A RP-HPLC method for the detection & quantification of Lactoferrin in milk of various species E. Tsakali1,2, K. Stamatopoulos1, S. Koulouris1, D. Houhoula1, J. Tsaknis1, J.F. Van Impe2, A. Chatzilazarou3 1 2
Department of Food Technology, Technological Educational Institute of Athens (T.E.I. –A)
Chemical and Biochemical Process Technology and Control (BioTeC), Department of Chemical Engineering, KU Leuven, Belgium 3Department
of Oenology and Beverage Technology, Technological Educational Institute of Athens (T.E.I.- A)
Introduction Lactoferrin (LF) is a multifunctional whey protein with unique properties. An existing one step RP-HPLC method previously designed for the quantification of LF in whey, was now evaluated for milk samples of various origin: ovine, caprine, bovine, donkey and human milk.
Materials & Method Sample Preparation • Milk samples were collected from local producers • All samples apart from human milk were pasteurized
Lactoferrin • Monomeric protein, family of transferrins • Antimicrobial properties • MW: ~80kDa
• Anti-inflammatory properties
• Strong basic character (8< pI< 9)
• Anti-carcinogenic properties
• Iron binding properties
• Immuno-modulatory properties
• Fat was removed from milk by centrifugation at 3000 g for 15 min • Separation of whey with precipitation using HCl 1M to pH 4.6 and centrifugation at 3000 g for 15 min
• Bone growth factor properties
Results & Discussion High Sensitivity
•
Good Repeatability (Intra day 1.88%RDS - Inter day 5.89% RDS)
•
Low Limit of Detection (35.46 μg/ml for 3S/N)
•
Low Limit of Quantification (50.86 μg/ml for 10S/N)
Flow rate (mL/ min) 0 67 33 1.0 10 62 38 1.0 20 62 38 1.5 29 61 39 1.5 30 67 33 1.0 *A: water/acetonitrile/trifluoroacetic acid (95:5:0.1) B: water/acetonitrile/trifluoroacetic acid (5:95:0.1) T (OC)
Injection Volume (μL)
DAD t (min)
50
10
30
Whey Chromatograph
nm
mAU
•
% B*
mAU
Method Validation
RP-HPLC analysis Gradient Elusion Profile t (min) % A*
Chromatographs of milk
Minutes
LF
LF
Ovine
Caprine
• All milk samples were re-tested with addition of LF standard in different concentrations and showed that the LF peak increased linearly with increasing LF concentration (no other peak was included)
Donkey
• Caprine, bovine and human milk samples gave LF peaks with good resolution and their LF content was found within the literature intervals
LF
Bovin e
• Ovine and donkey milk samples although they gave LF peaks with good resolution, their LF was significantly higher than the literature values
Human LF
LF
Conclusions Determined LF (μg/mL)
Reference LF (μg/mL)
Ovine Caprine
466.8 927.3
140.0 31.8-821.8
Bovine
188.4
80-500
Donkey Human
133.1 512.0
70-80 300-4200
• An existing, validated, one step RP-HPLC method for the determination of LF in whey was found applicable for the determination of LF in milk of various species • A larger number of samples (through different lactation stages) should be analysed in order to confirm the trends observed
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