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EDITORIAL Lactose (milk sugar) is a disaccharide of galactose and glucose occurring in milk. It represents approximately 1.5-8% in milk depending on species. Regulation (EC) No. 1169/2011 on provision of food information to consumers includes a mandatory labelling of nutritional values as of 2014 including declaration of carbohydrates and sugars. Enzymatic food testing for determining sugars goes back far into the past century. Food chemistry labs today are still using the various methods for determining sugars, acids and other metabolites. Traditionally, analytics were based on individual measurements that were performed in cuvettes. Thanks to the introduction of the microtiter plate, enzymatic testing can now be simplified and made more economical as well.

Testing for Lactose using a microtiter plate assay Tobias Hein*, Dr. Andrea Klink*, Elisabeth Halbmayr**, Lukas Frank** * ifp Institut für Produktqualität GmbH, Berlin (Germany) **Romer Labs Division Holding GmbH, Tulln (Austria)

In regulation (EU) No. 1169/2011 on the provision of food information, the term “sugar” covers all monosaccharides and disaccharides, except for polyvalent alcohols. Apart from this definition, it is common to speak of sugars even in the case of certain oligosaccharides, such as raffinose - which is composed of sucrose and galactose. However, in the history of food chemistry,



Elisabeth Halbmayr-Jech

non-specific methods were for a long time the only way to deal with the known variety of sugars.

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b) Lactose determination based on the glucose

The analytically relevant property of sugars, for in-

The enzymes’ extraordinary selectivity makes these

stance, was their ability to reduce heavy metals such

methods highly specific, hence permitting extremely

as copper. Hermann Christian von Fehling was the first

sensitive determination of the substance to be deter-

person to publish a method for the quantification of

mined. Enzymatic testing is therefore firmly establis-

sugar in urine [1] based on this property in the mid-

hed in food chemistry today. Common to all methods

19th century. The sample to be analysed was added

is the use of special enzymes that cleave the analyte

to a blue-coloured copper-tartrate complex solution.

and/or are involved in redox reactions, which leads

If reduction of the copper(II) ions to copper(I) ions

to the forming of the actual measurand. This finally

occurred during heating, this resulted in a change of

causes a change of colour or a change of absorbance

colour and reddish-brown precipitation.

in the ultraviolet range.

Sugar determination according to Luff-Schoorl is

Conventional enzymatic measurements are carried

based on a similar principle, while the excess non-

out one after the other in single cuvettes. Since two

reduced copper(II) ions remaining in the assay after

cuvettes are required for each sample, control and

reduction are determined iodometrically [2]. It is true

blank, testing of just three samples consumes 10 cu-

Regardless of whether method a) or b) is used to de-

that the amount of reduced sugar involved in the re-

vettes. This procedure is very labour-intensive and

termine lactose, there are always two assays perfor-

action could be derived from the amount of copper

uses a lot of material, especially with medium to large

med for each sample:

consumed. Yet, since there was no linear connection,

sample throughput, particularly because each sample

1. In the first assay (blue frame) with β-galactosidase,

both Luff-Schoorl and Fehling had to determine the

also requires a separate spatula to stir the reaction

lactose is split into galactose and glucose. Then the

sugar content from a table of empirical values.

assay by hand.

sucrose (being a non-reducing substance), for instance, could not be determined directly, but had to

copied to this file and the final result is automatically

Total glucose (1) Lactose + H2O

β-galactosidase

D-galactose D-glucose

+

Free glucose

(3) G6P + NADP+

hexokinase

EnzymeFast : The principle of microtiter plate based lactose assays ®

the

G6Pdehydrogenase

second

assay

sample weight. Sample preparation

D-glucose-6-phosphate (G6P) + ADP

thod and the lactose/glucose

D-gluconate-6-phosphate + NADPH + H+

rious food matrices and

Measurement A2 at 340 nm

2. In

calculated after entering the dilution factor and the

Although both the lactose/galactose me-

Measurement A1 at 340 nm (2) D-glucose + ATP

This evaluation is automated by the EnzymeFast® Excel Evaluation Sheet. The exported raw data is simply

method are suitable for vagive comparable results, the

total galactose (or glucose) content is determined.

