P1: SFK/UKS BLBS102-c26 P2: SFK BLBS102-Simpson March 21, 2012 13:51 Trim: 276mm X 219mm Printer Name: Yet to Come 507 26 Equid Milk: Chemistry, Biochemistry and Processing Table 26.6 Amino Acid Composition of Asinine and Equine Milk Expressed as g amino acid per 100 g Protein, with Comparative Data for Bovine and Human Milk Amino Acid Aspartic acid Serine Glutamic acid Glycine Histidine Arginine Threonine Alanine Proline Cystine Tyrosine Valine Methionine Lysine Isoleucine Leucine Phenylalanine Tryptophan Essential amino acids Asinine Equine Bovine Human 8.9 6.2 22.8 1.2 2.3 4.6 3.6 3.5 8.8 0.4 3.7 6.5 1.8 7.3 5.5 8.6 4.3 – 38.2 10.4 6.2 20.1 1.9 2.4 5.2 4.3 3.2 8.4 0.6 4.3 4.1 1.5 8.0 3.8 9.7 4.7 1.2 36.7 7.8 4.8 23.2 1.8 3.0 3.3 4.5 3.0 9.6 0.6 4.5 4.8 1.8 8.1 4.2 8.7 4.8 1.5 37.5 8.3 5.1 17.8 2.6 2.3 4.0 4.6 4.0 8.6 1.7 4.7 6.0 1.8 6.2 5.8 10.1 4.4 1.8 40.7 Source: Modified from Guo et al 2007 Table 26.7 Free Amino Acids (µM.L−1 ) of Equine, Bovine and Human Milk Amino Acid Equinea Bovinea Humanb Alanine Arginine Aspartic acid Cystine Glutamic acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Taurine Threonine Tyrosine Valine Total 105 14.0 40.0 2.0 568.0 485.0 100.0 46.0 8.0 16.0 26.0 ∼0 5.0 1.61 175 32.0 137.0 3.0 45.0 ∼1960.0 30.0 10.0 15.0 21.0 117.0 12.0 88.0 9.0 3.0 3.0 15.0 ∼0 3.0 – 23.0 13.0 16.0 0.3 5.0 578.0 227.5 35.4 183.2 56.0 1184.1 284.8 124.6 7.7 33.4 55.6 39.0 8.8 23.6 64.3 273.7 301.1 97.6 2.5 72.7 3019.7 a b Rassin et al 1978 Agostini et al 2000 P1: SFK/UKS BLBS102-c26 P2: SFK BLBS102-Simpson March 21, 2012 13:51 Trim: 276mm X 219mm 508 Printer Name: Yet to Come Part 4: Milk glycine, alanine and serine are the most abundant free amino acids in equine, bovine and human milk, and taurine also is exceptionally high in human milk (Rassin et al 1978, Sarwar et al 1998, Carrat`u et al 2003) Taurine is an essential metabolite for the human infant and may be involved in the structure and function of retinal photoreceptors (Agostini et al 2000) Compared to bovine milk, equine milk has an appreciable amount of taurine although it is ten times less than that of human milk (Table 26.7) In contrast to total amino acid composition, which is essentially similar in equine, bovine and human milks, free amino acids show a pattern characteristic of each species (Table 26.7), which may be important for early post-natal development in different animals Free amino acids are more easily absorbed than protein-derived amino acids and glutamic acid and glutamine, which comprise >50% of the total free amino acids of human milk, are a source of α-ketoglutaric acid for the citric acid cycle and also act as neurotransmitters in the brain (Levy 1998, Agostini et al 2000) Bioactive Peptides Both caseins and whey proteins are believed to contribute to human health through latent biological activity produced enzymatically during digestion, fermentation with specific starter cultures or enzymatic hydrolysis by microorganisms, resulting in the formation of bioactive peptides These peptides are important for their physiological roles, their opioid-like features, as well as their immunostimulating and anti-hypertensive activities and their ability to enhance Ca2+ absorption and are released or activated during gastrointestinal digestion Several peptides generated by the hydrolysis of milk proteins are known to regulate the overall immune function of the neonate (Baldi et al 2005) A detailed discussion on bioactive peptides in milk is outside the scope of this chapter, for reviews, see Donnet-Hughes et al (2000), Shah (2000), Malkoski et al (2001), Fitzgerald and Meisel (2003), Silva and Malcata (2005), Fitzgerald and Murray (2006), Lop´ez-Fandi˜no et al (2006), Michaelidou and Steijns (2006), Thomăa-Worringer et al (2006) and Phelan et al (2009) Research on the bioactive peptides derived from equid milk is very limited Peptides from the hydrolysis of equine βcasein may have a positive action on human health (Doreau and Martin-Rosset 2002) Chen et al (2010) reported the presence of four novel angiotensin-converting enzyme-inhibitory peptides in koumiss which may enhance the beneficial effects of koumiss on cardiovascular health Peptides with trophic or protective activity have been identified in asinine milk (Salimei 2011) Hormones and Growth Factors Leptin is a protein hormone of approximately 16kDa that has been discovered recently in human milk and plays a key role in the regulation of energy intake and energy expenditure, as well as functioning in mammary cell proliferation, differentiation and apoptosis Human-like leptin has been isolated from asinine milk at a level of 3.2–5.4 ng.mL−1 which is similar to levels reported for other mammals and showed little variation throughout lactation (Salimei et al 2002) Levels of the bioactive peptides, ghrelin and insulin growth factor I, which play a direct role in metabolism, body composition and food intake, have also been reported for asinine milk at 4.5 pg.mL−1 and 11.5 ng.mL−1 , respectively, similar to levels in human milk (Salimei 2011) Amyloid A Amyloid A3 (AA3) is a protein produced in the mammary gland and is encoded by a separate gene from that for serum amyloid A (serum AA) (Duggan et al 2008) AA3 is believed to prevent attachment of pathogenic bacteria to the intestinal cell wall (Mack et al 2003) and may prevent necrotising enterocolitis in human infants (Larson et al 2003) McDonald et al (2001) demonstrated the presence of AA3 in the colostrum of cows, ewes, sows and horses Bovine colostrum has a high concentration of AA3 but by approximately days post-partum the levels decline In bovine milk, the presence of serum AA in milk is an indicator of mastitic infection (Kaneko et al 2004, Winter et al 2006) In equine colostrum, the concentration of AA3 is considerably lower than in milk and consequently may play a crucial role in intestinal cell protection in the foal especially after gut closure (Duggan et al 2008) INDIGENOUS ENZYMES Milk contains many indigenous enzymes that originate from the mammal’s blood plasma, leucocytes (somatic cells), or cytoplasm of the secretory cells and the milk fat globule membrane (MFGM)(Fox and Kelly 2006) The indigenous enzymes in bovine and human milks have been studied extensively but the enzymes in the milk of other species have been studied only sporadically Equine milk probably contains all the enzymes that have been identified in bovine milk but relatively few studies have been reported Lysozyme Lyz (EC 3.1.2.17) occurs at high levels in equine, asinine and human milk (Table 26.2) Human, equine and asinine milk contain 3000, 6000 and >6000 times more Lyz, respectively, than bovine milk (Salimei et al 2004, Guo et al 2007) with levels as high as 0.4 g.100g−1 for Martina Franca donkeys (Coppola et al 2002) although 0.1 g/100 g is reported and more commonly found in asinine milk (Vincenzetti et al 2008) The concentration of Lyz in human milk increases strongly after the second month of lactation, suggesting that Lyz and Lf play major roles in fighting infection in breast-fed infants during late lactation, and protect the mammary gland (Montagne et al 1998) Equine milk Lyz is more stable to denaturation than human Lyz during pasteurisation at 62◦ C for 30 minutes but at 71◦ C for minutes or 82◦ C for 15 seconds, the inactivation of both were similar (Jauregui-Adell 1975) It has been suggested, but research is scarce, that while the composition of breast milk varies P1: SFK/UKS BLBS102-c26 P2: SFK BLBS102-Simpson March 21, 2012 13:51 Trim: 276mm X 219mm Printer Name: Yet to Come 26 Equid Milk: Chemistry, Biochemistry and Processing widely between well-nourished and poorly nourished mothers, the amount of Lyz is conserved Lyz found in egg white, tears and saliva not generally bind calcium but equine and canine milk Lyz and this is believed to enhance the stability and activity of the enzyme (Nitta et al 1987) The binding of a Ca2+ by Lyz is considered to be an evolutionary linkage between non-Ca2+ -binding Lyzs and α-La (Tada et al 2002, Chowdhury et al 2004) The conformation of the calcium-binding loop of equine Lyz is similar to that of α-La (Tsuge et al 1992, Tada et al 2002) and both equine Lyz and α-La form stable, partially folded, ‘molten globules’ under various denaturing conditions (Koshiba et al 2001,) with that of equine Lyz being considerably more stable than α-La (Lyster 1992, Morozova-Roche 2007) The molten state of canine Lyz is significantly more stable than that of equine Lyz (Koshiba et al 2000, Spencer et al 1999) Equine milk Lyz is very resistant to acid (Jauregui-Adell 1975) and proteolysis (Kuroki et al 1989), and may reach the gut relatively intact Asinine Lyz contains 129 amino acids, is a C-type Lyz, binds calcium strongly and has 51% homology to human Lyz (Godovac-Zimmermann et al 1988b) Two genetic variants of Lyz, A and B, have been reported in asinine milk (Herrouin et al 2000) but only one is found in equine milk Asinine Lyz is remarkably heat stable and requires 121◦ C for 10 minutes for inactivation The Lyz content of equid milks is one of the main attractions for use of these milks in cosmetology as it is reputed to have a smoothing effect on the skin and may reduce scalp inflammation when incorporated into shampoo Equid milk has very good antibacterial activity, presumably due to its high level of Lyz Other Indigenous Enzymes in Equine Milk Lactoperoxidase, catalase, amylase, proteinase (plasmin), lipase, lactate dehydrogenase and malate dehydrogenase have been reported in equine milk Bovine milk is a rich source of xanthine oxidoreductase (XOR) but the milk of other species for which data are available have much lower XOR activity, because in non-bovine species, most (up to 98% in human milk) of the enzyme molecules lack Mo and are inactive XOR has not been reported in equine milk, which is unusual considering the role of XOR in the excretion of fat globules from the secretory cells and also considering that equine milk contains quite a high level of molybdenum (Mo), which presumably is present exclusively in XOR Chilliard and Doreau (1985) characterised the lipoprotein lipase activity of equine milk and reported that the milk has high lipolytic activity, comparable to that in bovine milk and higher that in caprine milk There are no reports on hydrolytic rancidity in equine milk, which is potentially a serious problem in equine milk products and warrants investigation Plasmin, a serine proteinases, is one of a number of proteolytic enzymes in milk Visser et al (1982) and Egito et al (2002) reported γ -caseins in equine milk and, it is therefore assumed, that equine milk contains plasmin Humbert et al (2005) reported that equine milk contains five times more plasmin activity than bovine milk and 90% of total potential plasmin activity was plasmin, with 10% as plasminogen; the plasmin:plasminogen ratio in bovine and human milk is 18:82 and 28:72, respectively 509 Alkaline phosphatase (ALP) is regarded as the most important indigenous enzyme of bovine milk because ALP activity is used as the index of the efficiency of high-temperature short-time pasteurisation About 40% of ALP activity in bovine milk is associated with the MFGM Equine milk has 35–350 times less ALP activity than bovine milk and there are no reports on ALP in the equine MFGM Because of the low level of ALP in equine milk it has been suggested that it is not suitable as an indicator of pasteurisation efficiency of equine milk (Marchand et al 2009) although one would expect that once the exact initial concentration of ALP is known, the use of a larger sample size or a longer incubation period would overcome the low level of enzyme CARBOHYDRATES Lactose and Glucose The chemistry, properties and applications of lactose are described in Chapter 24 and have been reviewed extensively elsewhere for example Fox (1985, 1997) and McSweeney and Fox (2009) and will not be considered here The concentration of lactose in asinine milk is high (51–72.5 