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Università degli studi di Catania DOTTORATO DI RICERCA IN SCIENZE DELLE PRODUZIONI ANIMALI (XXV Ciclo) DIPARTIMENTO DI SCIENZE DELLE PRODUZIONI AGRARIE E ALIMENTARI DOCTORAL THESIS Investigation on donkey milk protein fractions: in vitro antimicrobial, antiviral and anti-proliferative activities and casein separation by cation exchange chromatography Coordinator: Prof. Marcella Avondo Supervisor: Prof. Donata Marletta Spingendo quotidianamente i nostri limiti riusciamo, a piccoli passi, a superare le paure che ci vietano il possesso della nostra esistenza Angelo D Arrigo 2 Table of Contents Acknowledgements... Abstract... Riassunto Chapter 1 Introduction to donkey milk.. Introduction Donkey milk composition Hygienic-Sanitary aspects of donkey milk Donkey milk in human nutrition The human immune system The human immuno system in relation to milk protein allergy.. 20 Chapter 2 Antimicrobial compounds in milk Introduction Antimicrobial milk compounds Antimicrobial compounds from whey fraction lactoglobulin lactoalbumin Immunoglobulins Lactoferrin Lactoferricin Lactoferrampin Lactoperoxidase Lysozime Antimicrobial compound from casein fraction Minor bioactive peptides milk compuond Antimicrobial peptide applications Conclusions Chapter 3 Antimicrobial properties of whey proteins in donkey milk. 58 Chapter 4 Antiviral activity of donkey milk on echovirus type Enterovirus echovirus. 4.2 Human Colon Adenocarcinoma Cell Line (CaCo-2) Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Chapter 5 A preliminary study on potential anti-proliferative effect of donkey milk on SK-N-BE cells line... Chapter 6 Separation of donkey s caseins by cation exchange chromatography: preliminary results... Abbreviations ACKNOWLEDGEMENTS The work presented in this thesis was carried out at the Dipartimento di Scienze delle Produzioni Agrarie e Alimentari, University of Catania, Italy; Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Aas, Norway; Department of Medical Microbiology, Akershus University Hospital, Oslo, Norway; and Dipartimento di Scienze Biologiche, Geologiche e Ambientali, Sezione di Biologia Animale, University of Catania, Italy. I would first like to thank my main supervisor Donata Marletta for being a guide and for supporting me throughout this work. Her smiling personality has been a constant and invaluable encouragement. I d like also to thank my unofficial Norwegian supervisor Gerd E. Vegarud for supporting both me and my project with enthusiasm. I also wish to thank the co-authors, who have been very interesting to work with and I have learned a lot from. I thank all the people that walked with me during this professional journey; in particular Andrea PhD Criscione, Antonio PhD Zuccaro, Bernardo PhD Valenti, Annamaria PhD Guastella, Salvatore PhD Bordonaro. Finally, a huge thank to my family for always being supportive and giving me encouragement. 5 Horses and donkeys at the first National cattle fair (beginning of twentieth-century, Catania) 6 ABSTRACT This thesis was aimed to study donkey milk protein fractions and their biological properties. In the first Chapter the donkey s milk gross composition and the related hygienicsanitary aspects are introduced. The main use of donkey milk in human nutrition, especially in infancy and in patients with cow milk protein allergy (CMPA), is also discussed. The second Chapter Antimicrobial compounds in milk was drafted after an intensive course at the University of Copenhagen: Functional milk compound with focus on milk proteins, and presented as the final report of the first year of PhD school. It deals with a review of the antimicrobial activity of milk components, with particular attention to whey proteins, as lactoferrin and lysozyme and their derived bioactive peptides, such as lactoferricin, which play a crucial role in human health and nutrition. The detailed description of the donkey whey proteins, which exhibit antimicrobial activity is presented in third chapter. Moreover, the antimicrobial activity of fresh, powdered, digested and fermented donkey milk has been discussed. The Fourth Chapter reports the studies on the antiviral activity of donkey milk and its fractions (skimmed milk, digested milk, casein, whey and low molecular weight whey fraction) that has been tested in vitro on an enterovirus, echovirus type 5, in two experimental trials. Among the others, we found that whey proteins showed the strongest inhibitory effects, probably thanks to the synergic actions of lactoferrin, lysozyme and lactoperoxidase. A preliminary investigation of anti-proliferative effect of skimmed donkey milk and its main protein fractions (casein and whey proteins), carried out through two assay performed in vitro on Human Neuroblastoma Cell Lines (SK-N-BE) is presented in the fifth Chapter. In our experimental conditions only the whey proteins, at the highest concentration, have shown anti-proliferative effect on cell growth. Finally, in the sixth Chapter, the preliminary results obtained from the analysis of the donkey s casein fraction by ion exchange chromatography, are described. By coupling chromatographic and mass spectrometry techniques, we obtained three different fractions from the separation of donkey s casein: pure β-casein, αscaseins and a pool containing κ-casein and αs-caseins. The survey was carried out with the aim to obtain protein standards to use in quantification analysis. 