The stated methods covered all reducing sugars (i.e. sugars containing a hemiacetal structure). However,

moiety (EnzymeFast Lactose/D-Glucose): ®

(green

frame)

without

β-galactosidase, only the free galactose (or glucose) content is determined. Both assays are pipetted and measured simultane-

galactose-based

method is usually used for most food matrices. The basic sample preparation method is the same for both methods. 1. Basic sample preparation for various foodstuffs 1 to 5 g (ml) of homogenised sample is extracted in 30 ml water at 60 °C for 15 minutes (sweets, chocolate and confectionery can be extracted at

be split into the reducing monosaccharides glucose

To make enzymatic testing more user-friendly and

ously in a microtiter plate. Photometrical measure-

and fructose by means of acid hydrolysis first.

more economical, ifp Institut für Produktqualität

ment at 340 nm is done in a UV/Vis microtiter plate

transferred the proven methods to a microtiter pla-

photometer (ELISA reader) prior to and after reaction

te based kit format, EnzymeFast®, which is available

2 (or 2 and 3 in the case of the glucose-based me-

world-wide through Romer Labs. The microtiter plate

thod). The NADH that forms can thus be determined

is already contained in each kit, so disposable cuvet-

based on the increase in absorbance (ΔA = A2 – A1

tes and spatulas no longer need to be purchased. Re-

– ΔA of the blank value) and is directly proportional to

action assays are mixed with the pipette tip simply by

the galactose (or glucose) concentration.

drawing solutions in and out during pipetting.

Quantification is done by means of a standard that is

The galactose-based method is also suitable for de-

There are two different methods that can be used to

included in the kit. The analyte’s concentration in the

termining lactose in dairy matrices such as yoghurt

determine the lactose content of a food sample. The

sample can be calculated based on the calibration line

and chocolate etc. However, dairy products that are

basic principle is the same, however, different en-

and the sample dilution factor F:

specifically labelled as “lactose-free”, thus targeting

Traditional enzymatic food testing In routine analysis, these elaborate and unspecific reductometric methods were finally superseded by enzymatic methods, which also permit linear quantification. With the help of specific enzymes, it is possible not only to determine the different sugar types separately, but also to test for a large number of different organic acids and other metabolites. Enzymatic food testing goes back far into the past century. An enzymatic method for the determination of glucose and fructose was known as early as 1961, for example [3]. The method for the determination of lactose in meat products specified in the German Official Collection of Examination Procedures as per § 64 of the German Foodstuffs, Consumer Goods and Animal Feed Code (LFGB), for instance, dates back to 1983 [4]. Today the range of enzymatic methods is vast: from ordinary household sugar (sucrose), to lactose and sulphite - both triggers of food intolerances – right up to substances such as ethanol and citric acid, which are widely used - not only in the beverage

zymatic reactions are available targeting either the molecule’s galactose or glucose moiety, respectively: a) Lactose determination based on the galacto-

(1) Lactose + H2O

β-galactosidase

D-galactose D-glucose

+

Free galactose Measurement A1 at 340 nm (2) D-galactose + NAD+

β-galactosedehydrogenase

D-galactonic acid + NADH + H+

rez clarification. Next, the solution is left to cool to room temperature in order to adjust the pH to 7 – 7.5, and then transferred quantitatively to a 100 ml volumetric flask. After topping up to the 100 ml mark, the entire extract is filtered through a pleated filter and diluted if necessary.