g.kg−1 ), probably marginally higher than equine milk (approximately 64 g.kg−1 ) (Table 26.2), which is similar to the level in human milk and significantly higher than that in bovine milk As an energy source, lactose is far less metabolically complicated than lipids but the latter provides significantly more energy per unit mass As well as being a major energy source for the neonate, lactose affects bone mineralisation during the first few months post-partum as it stimulates the intestinal absorption of calcium (Schaafsma 2003) Equine milk contains a significant concentration of glucose, approximately 50 mg/L in colostrum which increases to approximately 150 mg/L 10 days post-partum and then decreases gradually to approximately 120 mg/L (Enbergs et al 1999) Although the lactose content of equid milks is high, the physico-chemical properties of lactose that cause problems in the processing of bovine milk are of no consequence for equid milks, which are consumed either fresh or fermented In Mongolia, where approximately 88% of the population is lactose intolerant (Yongfa et al 1984), lactose intolerance is not a problem with fermented equine milk, koumiss, as approximately 30% of lactose is converted to lactic acid, ethanol and carbon dioxide during fermentation Oligosaccharides The milk of all species examined contains OSs but the concentration varies markedly (see Urashima et al 2009) The OSs in milk contain to 10 monosaccharides and may be linear or branched; they contain lactose at the reducing end and also contain fucose, galactosamine and N-acetylneuraminic acid The highest levels are in the milk of monotremes, marsupials, marine mammals, humans, elephants and bears OSs are the third most abundant constituent of human milk that has an exceptionally high content (approximately 20 g.L−1 in colostrum, which decreases to 5–10 g.L−1 in milk) and structural diversity of OSs (>200 molecular species), which have a range of functions, P1: SFK/UKS BLBS102-c26 P2: SFK BLBS102-Simpson March 21, 2012 510 13:51 Trim: 276mm X 219mm Printer Name: Yet to Come Part 4: Milk Table 26.8 Principal Oligosaccharides of Equine Colostrum Oligosaccharide (mg/L) Acidic Neu5Ac(α2–3) Gal(β1—4)Glc Gal(β1–4)GlcNAcα1-diphosphate (N-acetyllactosamine-α1-phospahte) N/a N/a Neutral Gal(β1–3)Gal(β1–4)Glc (β3’-galactosyllactose) Gal(β1–6)Gal(β1–4)Glc (β6’-galactosyllactose) Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (lacto-N-neotetraose) Gal(β1–4)GlcNAc(β1–6)Gal(β1–4)Glc (iso-lacto-N-neotetraose) Gal(β1–4)GlcNAc(β1–6)[Gal(β1–3)]Gal(β1–4)Glc (lacto-N-novopentanose 1) Gal(β1–4)GlcNAc(β1–6)[Gal(β1–4)GlcNAc(β1–3)]Gal(β1–4)Glc (lacto-N-neohexaose) Gal(β1–4)GlcNAc-1-phosphate (N-acetyllactosamine-1–0-phosphate) Neu5Ac(α2–3)Gal(β1–4)Glc (3’-N-acetylneuraminyllactose) 7.8 4.8 N/a 0.5 1.1 1.1 N/a N/a Source: From Urashima et al 1989, 2001, Nakamura et al 2001 Gal, d-galactose; Glc, d-glucose; GlcNAc, N-acetylglucosamine; Neu5A, N-acetylneuraminic acid; N/a, not available including as important components of our immune system and as prebiotics to promote a healthy gut microflora (Donovan 2009) Bovine, ovine, caprine and equine milk contain relatively low levels of OSs, which have been characterised (see Urashima et al 2001) The OSs identified in equine colostrum are summarised in Table 26.8 The OSs in mature equine milk have not been reported but it can be assumed that the level is considerably lower than in colostrum which has approximately 18.6 g/L (Nakamura et al 2001) The neutral OSs, lacto-N-neotetraose and lacto-Nneohexaose, in equine colostrum are also abundant in human milk, while iso-lacto-N-neotetraose and lacto-N-novopentanose are not, but have been identified in bovine colostrum; the latter has been identified also in the milk of the Tammar wallaby and brown capuchin monkey (Urashima et al 2009) LIPIDS Milk fat is important for the provision of energy to the newborn as well as being the vehicle for fat-soluble vitamins and essential fatty acids From a practical