Centre d’expertise en production laitière du Québec

Analytical Methods for Lactose Quantification in Milk and Milk Products
Rachid Kouaouci, Ing., M.Sc., Chemist Valacta, Ste-Anne de Bellevue, Québec, Canada

IDF International Symposium -Lactose and its Derivatives

METHODS Mid-infrared Polarimetry Gravimetry Enzymatic essay Differential pH Chromatography ( HPLC )

IDF International Symposium -Lactose and its Derivatives

16 Principle: Measurement of the absorption of the hydroxyl group (OH) at 9.MID-INFRARED IDF 141C:2000. AOAC 972. Advantages: Very fast (400 samples/hour) No sample preparation Widely used Disadvantages: Indirect method No differentiation between carbohydrates Limitation to fluid samples IDF International Symposium -Lactose and its Derivatives .6 µm.

POLARIMETRY AOAC 896.01 Principle: 1-Precipatation of fat and protein 2-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 carbohydrates Empirical calculation IDF International Symposium -Lactose and its Derivatives .

096 at 25 0C) F: %fat P: % protein IDF International Symposium -Lactose and its Derivatives .01 Calculation: C [g/100ml] = a[Vm + Vr – 0.01145Vm ( F+P) ] Vm (b) a: observed reading Vm: Volume of milk used Vr: Volume of reagent used b: rotation in a 40 cm tube of 100 ml solution containing 1 gram of lactose at t 0C ( b=2.POLARIMETRY AOAC 896.

GRAVIMETRY AOAC 930.28 Principle: Lactose + CuSO4 Advantages: Very simple procedure Costs Cu2O Disadvantages: Empirical calculation Interference with all reducing carbohydrates No differentiation between carbohydrates IDF International Symposium -Lactose and its Derivatives .

0 IDF International Symposium -Lactose and its Derivatives .GRAVIMETRY AOAC 930.0 .H2O (mg) 7.4 . 489.5 . 179.28 Hammond table for calculating Lactose Cu2O (mg) 11.7 Lactose. 342. 122.3 12.2 .7 8.

β-Galactose + β-Galactose dehydrogenase +NAD+ (nicotinamide Adenine-dinucleotide) + β-Galactosidase NADH 3-Measurment of the amount of NADH by absorbance at 340 nm IDF International Symposium -Lactose and its Derivatives .ENZYMATIC ESSAY IDF 79-1.Lactose Glucose + β-Galactose 2.2 (2002). AOAC 930.2/ISO 5765-1.28 Principle: 1.

AOAC 930.ENZYMATIC ESSAY IDF 79-1.2/ISO 5765-1.28 Advantages: Costs Solid samples can be used Disadvantages: More elaborate procedure No differentiation between carbohydrates IDF International Symposium -Lactose and its Derivatives .2 (2002).

Measurement of the pH + β-Galactosidase IDF International Symposium -Lactose and its Derivatives .Lactose 2.Differential pH IDF/ISO draft Principle: Glucose + β-Galactose 1.Glucose + ATP + Glucokinase Glucose-6P + ADP + H+ 3.

HPLC IDF 198 │ISO 22622 (FDIS) • Principle: + Biggs Solution2 1-Sample + Internal Std1 Precipitation of fat and protein 2-Filtration of the sample 3-Injection through an analytical column 4-Detection by a differential refractometer 5-Quantification 1Internal Std: Melezitose ( Glu-Fru-Fru ) 2Zinc acetate + Glacial acetic acid+ Phosphotungstic acid IDF International Symposium -Lactose and its Derivatives .

HPLC IDF 198 │ISO 22622 (FDIS) Chromatographic conditions: Mobile phase: degassed HPLC grade water Flow rate: 0.6ml / min Internal detector temperature: 350 C Column temperature: 850 C Volume to be injected: 20 µl Run time: 15 min Column type: Styrene divinylbenzene resin IDF International Symposium -Lactose and its Derivatives .

HPLC IDF 198 │ISO 22622 (FDIS) Chromatogram of raw milk sample IDF International Symposium -Lactose and its Derivatives .

HPLC IDF 198 │ISO 22622 (FDIS) Chromatogram of raw milk sample with added lactulose IDF International Symposium -Lactose and its Derivatives .

HPLC IDF 198 │ISO 22622 (FDIS) Chromatogram of lactose-reduced milk sample IDF International Symposium -Lactose and its Derivatives .

HPLC IDF 198 │ISO 22622 (FDIS) Chromatogram of raw milk sample with added sucrose and fructose IDF International Symposium -Lactose and its Derivatives .

HPLC IDF 198 │ISO 22622 (FDIS) Advantages: Direct method Differentiation between carbohydrates Automated method Reference method Flexibility Disadvantages: Costs IDF International Symposium -Lactose and its Derivatives .

HPLC IDF 198 │ISO 22622 (DIS) IDF International Symposium -Lactose and its Derivatives .

HPLC IDF 198 │ISO 22622 (DIS) IDF International Symposium -Lactose and its Derivatives .

HPLC IDF 198 │ISO 22622 (DIS) IDF International Symposium -Lactose and its Derivatives .

HPLC IDF 198 │ISO 22622 (DIS) IDF International Symposium -Lactose and its Derivatives .

HPLC IDF 198 │ISO 22622 (DIS) IDF International Symposium -Lactose and its Derivatives .

HPLC IDF 198 │ISO 22622 (DIS) IDF International Symposium -Lactose and its Derivatives .

C. Vujicic. 984.REFERENCES • • • • • • • Brons.. dried ice-mixes and processed cheese. 59. and Olieman. Y. J.Determination of milkfat. 1976. A.. IDF 106:2004│ISO 5548. 259. IDF 141C:2000 -Whole Milk. 386. I. T.Determination of lactose content. 1983. 1998. methods 896. AOAC INTERNATIONAL. 4th Revision.Dried milk.Dried milk.. Official Methods of Analysis. IDF 79-2:2002│ISO 5765-2. C.Part 2: Enzymatic method utilizing the galactose moiety of the lactose. A. Guidance on the operation of Mid-infrared instruments. 79. Nickerson. MD. F.16. 16th Ed. Study of the high performance liquid chromatography separation of reducing sugars applied to the determination of lactose in milk. Gaithersburg.. J. Colorimetric estimation of lactose and its hydrolytic products.01. IDF 79-1:2002│ISO 5765-1.28 and 972. Chromato.Determination of lactose content.. Dairy Sci. IDF International Symposium -Lactose and its Derivatives . and Lin. 930.15.Photometric method.Caseins and caseinates.Determination of lactose content. protein and lactose content. dried ice-mixes and processed cheese.Part 1: Enzymatic method utilizing the glucose moiety of the lactose.

I need to measure my lactose level! IDF International Symposium -Lactose and its Derivatives .Many thanks for your attention! Hi.