the lactose-intolerant consumer group, are usually Galactose (or glucose) content [g /100 g (ml)] =

treated enzymatically during processing in order to

(ΔA sample – y-intercept) × F

eliminate their natural lactose contents. As a result,

slope × weighed sample [g (ml)]

these products contain only minimal amounts of lac-

se moiety (EnzymeFast® Lactose/D-Galactose): Total galactose

80-100 °C for 5 minutes instead), followed by Car-

tose (< 0.1 g/100 g [ml]), but excess concentrations The galactose (or glucose) that is bound in lacto-

of its cleavage products galactose and glucose. Since

se can be derived from the difference between the

it is very difficult to determine the small galactose

two assays for total and free galactose (or glucose).

amounts bound in the residual lactose in the pre-

Based on its molecular weight, the bound galactose

sence of high concentrations of free galactose, the

(or glucose) is then converted to lactose in g/100 g

results achieved with the galactose-based assay are

(ml) by the multiplication factor 1.9.

not very reliable.

Measurement A2 at 340 nm

industry.

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Instead, EnzymeFast® Lactose/D-Glucose is the me-

If this sample preparation method is used in conjunc-

thod of choice in these cases. Unlike with galactose, it

tion with EnzymeFast® Lactose/D-Glucose, a factor 10

is possible to remove excess glucose from the sample

has to be taken into account during evaluation due to

as shown below. That way, prior to the analysis, the

the lactose determination.

the sample preparation in 10 ml instead of 100 ml.

Target concentration [mg / L]

Validation (excerpt)

Lactose/ D-Galactose (n=3) Lactose/ D-Glucose (n=6)

To determine the rates of recovery at low lactose con2. Sample preparation for lactose-free dairy

Spike recovery in water (lactose monohydrate) using the galactose-based and the glucose-based assay EnzymeFast® kit

sample’s high glucose content can be set back to a low level that does not have a negative influence on

EnzymeFast® Lactose/D-Glucose was used to analy-

Table 1

centrations, different spiking experiments were car-

Recovery

CV [%]

se dairy matrices whose natural lactose contents had been cleaved enzymatically during processing and which were labelled as “lactose-free”. The results are

[mg / L]

[%]

2.0

1.8

90.9

14.2

4.0

3.9

97.7

9.9

2.0

2.1

106.2

15.8

tioned before. The spiking level of 0.1 g/100 g (ml)

4.0

3.7

93.1

7.2

or 1000 ppm corresponds to the threshold that is ty-

shown in Table 3. Unless noted otherwise, triplicate sample portions of 0.5 g each were spiked and extracted using glucose oxidase and catalase as men-

pically used as the maximum lactose concentration

products

ried out. First, 100 µl of a lactose monohydrate solu-

In order to prepare a lactose-free dairy sample, 0.5

tion of 2 and 4 mg / L, respectively, were directly used

A second series of experiments was carried out to

in enzymatically treated dairy products labelled as

to 1 g (ml) of sample are pre-treated with trietha-

in both the galactose-based and the glucose-based

determine the recovery of low lactose monohydrate

“lactose-free”.

nolamine buffer, magnesium sulphate and an en-

assay with no further sample preparation or dilution.

spikes in lactose-free sample matrices. As shown in Ta-

The data show that all lactose monohydrate spikes

zyme mix containing glucose oxidase and catalase.

The data in Table 1 show that both EnzymeFast® kits

ble 2, EnzymeFast Lactose/D-Galactose was used on

are detected at recovery rates of at least > 86 % (ty-

During a 1 hour incubation step at 30 °C the free

are suitable to detect lactose monohydrate at such

a variety of sample matrices that were known to con-

pically > 90 %) and with variation coefficients of well

glucose present in the sample is thus converted

concentrations with recovery rates of 90-110 %. This

tain no significant amounts of lactose. This also inclu-

below 10 % in a variety of lactose-free dairy matrices.

to gluconate. This initial sample preparation step

is equivalent to a total amount of 200 ng lactose mo-

des two dairy products, gouda and parmesan cheese,

The results also demonstrate that the enzymatic clea-

is followed by a 15 min incubation step at 95 °C,

nohydrate, 190 ng lactose or 100 ng galactose (or

whose natural lactose contents are eliminated micro-

vage of lactose during the manufacture and proces-

Carrez clarification and pH adjustment. After top-

glucose) in the assay.

