metodos para cuantificacion de lactosa en leche (fil-idf)

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


METHODS

Mid-infraredPolarimetryGravimetryEnzymatic essayDifferential pHChromatography ( HPLC )


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


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


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


GRAVIMETRYAOAC 930.28

Principle: Lactose + CuSO4

Advantages:Very simple procedureCosts

Disadvantages:Empirical calculationInterference with all reducing carbohydratesNo differentiation between carbohydrates

Cu2O


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)


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


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


Differential pHIDF/ISO draft

Principle:1- Lactose Glucose + β-Galactose2- Glucose + ATP + Glucokinase

Glucose-6P + ADP + H+

3- Measurement of the pH

+ β-Galactosidase


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 )



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



Chromatogram of raw milk sample



Chromatogram of raw milk sample with added lactulose



Chromatogram of lactose-reduced milk sample



Chromatogram of raw milk sample with added sucrose and fructose



Advantages:Direct methodDifferentiation between carbohydratesAutomated methodReference methodFlexibility

Disadvantages:Costs


HPLCIDF 198 │ISO 22622 (DIS)


HPLC

IDF 198 │ISO 22622 (DIS)


HPLCIDF 198 │ISO 22622 (DIS)


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.


Many thanks for your attention!

Hi, I need to measure my lactose level!

metodos para cuantificacion de lactosa en leche (fil-idf)

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