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DETERMINING THE MILK CONTENT OF MILK-BASED FOOD PRODUCTS.

FSA Final Report Q01117.

Campden BRI Project 98164.

John J. Dooley, Piotr Jasionowicz, Brian Burch, Sophie Wellum & Helen M. Brown.

Executive Summary

The Food Standards Agency (FSA) has recently been focussing on methods that utilise

lab-on-a-chip capillary electrophoresis, which is a low-cost, easy-to-use technique that

enables profiling of analytes including protein and DNA. Such systems, for example the

Agilent 2100 Bioanalyzer, are ideally suited to authenticity applications and have already

been adopted by local government laboratories for DNA-based analysis of food.

Additional methods that can be applied using this platform will allow these laboratories

to widen their testing remit and utilise the full potential of lab-on-a-chip analysis. As

capillary electrophoresis techniques have been applied successfully for the quantitative

determination of milk proteins, the aim of the current work was to assess the feasibility

of transferring capillary electrophoresis-based methods to the simple lab-on-chip

platform to assess whether they could be used to determine whether composite food

products contain 50% or more of processed animal product, using milk as an example.

A protein profiling technique was evaluated for determining milk protein in food

products using the Agilent 2100 Bioanalyzer with the Protein 80 LabChip. Protein

separation on the 2100 Bioanalyzer can be achieved using one of two protein LabChips,

the Protein 80 or the Protein 230. For milk proteins, whilst profiles could be generated

on the Protein 230 LabChip, better resolution and sensitivity was achieved with the

Protein 80 LabChip and there was less interference from system peaks on this LabChip.

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Proteins were extracted under reducing conditions using a total protein solubilisation

(TPS) buffer. Alternative extraction methods were investigated but these caused

interference with protein separation on the LabChip.

Quantification of 1000µg/ml solutions of milk proteins prepared from commercial α-

casein, β-casein, κ-casein, α-lactalbumin and β-lactoglobulin gave recoveries of 103-

113% when calibrated against the equivalent protein standard curve. For quantification

of the amount of milk in milk-based products, β-casein (100-1500µg/ml) and α-

lactalbumin (50-300µg/ml) were selected and used in a mixed standard solution

(containing both proteins) to produce standard curves. Best results were achieved when

electropherograms were analysed manually rather than using the auto-analyse function

of the 2100 Bioanalyzer software. Manual peak analysis was required to correctly

identify some peaks or to define peaks which were not fully resolved.

When the method was applied to relatively complex mixtures of proteins (milk proteins

mixed with wheat flour or egg powder), milk proteins were not resolved from other

proteins, indicating that the method is only likely to be suitable for simple milk-based

products. Therefore method validation studies were performed using rice puddings and

body building powder (weight gain formula). These were produced at Campden BRI so

that the milk content was known on the basis of known weights of ingredients and by

compositional analysis of ingredients. These were compared with results generated

using the Bioanalyzer method.

Rice puddings containing 75%, 50% and 25% milk were produced using whole milk, semi-

skimmed milk, skimmed milk and Lactofree semi-skimmed milk. The milk content

calculated using compositional analysis and recipe data agreed with the actual milk

content in all cases except for rice puddings containing 75% whole milk, 75% Lactofree

semi-skimmed milk and 25% Lactofree semi-skimmed milk, where the calculated results

were 72%, 73% and 26%, respectively. The milk content determined using the

Bioanalyzer method was very variable (relative standard deviation of up to 60%) and in

all cases underestimated the milk content - determining between 30% and 80% of the

actual milk content. The lack of precision and accuracy was due to the unpredictable

performance of the upper marker used as an internal standard for quantification of

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proteins on the LabChips and to the poor resolution of the β-casein peak from α-casein.

Rice puddings were sampled by taking liquor only to avoid including rice proteins - this

may also have contributed to the lack of accuracy but was anticipated to result in

overestimation of milk content rather than underestimation.

