Abstract
The objective of this study was to investigate the effect of calcium content in raw donkeys’ milk (DM) on its antibacterial activity against Escherichia coli. Antibacterial assay was performed in artificially contaminated milk samples (without and with added CaCl2 and EDTA) incubated at 38 °C (donkeys’ body temperature). The EDTA was added to DM to bind its calcium ions, while CaCl2 was used as the donor of calcium ions. The content of calcium, lysozyme (LYZ) and lactoferrin (LF) as well as pH value were determined in all of the tested milk samples. DM samples showed varying degrees of antibacterial activity against the tested E. coli strains. The milk samples with higher calcium content possessed stronger antibacterial potential toward the tested bacteria. The determinated calcium-dependant antibacterial activity of DM was proved thorough addition of CaCl2 and EDTA to DM. Calcium content in tested samples was in the range from 377.1 to 1,231 mg/l. The LYZ and LF in tested DM were present at concentrations between 0.98–3.74 g/l and 2.2–54.3 mg/l, respectively. LF was not detected in five of the tested samples, and pH values of the samples were in range from 7.11 to 7.25.
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Andrea C, Vincenzo C, Salvatore B, Anna MG, Rosaria S, Antonio Z et al (2009) Donkeys’ milk protein fraction investigated by electrophoretic methods and mass spectrometric analysis. Int Dairy J 19:190–197
Bruhn O, Grötzinger J, Cascorbi I, Jung S (2011) Antimicrobial peptides and proteins of the horse e insights into a well-armed organism. Vet Res, 42: 98, (22 pages)
Christensen T, Gooden DM, Kung JE, Toone EJ (2003) Additivity and the physical basis of multivalency effects: a thermodynamic investigation of the calcium EDTA interaction. J Am Chem Soc 125:7357–7366
Conte F, Scatassa ML, Monsu G, Lo Verde V, Finocchiaro A, De Fino M (2006) Monitoring of safety and quality of donkey’s milk. In: Smulders FJM (ed) Food safety assurance and veterinary public health: Towards a risk-based chain control, 1st edn. Wageningen Academic Publishers, Wageningen
Coppola R, Salimei E, Succi M, Sorrentino E, Nanni M, Ranieri P (2002) Behaviour of Lactobacillus rhamnosus strains in ass’s milk. Ann Microbiol 52:55–60
Desmet J, Van Dael H, Van Cauwelaert F, Nitta K, Sugai S (1989) Comparison of the binding of Ca2+ and Mn2+ to bovine α-lactalbumin and equine lysozyme. J Inorg Biochem 37:185–191
EC (1995) Directive no 95/2/EC of the European Parliament and of the council of 20 February 1995 laying down directive on food additives other than colours and sweeteners (OJ No L 61, 18. 3. 1995, p. 1). http://ec.europa.eu/food/fs/sfp/addit_flavor/flav11_en.pdf Accessed 12.12.13
Etana M, Endeabu B, Jenberie S, Negussie H (2011) Determination of reference physiological values for working donkeys of Ethiopia. Ethiopian Vet J 15:79–86
Fantuz F, Ferraro S, Todini L, Piloni R, Mariani P, Salimei E (2012) Donkey milk concentration of calcium, phosphorus, potassium, sodium and magnesium. Int Dairy J 24:143–145
Farnaud S, Evans RW (2003) Lactoferrin-a multifunctional protein with antimicrobial properties. Mol Immunol 40:395–405
Floris R, Recio I, Berkhout B, Visser S (2003) Antibacterial and antiviral effects of milk proteins and derivatives thereof. Curr Pharm Des 9:1257–1275
Fotadar U, Zaveloff P, Terracio L (2005) Growth of Escherichia coli at elevated temperatures. J Basic Microb 45:403–404
Guo HY, Pang K, Zhang XY, Zhao L, Chen SW, Dong ML, Ren FZ (2007) Composition, physiochemical properties, nitrogen fraction distribution, and amino acid profile of donkey milk. J Dairy Sci 90:1635–1643
Haezebrouck P, Noppe W, Van Dael H, Hanssens I (1992) Hydrophobic interaction of lysozyme and alpha-lactalbumin from equine milk whey. BBA-Protein Struct M 1122:305–310
Iacono G, Carroccio A, Cavataio F, Montalto G, Soresi M, Balsamo V (1992) Use of ass’s milk in multiple food allergy. J Pediatr Gastr Nutr 14:177–181
Ibrahim HR, Higashiguchi S, Koketsu M, Juneja LR, Kim M, Yamamoto T, Sugimoto Y, Aoki T (1996) Partially unfolded lysozyme at neutral pH agglutinates and kills Gram-negative and Gram-positive bacteria through membrane damage mechanism. J Agr Food Chem 44:3799–3806
ISO (2001) Microbiology of food and animal feeding stuffs -Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli -Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide. ISO standard 16649-2. Geneva, Switzerland: International Organization for Standardization
Jirillo F, Jirillo E, Magrone T (2010) Donkey’s and goat’s milk consumption and benefits to human health with special reference to the inflammatory status. Curr Pharm Des 16:859–863
Lyster RLJ (1992) Effect of calcium on the stability of mares’ milk lysozyme. J Dairy Res 59:331–338
Monti G, Bertino E, Muratore MC, Coscia A, Cresi F, Silvestro L (2007) Efficacy of donkey’s milk in treating highly problematic cow’s milk allergic children: an in vivo and in vitro study. Pediatr Allergy Immunol 18:258–264
