Benefits of K-LOLAC over Common Enzymatic Assays

EFFICIENT First sequential enzymatic assay for lactose. This means reduced analyst hands-on time in the

laboratory and increased cost-effectiveness,

SELECTIVE Uses an exclusive enzyme which hydrolyses lactose more selectively than other enzymes

employed commercially.

SENSITIVE Can quantify lactose at lower levels than all other available enzymatic assays - essential for low-

lactose and lactose-free products.

ACCURATE Method includes 15 minute pre-incubation step to remove high background glucose levels,

allowing rapid and accurate measurement in low-lactose and lactose-free products.

Find out more or order online at www.megazyme.com

The very manufacturing process which breaks down the naturally

present lactose in the creation of lactose-free products also

results in the formation of di- and trisaccharides, which interfere

with most methods of lactose measurement.

Megazyme’s Lactose Assay Kit (K-LOLAC) addresses the key

challenges of accurately measuring lactose in low-lactose

samples, greatly reducing the over-reporting that occurs in

other lactose assay kits.

Lactose Thresholds in EU Member States

Labelling regulations vary from country to country, with

many requiring accurate detection of lactose at low levels.

Country “Lactose-free” “Low lactose”

Denmark 10 mg/100 g 1 g/100g

Estonia 10 mg/100 g 1 g/100g

Finland 10 mg/100 g 1 g/100g

Germany 100 mg/100 g N/A

Hungary 100 mg/100 g N/A

Ireland No lactose present

No galactose present

1 g/100g

Norway 10 mg/100 g 1 g/100g

Sweden 10 mg/100 g 1 g/100g

Slovenia 100 mg/100 g N/A

Source: European Food Safety Authority (EFSA), 2010

Current limitations with lactose detection methods 1. HPLC - insufficiently sensitive with refractive index (Figure 2). 2. Ion Chromatography - poor chromatographic baseline patterns. 3. Enzymatic Methods - lack of specificity for lactose measurement in real

samples with high levels of monosaccharides and other by-products produced during β-galactosidase treatment.

Megazyme, Bray Business Park, Bray, Co. Wicklow, Ireland

Lactose Measurement – A New Solution for the Dairy Industry

Lactose (1,4-β-D-galactosyl-D-glucose) is the natural sugar present in milk. Approximately 65% of the global human population is thought to be lactose intolerant as they are lacking or deficient in the enzyme lactase (β-galactosidase), which is required for its breakdown1,2.

To address the prevalence of lactose intolerance, dairy manufacturers have introduced “low-lactose” (< 1 g lactose/100 g) and “lactose-free” (10-100 mg lactose/100 g) dairy products 3, the production of which is outlined in Figure 1.

Megazyme has developed a novel enzymatic assay for the selective and accurate measurement of lactose in conventional, “low-lactose” and “lactose-free” dairy products. This kit is commercially available from Megazyme – K-LOLAC. British Patent Application No. GB1710170.0.

The Megazyme K-LOLAC test kit offers the following advantages over existing commercially available lactose detection methods: 1) Accuracy: Method includes a 15 minute pre-incubation step remove the

high levels of free D-glucose in samples, allowing accurate measurement of lactose in “low-lactose” and “lactose-free” dairy products.

2) Efficiency: World’s first sequential assay for lactose i.e. improves accuracy and efficiency while reducing cost and processing time.

3) Selectivity: MZ104 β-galactosidase hydrolyses lactose more selectively than the industry standard.

4) Sensitivity: Lower detection limit than all other commercially available enzymatic lactose detection.

Helena Culleton, David Mangan, Claudio Cornaggia, Ruth Ivory, Vincent McKie, Tadas Kargelis and Barry V. McCleary

1. High “background” levels of glucose: Megazyme’s K-LOLAC method has optimised an efficient enzymatic pre- treatment step (Figure 3) Improved efficiency and accuracy (Figure 4).

2. β-Galactosidase selectivity Megazyme’s K-LOLAC procedure employs MZ104 β-galactosidase which has higher selectivity than the industry standard (E. coli) (Figure 5).

3. Improvement of detection assay Megazyme’s K-LOLAC format allows for sequential measurement of lactose while having the lowest detection limit on the market i.e. detects lactose at 0.65 mg/L versus 8 mg/L for competitors (Figure 6).

Manufacture of “low-lactose” and “lactose-free” products D-Lactose

D-Galactose

D-Glucose

β-Galactosidase treatment

Hydrolysis Transglycosylation

Allolactose (β-1,6-lactose)

MAJOR PATHWAY MINOR PATHWAY

Large quantities

Trace quantities

Figure 1: Schematic representation of enzymatic reactions and products formed in the preparation of “low lactose” and “lactose-free” dairy products. The final concentration of these transglycosylation products vary considerably from product to product.

Figure 2: Example of a HPAEC-PAD chromatogram for the analysis of a commercially available “lactose free” milk using a gradient elution method on a CarboPac PA-1 column.

