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Metodos para Cuantificacion de Lactosa en Leche (FIL-IDF)

This document discusses and compares several analytical methods for quantifying lactose in milk and milk products, including mid-infrared polarimetry, gravimetry, enzymatic essay, differential pH, and high-performance liquid chromatography (HPLC). HPLC is identified as the reference method as it allows for direct, differentiated quantification of carbohydrates and is flexible. Other methods like mid-infrared and polarimetry are faster but less accurate, while enzymatic essay and gravimetry require more preparation but have fewer interference issues. The document provides detailed explanations of the principles, advantages, and disadvantages of each lactose quantification method.

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Ricardo Fogar
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734 views25 pages

Metodos para Cuantificacion de Lactosa en Leche (FIL-IDF)

This document discusses and compares several analytical methods for quantifying lactose in milk and milk products, including mid-infrared polarimetry, gravimetry, enzymatic essay, differential pH, and high-performance liquid chromatography (HPLC). HPLC is identified as the reference method as it allows for direct, differentiated quantification of carbohydrates and is flexible. Other methods like mid-infrared and polarimetry are faster but less accurate, while enzymatic essay and gravimetry require more preparation but have fewer interference issues. The document provides detailed explanations of the principles, advantages, and disadvantages of each lactose quantification method.

Uploaded by

Ricardo Fogar
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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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:
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

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

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 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

GRAVIMETRY
AOAC 930.28

Hammond table for calculating Lactose Cu2O (mg) 11.3 12.4 . 179.0 . 489.7 Lactose.H2O (mg) 7.7 8.5 . 122.2 . 342.0

IDF International Symposium -Lactose and its Derivatives

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

Principle:

1- Lactose Glucose + -Galactose 2- -Galactose + -Galactose dehydrogenase


+NAD+ (nicotinamide Adenine-dinucleotide)

+ -Galactosidase

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


IDF International Symposium -Lactose and its Derivatives

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

Advantages:
Costs Solid samples can be used

Disadvantages:
More elaborate procedure No differentiation between carbohydrates

IDF International Symposium -Lactose and its Derivatives

Differential pH
IDF/ISO draft

Principle: Glucose + -Galactose 1- Lactose 2- Glucose + ATP + Glucokinase Glucose-6P + ADP + H+ 3- Measurement of the pH
+ -Galactosidase

IDF International Symposium -Lactose and its Derivatives

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 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:
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

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

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