bially in the course of their ripening process. Unless

sing of dairy products has a limited efficiency; hence

ping up to 10 ml, the extract is filtered through a

noted otherwise, triplicate sample portions of 5 g each

the results of the original samples, analysed unspiked,

pleated filter (or centrifuged instead).

were spiked and extracted according to the kit insert.

range from approx. 15 to 60 mg/100 g (ml).

®

The data show that minimal amounts of lactose monohydrate are detectable at recovery rates of typically > 80 % (for samples spiked with 10 mg/100 g [ml]) and > 90 % (for samples spiked with 20 mg/100 g [ml]).

Table 2

Table 3

EnzymeFast® Lactose/D-Galactose: Spike recovery in various food matrices (lactose monohydrate; n=3) Sample matrix

Spike target concentration [mg / 100 g]

Assorted sliced roast pork, lactose-free Vienna sausage, poultry

CV [%]

[mg / 100 g]

[%]

unspiked

1.4

-

-

-

10.0

11.0

16.4

9.6

95.7

unspiked

0.3

-

-

-

10.0

8.5

18.3

8.2

81.7

20.0

19.9

6.1

19.6

97.8

unspiked (n=4)

1.2

-

-

-

10.0

9.8

16.6

8.6

85.7

Cooked ham

Salami, lactose-free

Gouda cheese, lactose-free

Parmesan cheese1

Bread, lactose-free

Muesli, lactose-free 1

Actual concentration [mg / 100 g]

EnzymeFast® Lactose/D-Glucose: Spike recovery in lactose-free dairy matrices (lactose monohydrate; n=3)

Recovery

20.0

20.6

10.0

19.4

97.0

unspiked

1.3

-

-

-

10.0

10.1

4.0

8.8

87.7

20.0

20.6

1.0

19.2

96.2

unspiked

0.1

-

-

-

10.0

9.9

9.7

9.7

97.4

20.0

18.5

6.6

18.3

91.7

50.0

49.6

3.4

49.5

99.0

unspiked

0.0

-

-

-

10.0

8.5

10.8

8.5

85.0

20.0

18.4

8.5

18.4

92.2

unspiked

1.7

-

-

-

20.0

25.2

13.7

23.5

117.5 97.7

50.0

50.6

0.8

48.9

unspiked

2.1

-

-

-

10.0

11.9

15.7

9.7

97.3

20.0

21.4

3.1

19.2

96.2

Actual concentration [mg / 100 g]

CV [%]

[mg / 100 g]

Strawberry yoghurt, lactose-free (n=2)

unspiked

34.7

10.1

-

-

100.7

128.2

2.5

93.6

92.8

Vanilla yoghurt, lactose-free

unspiked

25.3

5.5

-

-

118.7

2.6

93.4

92.8

Whole milk, 3.5 % fat, lactose-free

unspiked

Double cream cheese, lactose-free

unspiked

11.7

-

-

99.3

106.5

5.9

91.8

92.5

Cottage cheese, 20 % fat in dry matter, lactose-free (n=2)

unspiked

15.2

3.6

-

-

98.7

105.6

0.8

90.3

91.5

Mozzarella, lactose-free

unspiked

Greek brined curd cheese, lactose-free (n=2)

unspiked

Curd, low-fat, lactose-free (n=2)

unspiked

Milk chocolate with nuts, lactose-free

unspiked

100.6 100.5

96.5 105.2 100.7 101.4

61.3 159.5 14.7

24.7

[%]

10.3

-

-

2.6

98.2

97.7

14.6

-

-

115.3

6.3

90.6

93.9

22.2

4.4

-

-

115.2

3.4

93.1

88.4

15.8

8.0

-

-

107.1

1.4

91.3

90.7

60.9

2.8

-

-

148.7

4.4

87.7

86.6

Weighed sample portion 10 g, extraction at 100 °C

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Recovery

Spike target concentration [mg / 100 g]