7 RIASSUNTO L obiettivo principale di questa tesi è stato quello di mettere in luce alcune attività biologiche del latte di asina, con particolare riguardo alle proprietà antimicrobiche, antivirali e anti-proliferative delle sue frazioni proteiche. Nel primo Capitolo si introduce il latte di asina e se ne descrivono la composizione ed i relativi aspetti igienico-sanitari. Inoltre, il suo utilizzo nell alimentazione umana, specialmente nell infanzia e nei pazienti con allergia al latte vaccino è brevemente discusso. Il secondo Capitolo raccoglie le informazioni acquisite durante un corso intensive seguito presso l Università di Copenhagen su Functional milk compound with focus on milk proteins e costituisce il report del primo anno di dottorato. Esso riporta un accurata descrizione sull attività antimicrobica del latte, con particolare attenzione alle proteine del siero, quali la lattoferrina e il lisozima ed ai peptidi bioattivi da esse derivati, come la lattoferricina, i quali svolgono un ruolo cruciale per la salute umana e la nutrizione. Il terzo capitolo presenta una dettagliata descrizione delle proteine del siero di latte di asina che hanno mostrato azione antimicrobica nel latte fresco, latte digerito con enzimi proteolitici, ma anche in polvere e fermentato. Il quarto Capitolo presenta gli studi sull'attività antivirale del latte di asina e le sue frazioni (latte scremato, latte digerito con enzimi gastro-intestinali umani, caseina, proteine del e proteine del siero a basso peso molecolare), che è stata testata in vitro su un tipo di enterovirus, echovirus 5, in due prove sperimentali. I risultati ottenuti hanno mostrato un forte effetto inibente sulla replicazione virale delle proteine del siero, probabilmente dovuta al sinergismo tra tra i suoi componenti. Lo studio sull attività anti-proliferativa del latte di asina scremato e le sue frazioni proteiche (caseina e siero di latte), eseguito attraverso due prove sperimentali, in vitro, su Human Neuroblastoma Cell Lines (SK-N-BE), è presentato nel quinto Capitolo. Solo le proteine del siero alla più alta concentrazione hanno inibito la crescita cellulare. In fine, nel sesto Capitolo, sono riportati i risultati preliminari riguardanti l analisi della frazione caseinica mediante cromatografia a scambio ionico. Grazie all utilizzo combinato di tecniche di cromatografia e spettrometria di massa, sono state ottenute tre frazioni dalla separazione delle caseine del latte di asina: β- caseina pura, αs-caseine e un pool contenente κ-caseina e αs-caseine. Lo scopo della prova sperimentale era quello di ottenere standard proteici da utilizzare in successive analisi quantitative. 8 Chapter 1 Introduction to Donkey milk 9 10 Introduction The donkey (Equus asinus) domestication began about 6000 BC in present-day Libya, starting from one or two subspecies of African wild asses (E. africanus). Over the centuries donkeys have spread in Asia, India, South-America and south Europe, being used as beast of burden. Today donkeys still have this role but only in the poorest regions of the world (Bordonaro et al., 2012). During the 20 th Century, in Europe donkey population was reduced by 80-90% because of growing mechanization in agriculture that severely affected the use of this specie (Colli et al., 2012). To date, in Italy, six donkey breeds are already extinct and eight autochthonous breeds (Asinara, Pantesco, Ragusano, Grigio Siciliano, Romagnolo, Amiatino, Sardo Grigio and Martina Franca) are still reared. They have been classified as critically endangered by Food and Agriculture Organization (FAO). Despite this considerable loss, during the last few years, some local breeds and populations are growing thanks to new and rediscovered traditional use of donkeys. This animal can be used for meat (e.g. stew or salame) and milk production (used in human nutrition and in cosmetic industry), for onotherapy (a method of using contact and educational techniques with donkeys to help people with challenges in the relational and emotional areas) and also for recreational purposes such as ecotourism and trekking (Colli et al., 2012; Bordonaro et al., 2011). General legislative rules regulate the donkey welfare and the commercialization of ass milk (e.g. Regio decreto of the 9 th of May 1929, n artt. 15 and 43- and Directive 98/58/CE, adopted in Italy by Legislative Decree n.146), but there are not specific rules for donkey breeding and milk production. This thesis was aimed at the study of donkey milk s protein fractions and its antimicrobial, antiviral, anti-proliferative properties through a detailed review on antimicrobial whey protein properties and in vitro experimental tests to verify the antiviral effects of donkey milk fractions (whole, skimmed, digested milk, whey and casein protein fractions) and potential anti-proliferative effects of skimmed donkey milk, whey protein fraction and casein. Finally, a preliminary work was 11 carried out in order to separate the donkey s casein fractions by cationic exchange chromatography. 