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IDF International Symposium -Lactose and its Derivatives
Analytical Methods for Lactose Quantification in Milk and Milk Products
Rachid Kouaouci, Ing., M.Sc., ChemistValacta, Ste-Anne de Bellevue, Québec, Canada
Centre d’expertise en production laitière du Québec
IDF International Symposium -Lactose and its Derivatives
METHODS
Mid-infraredPolarimetryGravimetryEnzymatic essayDifferential pHChromatography ( HPLC )
IDF International Symposium -Lactose and its Derivatives
MID-INFRAREDIDF 141C:2000; AOAC 972.16
Principle: Measurement of the absorption of the hydroxyl group (OH) at 9.6 µm.
Advantages:Very fast (400 samples/hour)No sample preparationWidely used
Disadvantages:Indirect methodNo differentiation between carbohydratesLimitation to fluid samples
IDF International Symposium -Lactose and its Derivatives
POLARIMETRYAOAC 896.01
Principle:1-Precipatation of fat and protein2-Measurement of the specific rotation of the polarized
light due to the asymmetric carbon of lactose
Advantages:Costs
Disadvantages:Interference with optically active components No differentiation between carbohydratesEmpirical calculation
IDF International Symposium -Lactose and its Derivatives
POLARIMETRYAOAC 896.01
Calculation:C [g/100ml] = a[Vm + Vr – 0.01145Vm ( F+P) ]
Vm (b)a: observed readingVm: Volume of milk usedVr: Volume of reagent usedb: rotation in a 40 cm tube of 100 ml solution
containing 1 gram of lactose at t 0C ( b=2.096at 25 0C)
F: %fat P: % protein
IDF International Symposium -Lactose and its Derivatives
GRAVIMETRYAOAC 930.28
Principle: Lactose + CuSO4
Advantages:Very simple procedureCosts
Disadvantages:Empirical calculationInterference with all reducing carbohydratesNo differentiation between carbohydrates
Cu2O
IDF International Symposium -Lactose and its Derivatives
GRAVIMETRYAOAC 930.28
Hammond table for calculating Lactose
7.78.5.
122.2.
342.0
11.312.4
.179.0
.489.7
Lactose.H2O(mg)
Cu2O(mg)
IDF International Symposium -Lactose and its Derivatives
ENZYMATIC ESSAYIDF 79-1,2/ISO 5765-1,2 (2002); AOAC 930.28
Principle: 1- Lactose
2- β-Galactose + β-Galactose dehydrogenase
+NAD+ (nicotinamide Adenine-dinucleotide)
NADH
3-Measurment of the amount of NADH by absorbance at 340 nm
+ β-GalactosidaseGlucose + β-Galactose
IDF International Symposium -Lactose and its Derivatives
ENZYMATIC ESSAYIDF 79-1,2/ISO 5765-1,2 (2002); AOAC 930.28
Advantages:CostsSolid samples can be used
Disadvantages:More elaborate procedureNo differentiation between carbohydrates
IDF International Symposium -Lactose and its Derivatives
Differential pHIDF/ISO draft
Principle:1- Lactose Glucose + β-Galactose2- Glucose + ATP + Glucokinase
Glucose-6P + ADP + H+
3- Measurement of the pH
+ β-Galactosidase
IDF International Symposium -Lactose and its Derivatives
HPLCIDF 198 │ISO 22622 (FDIS)
• Principle: 1-Sample + Internal Std1
2-Filtration of the sample3-Injection through an analytical column4-Detection by a differential refractometer5-Quantification
+ Biggs Solution2Precipitation of fat
and protein
2Zinc acetate + Glacial acetic acid+ Phosphotungstic acid1Internal Std: Melezitose ( Glu-Fru-Fru )
IDF International Symposium -Lactose and its Derivatives
HPLCIDF 198 │ISO 22622 (FDIS)
Chromatographic conditions:Mobile phase: degassed HPLC grade waterFlow rate: 0.6ml / minInternal detector temperature: 350 CColumn temperature: 850 CVolume to be injected: 20 µlRun time: 15 minColumn type: Styrene divinylbenzene resin
IDF International Symposium -Lactose and its Derivatives
HPLCIDF 198 │ISO 22622 (FDIS)
Chromatogram of raw milk sample
IDF International Symposium -Lactose and its Derivatives
HPLCIDF 198 │ISO 22622 (FDIS)
Chromatogram of raw milk sample with added lactulose
IDF International Symposium -Lactose and its Derivatives
HPLCIDF 198 │ISO 22622 (FDIS)
Chromatogram of lactose-reduced milk sample
IDF International Symposium -Lactose and its Derivatives
HPLCIDF 198 │ISO 22622 (FDIS)
Chromatogram of raw milk sample with added sucrose and fructose
IDF International Symposium -Lactose and its Derivatives
HPLCIDF 198 │ISO 22622 (FDIS)
Advantages:Direct methodDifferentiation between carbohydratesAutomated methodReference methodFlexibility
Disadvantages:Costs
IDF International Symposium -Lactose and its Derivatives
HPLCIDF 198 │ISO 22622 (DIS)
IDF International Symposium -Lactose and its Derivatives
HPLC
IDF 198 │ISO 22622 (DIS)
IDF International Symposium -Lactose and its Derivatives
HPLCIDF 198 │ISO 22622 (DIS)
IDF International Symposium -Lactose and its Derivatives
HPLCIDF 198 │ISO 22622 (DIS)
IDF International Symposium -Lactose and its Derivatives
HPLCIDF 198 │ISO 22622 (DIS)
IDF International Symposium -Lactose and its Derivatives
HPLCIDF 198 │ISO 22622 (DIS)
IDF International Symposium -Lactose and its Derivatives
REFERENCES
• Brons, C, and Olieman, C, 1983. Study of the high performance liquid chromatography separation of reducing sugars applied to the determination of lactose in milk, J. Chromato., 259, 79.
• IDF 141C:2000 -Whole Milk- Determination of milkfat, protein and lactose content, Guidance on the operation of Mid-infrared instruments.
• IDF 79-1:2002│ISO 5765-1- Dried milk, dried ice-mixes and processed cheese- Determination of lactose content- Part 1: Enzymatic method utilizing the glucose moiety of the lactose.
• IDF 79-2:2002│ISO 5765-2- Dried milk, dried ice-mixes and processed cheese- Determination of lactose content- Part 2: Enzymatic method utilizing the galactose moiety of the lactose.
• IDF 106:2004│ISO 5548- Caseins and caseinates- Determination of lactose content- Photometric method.
• Official Methods of Analysis, 16th Ed., 4th Revision, 1998, AOAC INTERNATIONAL, Gaithersburg, MD, methods 896.01, 984.15, 930.28 and 972.16.
• Nickerson, T. A., Vujicic, I. F., and Lin, A. Y, 1976. Colorimetric estimation of lactose and its hydrolytic products. J. Dairy Sci., 59, 386.
IDF International Symposium -Lactose and its Derivatives
Many thanks for your attention!
Hi, I need to measure my lactose level!