Sample matrix

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Summary Both lactose methods have shown to give reliable re-

• no more individual measurements in single cuvettes

sults even at low concentration levels. The galactose-

• simultaneous measurement of up to 90 samples plus blank value, standards and control

based kit is suitable for routine lactose testing in all kinds of foods. The analytical challenges posed by en-

• uniform pipetting volumes for blank values, stan-

zymatically treated, lactose-free dairy products are

dards, controls and samples

met by a specific sample preparation method com-

• minimal material consumption

bined with the glucose-based kit format. Food ma-

• reduced workload

nufacturers and testing labs thus have a selection of

• use of multi-channel pipettes possible

useful and precise methods at hand for the determi-

• microtiter plate photometer also suitable for ELISAs

nation of lactose in a broad range of sample matrices. From a practical and economical point of view, there are various advantages of the microtiter plate kit format as compared to conventional cuvette methods: EnzymeFast® Test Kits available:

Bibliography

Item No.

Product

No. of Reactions

COKEF0100

EnzymeFast Lactose/D-Galactose

140

COKEF0200

EnzymeFast® Sucrose/D-Glucose/D-Fructose

140

COKEF0300

EnzymeFast® Sucrose/D-Glucose

140

COKEF0400

EnzymeFast® D-Glucose/D-Fructose

COKEF0700

EnzymeFast Lactose/D-Glucose

®

®

[1] Fehling, H.C. (1848) Quantitative Bestimmung des Zuckers im Harn, Archiv für physiologische Heilkunde, 7, 64-73. [2] BVL L 31.00-11 (1984): Bestimmung des Zuckergehaltes vor und nach Inversion in Fruchtsäften (Luff-Schoorl-

70

Methode), 11-1983, Amtliche Sammlung von Untersuchungsverfahren nach § 64 LFGB (in the German Offical

140

Collection of Examination Procedures (ASU) as specified in § 64 of the German Foodstuffs, Consumer Goods and Animal Feed Code (LFGB)).

Other parameters, such as Raffinose/D-Galactose, Maltose/Sucrose/D-Glucose, D-sorbitol / xylitol and various organic acids, are currently in the development stage.

[3] Schmidt, F.H. (1961) Die enzymatische Bestimmung von Glucose und Fructose nebeneinander, Klinische Wochenschrift 39, 1244-1247. [4] BVL L 07.00-23 (1983): Bestimmung von Lactose in Fleischerzeugnissen, 05-1983, Amtliche Sammlung von

In cooperation with IFP

Untersuchungsverfahren nach § 64 LFGB (in the German Offical Collection of Examination Procedures (ASU) as specified in § 64 of the German Foodstuffs, Consumer Goods and Animal Feed Code (LFGB)). [5] BVL L 07.00-24 (1983): Bestimmung von Sucrose in Fleischerzeugnissen, 05-1983, Amtliche Sammlung von Untersuchungsverfahren nach § 64 LFGB (in the German Offical Collection of Examination Procedures (ASU) as specified in § 64 of the German Foodstuffs, Consumer Goods and Animal Feed Code (LFGB)).

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ABOUT THE AUTHOR Name

Tobias Hein

Position

Productmanagement and Marketing at ifp Institut für Produktqualität

Education

Tobias Hein studied biotechnology at the University of Cooperative Education Riesa. After achieving his diploma degree for a thesis dealing with the quantification of genetically modified maize, he worked for a company focused on the production of molecular biological detection systems for the food industry.

Address

ifp Institut für Produktqualität GmbH, Teltowkanalstraße 2, 12247 Berlin, Germany, Phone +49 (0)30 / 76 68 60 – 0; Fax +49 (0)30 / 76 68 60 – 50; email: hein@produktqualitaet.com

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