1.1 Donkey milk composition Since ancient times donkey milk (DM) was used as substitute for babies which could not be breastfeed because among different species, equine milk the most similar to human milk (Swar 2011), except for the lipid content (see table1). Table 1. Gross milk composition from different species. a Species Total Solids Milk components Casein/Whey Protein ratio Fat Lactose Ash Protein Human a :1 Donkey a :1 Mare a :1 Camel b :1 Cow b :1 Goat a :1 a Data from (human, cow, goat and camel) Uniacke-Lowe et al., (2010), (donkey) Salimei et al., (2004), Guo et al., (2007), Tidona et al., (2011a), (mare) Malacarne et al., (2002). Mean value expressed as: a g Kg -1 and b g L -1. DM composition changes during lactation (about 150 days). The amount produced ( kg d -1, not including the part sucked by foal) shows a nonlinear trend after 45 and 90 days from partum or with a single peak after 90 days (see figure 1). Some authors showed that milk production was affected both by breed and the season of parturition. The donkeys who gave birth in a autumnwinter period yielded more milk (Salimei et al.,2004; Guo et al., 2007; Tidona et. al., 2011a; Cosentino et al., 2012). Moreover a difference was observed between the milked amount in the morning (about 0.67 kg) and that milked in the afternoon (about kg) (Tidona et al., 2011a). 12 Figure 1. Donkey lactation curve for milk yield, protein and fat percentuage (from Cosentino et al., 2012). The ph value is around 7.2, which is slightly higher than cow milk (6.7), but closer to human (7.3) and mare milk (7.18) (Guo et al. 2007). It decreases in the late lactation, even if the difference is not statistically significant (Guo et al., 2007; Tidona et al., 2011a). The highest value with respect to cow milk might be due to low content of casein and phosphates, which is also common to human and mare milk (Guo et al., 2007; Salimei et al., 2004). Proteins content ranges from 1.3% to 2.0%, lower than in cow milk (3.2%), and with a low casein/whey ratio (1.04 on average) (Guo et al., 2007). Whey protein fractions are 35-50% of the nitrogen fraction, while in cow milk it represents only 20%. Casein represents about 47 % of crude protein (on average, because the casein amount decreases during lactation) (see figure 2 and 3). The casein/whey protein ratio, which change during the lactation, results to have decreasing value between (Tidona et al., 2011a). This low casein/whey protein ratio plays a crucial role in the sensitization to cow milk protein fraction, acting on the allergenic properties. A relevant heterogeneity was reported for donkey milk protein profiles; in Ragusano donkey it was shown that 35.7% of individual milks had a IEF pattern characterized by the absence of some protein bands with respect to a reference (which consisted of samples with a common protein profile (64.3%) (Criscione et 13 al., 2009). This observed quantitative polymorphism, especially if it will be confirmed at the genetic level, could influence donkey s milk protein allergenicity. Figure 2. Trend of total amount of protein during lactation (from Cosentino et al., 2012). Figure 3. a) Trend of total amount of proteins, casein and whey protein; b) trend of casein/whey protein during lactation (from Tidona et al., 2011a). The lipids amount is low in comparison with the other species. The lipid composition of cow and human milk constitutes 98-99% of triglycerides, 1-2% of phospholipids, sterols, monoglycerides, wax, squalene, carotene and fat-soluble vitamins (A, D, E, K), and traces of free fatty acids. In equine milk the 14 triglyceride content is lower than in cow and human milk, in mare milk triglyceride content is around 80% (Malacarne et al., 2002). Figure 4. Lipid trend during lactation (Cosentino et al., 2012). Although the lipid content value in donkey milk could be affected by breed, it is certainly influenced by breeding system, milking technique and interval between milking (Guo et al., 2007). It ranges between 0.03 and 1.18 g Kg -1 (see figure 4) (Tidona et al., 2009) with an increasing, not-linear, trend from partum to the end of lactation (Salimei et al., 2004; Guo et al., 2007; Tidona et al., 2011a). That amount is lower than in cow, goat, sheep and human milk. Even if it implies a lipid deficiency in an infant diet, which must be filled by supplements, it is a benefit in the diet therapy to prevent cardiovascular, autoimmune and inflammatory diseases. In particular, the high value of polyunsaturated fat acids (PUFA) (ω6 and ω3) (52.2%), the low ω6 to ω3 ratio, and the advantageous values of atherogenic and thrombogenic indices (see table 2 and figure 5) (Martemucci & D Alessandro, 2012) tend, in human diet, to lower the level of cholesterol in blood, to prevent the formation of atherosclerotic plaques, removing the risk of coronary heart disease, hypertension and thrombosis (D amico et al., 2007; Agostino et al., 2007), suggesting the DM as a functional food for infant nutrition, but also for adults that have to follow particular diets (Martemucci. & D Alessandro, 2012). 