point of view, milk lipids are important as they confer distinctive nutritional, textural and organoleptic properties on dairy products Dietary composition is considered one of the major determinants of the fatty acid composition of equid milk and non-dietary factors such as stage of lactation, age and parity of the mare play minor roles Triglycerides (TGs) represent approximately 80–85% of the lipids in equine and asinine milk, while approximately 9.5% are free fatty acids (FFAs) and approximately 5–10% are phospholipids (Jahreis et al 1999) In contrast, approximately 97–98% of the lipids in bovine and human milk are TGs, with low levels of phospholipids and free fatty acids, 1.3 and 1.5 g.100g−1 , respectively The high level of free fatty acids in equid milk implies that rancidity is a problem with these milks and is dealt with in Section ‘Stability of Equine Milk Fat’ The relatively high content of phospholipids in equid milk is thought to contribute to its buffering properties TGs, the primary transport and storage form of lipids, are synthesised in the mammary gland from fatty acids that originate from three sources: de novo synthesis (C8:0, C10:0 and C12:0), direct uptake from the blood (>14 carbons) and modification of fatty acids in the mammary gland by desaturation and/or elongation Circulating fatty acids in the blood may originate from dietary fat or from lipids mobilised from body fat stores The principal phospholipids of equid milk are phosphatidylcholine (19%), phosphatidylethanolamine (31%), phosphatidylserine (16%) and sphingomylin (34%); the corresponding values for bovine milk are 35%, 32%, 3% and 25% and for human milk are 28, 20, and 39%, respectively The high level of FFAs in equid milk implies considerable lipolysis but this has not been suggested; if lipolysis was responsible for the high level of FFAs, they should be accompanied by high levels of mono- and di-glycerides but these are reported to be quite low at approximately 1.8% of total lipids Table 26.9 shows the monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in the milk fat of some ruminant and non-ruminant species The milk fat of nonruminants contains substantially higher levels of PUFAs than ruminant milks due to the lack of biohydrogenation of fatty acids in the former, and for the two-equid species shown, the horse has considerably more PUFAs in its milk than the donkey Saturated fatty acids are the dominant class in asinine milk and levels are significantly higher than those in equine or human milk (Table 26.9) Fatty Acids in Equid Milks The fatty acid profile of equid milk (Table 26.10) differs from that of bovine and human milk fat in a number of respects Like human, and unlike bovine milk, equid milk is characterised by low proportions of saturated fatty acids with low or higher numbers of carbons, that is, C4:0 , C6:0 , C16:0 and C18:0 (Pikul and W´ojtowski 2008) Butyric acid (C4:0 ) is present at high levels in bovine and other ruminant milk fats, produced from P1: SFK/UKS BLBS102-c26 P2: SFK BLBS102-Simpson March 21, 2012 13:51 Trim: 276mm X 219mm Printer Name: Yet to Come 511 26 Equid Milk: Chemistry, Biochemistry and Processing Table 26.9 Monounsaturated and Polyunsaturated Fatty Acids (Percent of Total Fatty Acids ± Standard Deviations) in the Milk Fat of Some Ruminants and Non-Ruminants MUFAs PUFAs CLA 20.70 15.30 51.80 33.20 36.80 16.00 12.40 12.50 0.09 – 0.23 0.39 26.90 23.20 23.00 2.58 2.42 3.85 0.65 1.01 1.08 Non-ruminants Equine Asinine Porcine Human Ruminants Caprine Bovine Ovine Data from Jahreis et al 1999, Salimei et al 2004 MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; CLA, conjugated linoleic acid 3-hydroxybutanoic acid, which is synthesised by bacteria in the rumen (Pikul and W´ojtowski 2008) Caprylic acid, C8:0 , is very high in equid milk compared to the level in human and bovine milk (Table 26.10) Levels of middle chain-length FAs, especially C10:0 and C12:0 , are high in equid milk (20–35% of all FAs contain