Centre dexpertise en production laitire du Qubec

Analytical Methods for Lactose

Quantification in Milk and Milk Products

Rachid Kouaouci, Ing., M.Sc., Chemist

Valacta, Ste-Anne de Bellevue, Qubec, 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

 MID-INFRARED
IDF 141C:2000; AOAC 972.16

Principle: Measurement of the absorption of the hydroxyl

group (OH) at 9.6 m.

Advantages:
9 Very fast (400 samples/hour)
9 No sample preparation
9 Widely used

Disadvantages:
9 Indirect method
9 No differentiation between carbohydrates
9 Limitation to fluid samples

IDF International Symposium -Lactose and its Derivatives

 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:
9 Costs

Disadvantages:
9 Interference with optically active components
9 No differentiation between carbohydrates
9 Empirical calculation

IDF International Symposium -Lactose and its Derivatives

 POLARIMETRY
AOAC 896.01

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=2.096
at 25 0C)
F: %fat
P: % protein

IDF International Symposium -Lactose and its Derivatives

 GRAVIMETRY
AOAC 930.28

Principle: Lactose + CuSO4 Cu2O

Advantages:
9 Very simple procedure
9 Costs

Disadvantages:
9 Empirical calculation
9 Interference with all reducing carbohydrates
9 No differentiation between carbohydrates

IDF International Symposium -Lactose and its Derivatives

 GRAVIMETRY
AOAC 930.28

Hammond table for calculating Lactose

Cu2O Lactose.H2O
(mg) (mg)
11.3 7.7
12.4 8.5
. .
179.0 122.2
. .
489.7 342.0

IDF International Symposium -Lactose and its Derivatives

 ENZYMATIC ESSAY
IDF 79-1,2/ISO 5765-1,2 (2002); AOAC 930.28

Principle:
+ -Galactosidase
1- Lactose Glucose + -Galactose
2- -Galactose + -Galactose dehydrogenase

+NAD+ (nicotinamide Adenine-dinucleotide)

NADH
3-Measurment of the amount of NADH by absorbance
at 340 nm

IDF International Symposium -Lactose and its Derivatives

 ENZYMATIC ESSAY
IDF 79-1,2/ISO 5765-1,2 (2002); AOAC 930.28

Advantages:
9 Costs
9 Solid samples can be used

Disadvantages:
9 More elaborate procedure
9 No differentiation between carbohydrates

IDF International Symposium -Lactose and its Derivatives

 Differential pH
IDF/ISO draft

Principle:
+ -Galactosidase
1- Lactose Glucose + -Galactose
2- Glucose + ATP + Glucokinase

Glucose-6P + ADP + H+

3- Measurement of the pH

IDF International Symposium -Lactose and its Derivatives

 HPLC
IDF 198 ISO 22622 (FDIS)

Principle: + Biggs Solution2

Precipitation of fat
1-Sample + Internal Std1 and protein

2-Filtration of the sample

3-Injection through an analytical column
4-Detection by a differential refractometer
5-Quantification

1InternalStd: 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 l
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:
9 Direct method
9 Differentiation between carbohydrates
9 Automated method
9 Reference method
9 Flexibility

Disadvantages:
9 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

 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 79-1:2002ISO 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 79-2:2002ISO 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 106:2004ISO 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!

IDF International Symposium -Lactose and its Derivatives