15 Table 2. Fatty acid composition: saturated fatty acids (SFA); unsaturated fatty acids (UFA); monounsaturated fatty acids (MUFA); polyunsaturated fatty acids (PUFA). Mean values influenced by the lactation stage (data from Martemucci. & D Alessandro, 2012). Fatty acid composition % Saturated (SFA,) Unsaturated (UFA) Monounsaturated (MUFA) Polyunsaturated (PUFA) of which PUFA ω PUFA ω Figure 5. Fatty acids tred during lactation: saturated fatty acids (SFA); unsaturated fatty acids (UFA); monounsaturated fatty acids (MUFA); polyunsaturated fatty acids (PUFA) (from Martemucci. & D Alessandro, 2012). Carbohydrates are mainly represented by lactose, which content is generally around 6% (see figure 6) (Salimei et al., 1999; Polidori 1994; Polidori and Vincenzetti 2006). The high lactose content promotes osteogenesis processes improving the intestinal absorption of calcium, phosphorus and influencing the mineral accumulation in bone structure, which is useful for the prevention of osteoporosis (Borrello, 2007). Lactose gives good taste to DM (Iacono et al., 16 1992; Monti et al., 2005; Paolicelli 2005), and is also a precious source of galactose, essential for the development of the nervous system. Figure 6. Lactose trend during lactation (from Cosentino et al., 2012). Vitamins Although the amount of several vitamins has not yet been detected in donkey milk, it is known that mare milk contains a significantly higher level of vitamin C than cow milk. In DM, with exception for niacin (vitamin B3), the amount of thiamine (vitamin B1), riboflavin (vitamin B2) and cobalamin (B3) is higher than in human milk (see table 3). The level of vitamin E is low in asinine milk (~0.05 mg L -1 ). 17 Table 3 Vitamins in donkey, mare, human and cow milk. a Vitamins Donkey 1 Mare 1 Human 1 Cow 1 Vitamin A b Vitamin E b Vitamin C c Vitamin B d Vitamin B d Vitamin B d Vitamin B d a Data from (donkey b, mare and human) Salimei et al., (2012), (donkey c ) Beghelli et al., (20102), (donkey d and cow) Uniacke-Lowe (2011). Value are expressed as: 1 mg L -1, 2 mg ml -1. Mineral The importance of minerals in human nutrition is well known, because they play a fundamental role in growth and skeletal structure development; nevertheless little information about the mineral composition of DM has been reported in literature so far, Despite some inconsistencies, which might due to differences in breed, stage of lactation and analytical methods applied, mineral fraction represents about 0.39% (mean value) of total solids in donkey milk (Fantuz et al., 2012). Mineral amount is higher in early lactation (0.51 g kg -1 ), during which the milk is the only source of minerals for the growth of the foal (Salimei et al., 2000; Guo et al., 2007). The reduction observed of Ca, P and Mg, during the lactation, could be explained by the contemporaneous decline of casein amount since those minerals are mainly associated to the casein micelles (Giosuè et al., 2008; Fantuz et al., 2012). DM mineral content is similar to that of human and mare milk, and lower than that of cow milk (Doreau & Martin-Rosset, 2011; Gaucheron, 2005; Fantuz et al., 2012) (see table 4). 18 DM contains higher amount of calcium and phosphorus (Belli Blanes, 2001) than HM, even if the amount is still lower than that of cow milk (1.17 g kg -1 ) (Salimei et al., 2000). Table 4. Minerals in donkey, mare, human and cow milk. a Minerals Donkey b Mare b Human c Cow c Ca P K Na Mg Fe Zn Cu Mn trace a Data from (donkey and mare) Salimei et al., (2012), Csapò et al., (1995); (human and cow) from Park et. al., (2007). Mean values expressed as: b mg L -1 and c mg g. 1.2 Hygienic-sanitary aspects of donkey milk Milk, for its gross composition and ph value, is a good medium for the growth of microorganisms. Moreover, the health and hygiene of animal and the temperature of milk can aid pathogen microorganisms proliferation. In donkey milk, the somatic cell count (SCC) resulted to be lower than in ruminants milk, comparing health animals (Salimei & Chiofalo, 2006; Salimei, & Fantuz, 2010), as well as the total bacterial count: it was reported to be lower ( log CFU ml -1 ) (Salimei & Chiofalo, 2006; Salimei, & Fantuz, 2010) respect that of the cow milk (7.58 log CFU ml -1 ) (Tassew & Seifu, 2010). Besides, a lower total bacterial count and a longer she
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