk is WATER (87%) in which other components are dispersed. Milk components as sugars, minerals and vitamins are dissolved there. In water emulgated milk fat and suspended proteins are present. [E-B 2] LACTOSE, or milk sugar (5%) is a disaccharide consisting of glucose and galactose. The amount of other carbohydrates in milk is presented in milligrams. Lactose is the energy and carbon source for most microbes growing in milk. The osmotic pressure of milk, the decrease of freezing point and the increase of boiling temperature depend mainly on lactose. During heating, lactose reacts with the proteins’ amino groups. Lactose forms soluble complexes with Ca-salts, thus favouring Ca assimilation. Fermentation of lactose by lactic acid bacteria forms basis for the technologies for producing sour milk products. [E-B 3] Milk FAT (4%) is composed of many different lipophilic substances. Fat content can vary from 2 to 8%, according to nutrition and breed. 1

Estonian University of Life Sciences, Institute for Veterinary Medicine, Tartu, Estonia

Table 1 Composition and structure of milk (approximate average quantities in 1 kg milk) (Walstra and Jenness, 1984) Fat Globule Glycerides triglycerides diglycerides monoglycerides Fatty acids Sterols Carotenoids Vitamins A, D, E, K Water Others Serum Water Carbohydrates lactose others Minerals Ca Mg K Na chloride phosphate sulphate bicarbonate Trace elements Zn Fe Cu many others

Globule Membrane Casein Micelle Water 80 mg Protein 38 g Protein 350 mg casein 0.1 g Lipids proteose peptone 10 mg phospholipides 210 mg Salts 25 mg cerebosides 30 mg Ca 100 mg gangliosides 5 mg phosphate 0.4 mg sterols 15 mg citrate 2 mg natural gly+ Mg, K, Na, Zn, cerides etc. 60 mg Enzymes + Enzymes 30 mg Cu 4 μg Water Fe 100 μg 870 g Organic acids citrate 46 g formate 0.1 g acetate lactate 370 mg oxalate 75 mg

others

1340 mg Gases 460 mg oxygen 1060 mg nitrogen 1080 mg Lipides 100 mg natural glycerides 100 mg fatty acids phospholipides 400 μg cerebosides 100 μg sterols 20 μg others Vitamins B vitamins ascorbic acid

Leucocytes Enzymes Nucleic acids

26 g 0.4 g 800 mg 950 mg 140 mg 150 mg + +

Protein casein + β-lactoglobulin 3200 mg α-lactalbumin 1200 mg serum albumin 400 mg immuno750 mg globulins 20 mg proteose pep200 mg tone others 400 mg 6 mg Nonprotein nitrogenous 15 mg compounds urea 300 mg + peptides 200 mg

1600 mg 40 mg 30 mg 30 mg 20 mg

15 mg amino acids 110 mg others 10 mg Phosphoric esters 15 mg Enzymes Alcohol

300 mg 300 mg + 3 mg

200 mg 20 mg Lipoprotein Particle Polar lipids Protein Enzymes

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Estonian University of Life Sciences, Institute for Veterinary Medicine, Tartu, Estonia