1

β-1,4

D-Galactose D-Glucose

β-1,2-lactose

β-1,3-lactose

Galacto-oligosaccharides (GOS) 1

β-1,6

1

β-1,2

1 β-1,3

n

D-Galactose

D-Glucose

1,6-Galactobiose

Allolactose

Lactose

1,4-Galactobiose

β-1,6

1

β-1,6

1

1

β-1,4

β-1,4

1

Untreated Sample Treated Sample

A1 A2

A1 A2

A2 – A1

A2 – A1

Untreated Samples: A2 – A1 is very low compared to A1 which leads to large errors. Treated Samples: A2 – A1 is now substantial compared to A1 which greatly reduces errors.

Two kit formats have been developed by Megazyme: K-LOLAC

contains 6-phosphogluconate dehydrogenase and produces 2 moles of NADPH for each molecule of lactose – Sensitivity x 2 K-LOLACHS

adds an additional reagent (INT/diaphorase) that produces a further 2.5 fold increase in sensitivity – Sensitivity x 5

Principle of novel K-LOLAC lactose assay with 3 key features 1. Efficient sample preparation procedure to remove excess levels of “free”

glucose from sample prior to lactose measurement. 2. Selective hydrolysis of lactose in the presence of other galacto-

oligosaccharides (GOS) and β-D-galactosyl-D-glucose compounds (especially allolactose), with subsequent detection of glucose released.

3. Improved detection assay – increased sensitivity and reduced assay times.

Glucose + O2 + H2O

Gluconolactone + H2O2

Glucose oxidase Catalase

Figure 3: Schematic representation of enzymatic pre-treatment reaction involving glucose oxidase and catalase to clarify and remove the high levels of free D-glucose in “low-lactose” and “lactose-free” samples.

Steps in enzymatic measurement of lactose 1) Measure free glucose in the sample = A1 2) Hydrolyse the lactose present with β-galactosidase 3) Measure free glucose plus glucose released from

lactose = A2 4) Calculate lactose content by subtracting A1 from A2

Figure 4: Effect of glucose removal pre-treatment on accuracy of lactose detection.

0

0.4

0.8

1.2

1.6

2

0 5 10 15 20 25 30

Abso

rban

ce (O

D @

340

nm)

Incubation Time (mins)

Addition of E.coli β-galactosidase

0

0.4

0.8

1.2

1.6

2

0 5 10 15 20 25 30

Incubation Time (mins)

Addition of MZ104 β-galactosidase

Blank

50ug Lactose

50ug Allolactose

Figure 5: Comparison of lactose and allolactose hydrolysis with MZ104 and E.coli β-galactosidase.

Figure 6: Alternative assay format biochemistries

Figure: The array above demonstrates the

difficulty of detecting lactose (yellow marker).

This is a a HPAEC-PAD chromatogram for the

analysis of a commercially available “lactose-

free” milk using a gradient elution method on a

CarboPac PA-I column.

Measurement of Lactose in Lactose-Free

and Low-Lactose Products

Enzymes for Dairy Laboratories

Megazyme is world-renowned for producing extremely pure

enzyme preparations, offering reliable results with every use.

Product

Code

Product Name Pack Size

E-AMGDF Amyloglucosidase (A. niger) - 10 mL 32,600 U

E-AMGPU Amyloglucosidase (Rhizopus sp.) 5000 U

E-ISAMY Isoamylase 600 U

E-ENDOIAN endo-Inulinase (A. niger) 500 U

E-EXOIAN exo-Inulinase (A. niger) 5000 U

E-FRMXLQ Fructanase Mixture (Purified Liquid) 20 mL

E-FRMXPD Fructanase Mixture (Purified Powder) 20,000 U

Why choose our Lactose Assay Kit? The K-LOLAC Difference

Our new Lactose Assay Kit (K-LOLAC) joins our bestselling Lactose/Galactose Assay Kit (K-LACGAR) as part of

the Megazyme range for carbohydrate analysis. The Lactose Assay Kit is specifically designed for manufacturers

testing low-lactose and lactose-free foods and beverages.

K-LACGAR K-LOLAC Competitor Kit

Sequential Assay Format

Uses selective β-galactosidase

Suitable for Standard Dairy Products

Suitable for Low-Lactose and Lactose-Free *

* Lactose overestimation is likely due to unselective β-galactosidase mediated hydrolysis of various transglycosylation products

typically present in “low-lactose” food products.

Measurement of Other Sugars

Product

Code

Product Name No.

Assays

K-MANGL Mannose/Fructose/Glucose Assay Kit 55

K-MASUG Maltose/Sucrose/Glucose Assay Kit 100

K-SUFRG Sucrose/Fructose/Glucose Assay Kit 300

Measurement of Acids

Product

Code

Product Name No.

Assays

K-DLATE D-/L-Lactic Acid Assay Kit 100

K-LATE L-Lactic Acid Assay Kit 50

K-DATE D-Lactic Acid Assay Kit 50

Measurement of Lactose

Product

Code

Product Name No.

Assays

K-LOLAC Lactose Assay Kit

Developed for use with low-lactose

or lactose-free samples.

*Due to sequential measurement, ‘65

assays’ means 65 samples analysed.

65

K-LACGAR Lactose/Galactose Assay Kit

Rapidly and accurately measures

lactose, D-galactose and L-arabinose.

115

Your Trusted Partner for Quality Control

in the Dairy Industry

Find out more or order online at www.megazyme.com