Malisauskas M, Zamotin V, Jass J, Noppe W, Dobson CM, Morozova-Roche LA (2003) Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration. J Mol Biol 330:879–890
Mao X, Gu J, Sun Y, Xu S, Zhang X, Yang H, Ren F (2009) Anti-proliferative and anti-tumour effect of active components in donkey milk on A549 human lung cancer cells. Int Dairy J 19:703–708
Mariani PL (2010) Donkey milk nutraceutical characteristics: a biochemical evaluation of nutritious and clinical properties. http://cameprints.unicam.it/284/1/Donkey_milk.pdf Accessed 12.12.13
Masschalck B, Michiels CW (2003) Antimicrobial properties of lysozyme in relation to foodborne vegetative bacteria. Crit Rev Microbiol 29:191–214
Morozova-Roche LA (2007) Equine lysozyme: the molecular basis of folding, self-assembly and innate amyloid toxicity. FEBS Lett 581:2587–2592
Nitta K, Tsuge H, Sugai S, Shimazaki K (1987) The calcium-binding property of equine lysozyme. FEBS Lett 223:405–408
Noppe W, Hanssens I, De Cuyper M (1996) Simple two-step procedure for the preparation of highly active pure equine milk lysozyme. J Chromatogr A 719:327–331
Permyakov SE, Khokhlova TI, Nazipova AA, Zhadan AP, Morozova-Roche LA, Permyakov EA (2006) Calcium-binding and temperature induced transitions in equine lysozyme: new insights from the pCa–temperature ‘‘phase diagrams’’. Proteins 65:984–998
Pilla R, Daprà V, Zecconi A, Piccinini R (2010) Hygienic and health characteristics of donkey milk during a follow-up study. J Dairy Res 77:392–397
Purdy SR (2006) A donkey is not a horse: The differences from a practical veterinary standpoint. http://www.vasci.umass.edu/sites/vasci/files/donkeynotahorse.pdf Accessed 12.12.2013
Salimei E, Fantuz F, Coppola R, Chiofalo B, Polidori P, Varisco G (2004) Composition and characteristics of ass’s milk. Anim Res 53:67–78
Salimei E, Fantuz F (2012) Equid milk for human consumption. Int Dairy J 24:130–142
Šarić Lj, Šarić B, Mandić A, Torbica A, Tomić J, Cvetković D, Okanović Đ (2012) Antibacterial properties of domestic Balkan donkeys’ milk. Int Dairy J 25:142–146
Sarwar A, Enbergs H, Klug E (2001) Influences of parity, age and mineral and trace element mixture on lysozyme activity in mare’s milk during early lactation period. Vet Arhiv 71:139–147
Summer A, Sabbioni A, Formaggioni P, Mariani P (2004) Trend in ash and mineral element content of milk from Haflinger nursing mares throughout six lactation months. Livest Prod Sci 88:55–62
Tafaro A, Magrone T, Jirillo F, Martemucci G, D’Alessandro AG, Amati L, Jirillo E (2007) Immunological properties of donkey’s milk: its potential use in the prevention of atherosclerosis. Curr Pharm Des 13:3711–3717
Tidona F, Sekse C, Criscione A, Jacobsen M, Bordonaro S, Marletta D et al (2011) Antimicrobial effect of donkeys’ milk digested in vitro with human gastrointestinal enzymes. Int Dairy J 21:158–165
Torbica A, Živančev D, Nikolić Z, Ðorđević V, Nikolovski B (2010) The advantages of lab-on-a-chip method in determination of Kunitz trypsin inhibitor in soybean varieties. J Agr Food Chem 58:7980–7985
Tsuge H, Koseki K, Miyano M, Shimazaki K, Chuman T, Matsumoto T, Noma M, Nitta K, Sugai S (1991) A structural study of calcium-binding equine lysozyme by two-dimensional 1H-NMR. Biochim Biophys Acta 1078:77–84
Van Dael H, Haezebrouck P, Morozova L, Arico-Muendel C, Dobson CM (1993) Partially folded states of equine lysozyme. Structural characterization and significance for protein folding. Biochem 32:11886–11894
Van Herreweghe JM, Michiel CW (2012) Invertebrate lysozymes: diversity and distribution, molecular mechanism and in vivo function. J Biosci 37:327–348
Vincenzetti S, Polidori P, Mariani P, Cammertoni N, Fantuz F, Vita A (2008) Donkey’s milk protein fractions characterization. Food Chem 106:640–649
Wilhelm KR (2009) Protein Complexes: Assembly, Structure and Function. http://umu.diva-portal.org/smash/get/diva2:278077/FULLTEXT02 Accessed 12.12.2013
Wu H, Kuzmenko A, Wan S, Schaffer L, Weiss A, Fisher JH, Kim KS, McCormack FX (2003) Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability. J Clin Invest 111:1589–1602
Xu X, Gullberg A, Arnason U (1996) The complete mitochondrial DNA (mtDNA) of the donkey and mtDNA comparisons among four closely related mammalian species-pairs. J Mol Evol 43:438–446
Zhang XY, Zhao L, Jiang L, Dong ML, Ren FZ (2008) The antimicrobial activity of donkey milk and its microflora changes during storage. Food Control 19:1191–1195
Acknowledgments
This work is a part of the National Project (TR–31029) financially supported by the Ministry of Education and Science, Republic of Serbia. Authors are grateful to Slobodan Simić and Nikola Nilić (Special Nature Reserve “Zasavica”, Serbia) for providing the milk samples.
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Šarić, L.Ć., Šarić, B.M., Mandić, A.I. et al. Role of calcium content in antibacterial activity of donkeys’ milk toward E. coli . Eur Food Res Technol 239, 1031–1039 (2014). https://doi.org/10.1007/s00217-014-2299-4
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DOI: https://doi.org/10.1007/s00217-014-2299-4