Triglycerides form more than 98% of total fat weight. Of other components free fatty acids, cerebrosides, phospholipids and cholesterol should be mentioned. In milk fat about 400 different fatty acids are represented, however the content of only 14 of them is over one per cent in total fatty acids by weight. As compared to other animal fats, milk fat content of long and medium C-chain fatty acids and polyunsaturated fatty acids is relatively high. Short chain fatty acids inhibit the development of pathogenic microbes. As to non-essential fatty acids, arachidonic and linolenic acids should be pointed out. The content of individual fatty acids in milk fat depends on ration, lactation stage, season, breed etc. Altering of the fatty acid composition of milk fat affects favour, odour, consistence and shelf life of butter. In milk fat the contents of myristic, palmitic, stearic and oleic acids are the highest. Milk fat includes soluble vitamins A, D and E as well. Milk fat is dispersed in the plasma in the form of small globules. Each fat globule (0.1-10µm) is surrounded by a membrane. The membrane of fat globule is consisting of phospholipids, lipoproteins, cerebrosides, proteins, nucleic acids, enzymes, trace elements and water. Lipids comprise 60% and proteins 40% of the membrane. In natural untreated milk the membrane inhibits lipolysis. When the structure of the membrane is broken, degradation of fat into free fatty acids quickly begins, causing rancid flavour of milk. Thus, before homogenisation milk must be pasteurised in order to inactivate active lipoprotein lipases. [E-B 4] Cow’s milk contains 2.8 to 3.5% PROTEIN (Table 2). As milk also contains non-protein nitrogen compounds (urea, creatin, free amino acids), the protein content cannot be directly determined by nitrogen content. Cow’s milk content of protein hormone prolactin is 50µg/kg. In milk also steroid hormones cortisol, corticosterol, progesterone and estradiol can be found. The content of steroid hormones do not exceed one microgram per litre. Milk proteins are divided into caseins (precipitate at pH 4.6) and whey proteins. Casein is a phosphoprotein consisting of 4 gene products: αs1-, αs2-, β- and κcaseins. Isoelectric point of caseins is pH 4.6, at which micelles lose their electric stability and aggregate. Solubility of caseins at this pH value differs from that of whey proteins so that they can be separated. In all caseins there is at least one phosphate group attached by ester bonds, whey proteins are not phosphorylized. Caseins contain quite few sulphur-containing amino acids: αs1-casein and β-casein do not contain cystein, αs2-casein and κ-casein both contain two cystein molecules. The content of proline in caseins is quite high and thus no ordered secondary sructure exists. κ-casein (MW 19025) is the only protein in casein complex containing carbohydrates. κ-casein binds about 2 mol of calcium per 1 mol of protein. As to technological aspect, it is important that κ-casein is hydrolysed by renneting enzyme

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Estonian University of Life Sciences, Institute for Veterinary Medicine, Tartu, Estonia

chymosin. In technologies caseins are converted to coagulants by acids (sour milk products) and by renneting enzymes (cheeses). Table 2 Approximate composition of milk protein Protein Casein αs1-casein αs2-casein β-casein κ-casein Whey proteins α-lactalbumin β-lactoglobulin Serumalbumin Immunoglobulins Other whey proteins Globule membrane proteins Total

Content g/kg 26.0 10.0 2.6 10.1 3.3 6.3 1.2 3.2 0.4 0.7 0.8 0.4 32.7

% of total protein 79.5 30.6 8.0 30.8 10.1 19.3 3.7 9.8 1.2 2.1 2.5 1.2 100

Whey proteins consist of two proteins synthesized in the mammary gland – βlactoglobulin and α-lactalbumine. Serum albumine and immunoglobulines originate from the blood (Table 2). Whey proteins include some products of β-casein decomposition known as proteoses and peptones. Whey proteins are thermolabile – it is possible to precipitate them by heating whey. As to technological aspect, the most important whey protein is β-lactoglobulin that exists in two main genetic variants – A and B. In the milk of some cow breeds variants C and D exist. Genetic variants differ from each other mainly by the composition of amino acids sequence. β-lactoglobulin comprises up to 50% of the total whey proteins. The other important whey protein is α-lactalbumin, comprising about 20% of whey proteins in cow’s milk. This protein is with higher thermostability than β-lactoglobulin. Serum albumine takes the third place by amount, originating from the blood. Albumines differ from caseins mainly due to comparatively high sulphur content in their molecule, phosphor is practically lacking. The content of immunoglobulines, important if immunity is considered, is high in colostrum that is necessary for passive immunisation. In the milk of the first milking in which protein content may reach to 16%, immunoglobulines comprise

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Estonian University of Life Sciences, Institute for Veterinary Medicine, Tartu, Estonia

about 50% of the protein. Another essential whey protein is lactoferrin, bactericidial iron-binding glycoprotein, which content in milk is 0.2 mg per litre. Its content is considerably higher in colostrum and mastitic milk. Lactoferrin binds 2 mol of Fe3+ ions per 1 mol. [E-B 5] Cow’s milk contains 7 to 8 g MINERALS per one litre. The salts of organic as well as anorganic acids are presented in milk. The majority of them are potassium-, calcium-, magnesium- and sodium phosphates, citrates, chlorides, hydrogen carbonates (Fox, McSweeney, 1998; Laht, Olkonen, 2001). Table 3 represents macro elements found in milk and their content. Table 3 Mineral content of milk, macro elements (Fox, McSweeney, 1998) Element

Average, g/l

Range, g/l

Calcium Phosphorus Potassium Sodium Chlorine Magnesium

1.20 0.95 1.45 0.50 1.00 0.13

0.65...2.65 0.47...1.44 1.15...2.00 0.11...1.15 0.54...2.42 0.02...0.23

Micro amounts of more than 20 chemical elements can be found in milk. Most of them are very important as to metabolic aspect. Milk is a good source of Zn and I, covering the needs of the organism. Besides them, milk may contain other minerals, which are not very important in nutrition (Li, B, Si, Br, Al, Sr, Co, As, Ag, Pb, Hg, Cd, Rb, Cs, V), some of them may be toxic. The concentration of toxic elements exceeds the limits very rarely as the udder serves as a efficient biofilter (Laht, Olkonen, 2001). Mineral components are in the form of salts or are bound to casein micelles. Very small part of minerals is bound to fat globules (Walstra, Jenness, 1984) and about 0.15% of Ca is attached to α-lactalbumin. One-valence cations are mostly in solution, 66% of Ca and 57% of P is in the composition of colloids (Walstra, Jenness, 1984; Fox, McSweeney, 1998). Phosphorus is present in milk only in several compounds. Phosphate group has an important role in giving milk its buffering capacity (Nielsen, Ullum, 1989). Equilibrium between colloidal and dissolved salts in milk is affected by several factors as acidity of milk, temperature, dilution and freezing (Fox, McSweeney, 1998).

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Estonian University of Life Sciences, Institute for Veterinary Medicine, Tartu, Estonia

1. Acidifying milk, colloidal calcium phosphate dissolves from casein (at pH≤ 4.9 whole calcium phosphate is dissolved) (Walstra, Jenness, 1984; Fox, McSweeney, 1998). 2. With the increasing of temperature the solubility of calcium phosphate decreases and in cooling the percentage of calcium phosphate increases at the expense of colloidal calcium phosphate. At high temperatures this reaction is irreversible. Calcium phosphate precipitates if milk is heated at high temperature. The dependence of the equilibrium of Na, K, Mg and citrate on temperature has not been found (Walstra, Jenness, 1984; Fox, McSweeney, 1998). 3. Dilution of milk decreases the concentration of dissolved calcium phosphate and this is compensated at the expense of colloidal calcium phosphate. With the increase of milk concentration the percentage of colloidal calcium phosphate increases as well as milk acidity (Walstra, Jenness, 1984; Fox, McSweeney, 1998). 4. Freezing of milk results in water crystallization and increase of salt concentration. The co-effect of the decreased percentage of Ca-ions caused by concentration and the decrease of milk pH (pH 5.8 at -20°C) result in the destabilization of casein micelles (Fox, McSweeney, 1998). Milk contains all known VITAMINS that are soluble in fat or water. Usually raw milk contains carbon dioxide, nitrogen and oxygen. The contents of oxygen and nitrogen increase when milk is exposed to air. Oxygen affects the development of starter culture, in modern yoghurt manufacturing systems milk is degased. SOMATIC CELLS are leucocytes and different types of epithelial cells. The number of them in the milk of healthy cows ranges from a thousand to several hundred thousand per ml of milk. Inflammatory processes increase the number of somatic cells to millions.

References and recommended reading 1. Fox P.F., McSweeney P.L.H. Dairy Chemistry and Biochemistry. Chapman & Hall, 1998, 465. 2. Laht, T.-M., Olkonen, A. Piima koostis, füüsikalis-keemilised omadused. Piimanduse Käsiraamat. Koost. A. Olkonen. EPMÜ LKI, Tartu, 2001, 102–130. 3. Nielsen, E.W., Ullum, J.A. Dairy technology I. Danish Turnkey Dairies Ltd, 1989, 110. 4. Roginski, H., Fuquay, J.F., Fox, P.F. Encyclopedia of Dairy Sciences. 2002, Vol. 3, Academic Press. 6

Estonian University of Life Sciences, Institute for Veterinary Medicine, Tartu, Estonia

5. Walstra, P., Jenness, R. Dairy Chemistry and Physics. John Wiley & Sons, 1984, 467.

Part II. Nutritional role of milk and milk products Foodstuff’s nutritional quality is assessed and evaluated by • • •

amount of energy derived from it; content of essential amino acids, fatty acids, minerals and vitamins; biological availability of nutrients.

[E-B 6] Milk is the mammary gland secret of the mammals which contains all nutrients needed by offspring in a balanced proportion. Over thousands of years people have learned to use animal milk as a valuable food. In Northern countries cow’s milk plays a central role, in other cultures milk of goat, sheep, camel and horse are used as well. Milk and sour milk contain about 3.2% protein. The main milk proteins are casein (80%) and serum (or whey) proteins (20%). Casein in milk is in the form of small globules, which are containing about 80% of calcium found in milk and also much phosphorus. The primary function of casein is to supply offspring with amino acids needed for building up body proteins. Casein is rich in essential amino acids in favourable proportions. Half a litre of milk per day almost covers the minimal need for essential amino acids. [E-B 7] Milk carbohydrate − lactose − comprises about 30% of the amount of energy assimilated from milk. Lactose is a disaccharide consisting of galactose and glucose. In nature it can be found in milk only. Lactose • •

favours the absorption of calcium and many other minerals in the digestive tract, favours the development of lactic acid bacteria which are favourable for the digestive tract, lactic acid derived from lactose increases the level of acidity in the digestive tract and inhibits the development of pathogenic bacteria.

Some people may have lactase deficiency or hypolactasia. In the digestive tract such people lack the lactose degrading enzyme lactase, or it is synthesized in small amounts. Undegraded lactose goes into the large intestine where microbes use it for nutrition, resulting in pain in the stomach, diarrhoea and formation of gases. Sour milk products cause fewer symptoms than milk as lactose in them

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Estonian University of Life Sciences, Institute for Veterinary Medicine, Tartu, Estonia

has turned into lactic acid. People having hypolactasia are recommended to use kefir, sour milk, yoghurt or cheese. [E-B 8] Milk fat is primarily source of energy. Compared to other animal origin fats, the content of short and medium C-chain fatty acids and polyunsaturated fatty acids in milk is comparatively high. Butyric acid is found only in milk fat of the ruminants. Short chain fatty acids inhibit the development of pathogenic microbes. As to non-essential amino acids, in milk fat arahhidonic and linolenic acids can be found which are mainly needed to build up nervous tissue. Milk fat contains fat-soluble vitamins A, D, E, F and K. Due to the high content of short chain and unsaturated fatty acids, milk fat is in a liquid state and thus easily assimilated as liquid triglycerides are hydrolyzed in the small intestine by pancreatic lipase. The digestibility of milk fat is 99%. Comparing to other animal fats, the cholesterol content of milk fat is low. Nowadays researchers are in opinion that 45% of fatty acids of milk fat reduce cholesterol level and only 14% increase it. The effect of 41% fatty acids on plasma cholesterol is not known yet. The effect of the most important milk fatty acids on blood cholesterol level is presented in Table 1. Table 1 The effect of most important milk fatty acids on blood cholesterol level (Laht, 2001) Concentration % Effect* Butyric acid C 4:0 4 0 Caproic acid C 6:0 2 0 Caprylic acid C 8:0 1 0 Capric acid C 10:0 3 0 Lauric acid C 12:0 3 + Myristic acid C 14:0 11 + Palmitic acid C 16:0 28 0 Stearic acid C 18:0 10 Oleic acid C 18:1 26 Linoleic acid C 18:2 3 * 0 = neutral, + = increases cholesterol level, - = decreases cholesterol level Fatty acid

[E-B 9] Milk contains almost all micro- and macro elements needed for the human organism. Milk and milk products are very good sources of calcium, phosphorus, sodium, potassium, zinc and iodine in our food. This is not only due to their high mineral content but also due to the fact that minerals present in these products are easily absorbable. By the data of different researchers, milk and milk products should cover 55 to75% of our daily calcium requirement (Schaafsma, 1984; Hazell, 1985) and 30 to 45% of daily phosphorus requirement (Hazell, 1985; Flynn, Cashman, 1997). Figure 1 shows the percentage of our

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Estonian University of Life Sciences, Institute for Veterinary Medicine, Tartu, Estonia

daily calcium and phosphorus content, which milk and milk products should cover.

Fish, meat, eggs 7% Vegetable food 7%

A

Other sources 11 %

Eggs 5% Vegetables 8% Beans 6% Grain Milk and 12 % milk products 75 %

Other sources 9%

Milk and milk products 35 %

Meat, fish 25 %

B

Figure 1 Sources of calcium (A) and phosphorus (B) in average mixed foods (Teesalu, 1997) Without consuming milk it is almost impossible to cover the daily calcium requirement. For skeleton formation 100 to 150 mg calcium per day is needed up to the age of 20. The critical minimum limit per day is 600 mg. Consumption of calcium below that limit directly increases risk of thinning of bones or osteoporosis (Teesalu, 1998). Very intense muscle work, stress and depression increase calcium requirement even more (Zilmer et al., 1996). Absorption of calcium from milk is favoured by other milk components as lactose, vitamin D and phosphorus, but also vitamins A and C. If the calcium requirement of the organism is high, all calcium present in cow’s milk is absorbable. Although in skimmed milk almost all calcium is remained, calcium absorption from skimmed milk and the products made from it is low. This is caused by removing fat and together with it fat-soluble vitamin D (Teesalu, 1998). Milk contains all known vitamins and is an important source of them in our food. Milk is especially rich in vitamins B2 and B12, but also covers our daily need for vitamins A, K and B7 (Schaafma, 2002) Pasteurization of milk can result in the loss of temperature-sensible vitamins (C, B1, B6, folic acid) by 5 to 20%. Further loss (oxidation) of vitamins from milk products depends on storage conditions (exposure to light, temperature, and wrapping material). By removing fat, fat-soluble vitamins are removed from milk as well, thus milk products with low fat content are poor in fat-soluble vitamins.

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Estonian University of Life Sciences, Institute for Veterinary Medicine, Tartu, Estonia

References 1. Flynn, A., Cashman, K. Nutritional aspects of minerals in bovine and human milk. Ed. by In: Fox, P. F. (Ed.). Advanced Dairy Chemistry. Volume 3: Lactose, water, salts and vitamins, 2nd Edition. Chapman & Hall, London, 1997, 257–302. 2. Hazell, T. Minerals in foods: dietary sources, chemical forms, interactions, bioavailability. World Rev. Nutr. Diet., 46, 1985, 1–123. 3. Laht, T.-M. Piim ja piimatooted toiduna. Piimanduse Käsiraamat. Koost. A. Olkonen. EPMÜ LKI, Tartu, 2001, 75–101. 4. Schaafma, G. The significance of milk as a source of dietary calcium. IDF Bulletin 166, 1984, 19–32. 5. Schaafma, G. Vitamins. General introduction. In: Roginski, H., Fuquay, J. F., Fox, P. F. (Eds.). Encyclopaedia of Dairy Sciences, 4. Ed by Roginski, H., Fuquay, J.F., Fox. Academic Press, 2002, 2653–657. 6. Teesalu, S. Piim ja piimatooted meie toidulaual. Piimast ja eesti piimandusest. Koost. M. Karelson, S. Teesalu, A. Tammisto. EPMÜ LKI, Tartu, 1997, 7–19. 7. Teesalu, S. Laste ja noorukite osteoporoos - luude hõrenemine. Tartu, 1998, 110. 8. Zilmer M., Karelson E., Vihalem T. Meditsiiniline biokeemia I. Biomolekulid: biokeemilised ja meditsiinilised aspektid. Tartu, 1996, 321.

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