For the body building powder, the protein profile generated using the Bioanalyzer

showed the presence of whey proteins α-lactalbumin and β-lactoglobulin and only

traces of casein, showing that for this sample it was more relevant to quantify whey

protein than milk content. Quantification using the Bioanalyzer method was based on

using α-lactalbumin as the calibrant. The high α-lactalbumin content of the body

building powder required that the sample be tested using 2mg body building powder/ml

solubilisation buffer to ensure that the protein concentration fell within the range of the

standard curve appropriate for the Bioanalyzer. The measured α-lactalbumin content

and total whey protein content calculated from it indicated that this product contained

less than 50% whey protein. However, on the basis of the known weight of ingredients

and their composition, the body building powder contained 74.25% whey protein and,

on the basis of compositional analysis, 74.9% protein. The underestimate when using

the LabChip method may be due to a lack of linearity at the high whey protein

concentrations in the body building powder and/or due to the proportion of α-

lactalbumin in whey isolates and concentrates differing from that found in milk, due to

separation and manufacturing technologies employed in the preparation of whey

proteins.

In conclusion, the validation study showed that the lack of precision and accuracy of the

method in its current form makes it unfit for estimating whether the milk content of

even simple milk-based products exceeds 50%. For very simple milk protein based

products, it may be possible to use the current LabChip technology as a relatively simple,

rapid screening method to indicate the presence of a biased peak ratio, i.e. very large α-

lactalbumin peak compared to β-casein peak, for detecting samples that have been

bulked with added whey protein or other milk protein(s), but the LabChip technology in

its current form is unsuitable for estimating the milk content of unknown samples to

determine whether they contain 50% or more milk.

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TABLE OF CONTENTS

TABLE OF CONTENTS ............................................................................................................................... IV

GLOSSARY ................................................................................................................................................ V

ABBREVIATIONS ...................................................................................................................................... VI

1. INTRODUCTION ............................................................................................................................... 1

2. MATERIALS AND METHODS ............................................................................................................. 2

2.1. CHEMICALS AND REAGENTS ........................................................................................................... 2

2.2. PREPARATION OF SAMPLES ........................................................................................................... 2

2.3 PROTEIN EXTRACTION .................................................................................................................. 3

2.4 PREPARATION OF MILK PROTEIN STANDARDS ................................................................................... 3

2.4.1 Preparation of milk protein stock solutions ................................................................................. 3

2.4.2 Preparation of F-milk protein standards ...................................................................................... 5

2.4.3 Preparation of T-milk protein standards ...................................................................................... 5

2.5 ANALYSIS OF SAMPLES TO DETERMINE MILK CONTENT ON THE 2100 BIOANALYZER ............................... 6

2.5.1 Sample denaturation and dilution ............................................................................................... 6

2.5.2 Separation of protein on the LabChip and data analysis ............................................................. 7

2.5.3 Calculation of the milk content .................................................................................................... 8

2.6 COMPOSITIONAL ANALYSIS ........................................................................................................... 8

3 RESULTS AND DISCUSSION ............................................................................................................... 9

3.1. METHOD DEVELOPMENT .............................................................................................................. 9

3.1.1. Modified De Jong extraction buffer ........................................................................................... 10

3.1.2. Total protein solubilisation buffer (TPS buffer) .......................................................................... 10

3.1.3 BMR extraction buffer ................................................................................................................ 11

3.1.4 Protein molecular weights ......................................................................................................... 12

3.2. QUANTIFICATION OF MILK PROTEINS ............................................................................................ 13

3.2.1 Composition of milk and milk protein ........................................................................................ 13

3.2.2 Quantification of milk proteins using the Bioanalyzer ............................................................... 14

3.3. ANALYSIS OF MILK..................................................................................................................... 16

3.4 IN-HOUSE VALIDATION USING MILK PRODUCTS PREPARED AT CAMPDEN BRI ...................................... 17

3.4.1. Production of rice puddings ....................................................................................................... 17

3.4.2. Calculation of milk content from compositional analysis of rice puddings ................................ 18

3.4.3. LabChip analysis of rice puddings and calculation of milk content ............................................ 20

3.4.4. LabChip analysis of body building powder ................................................................................. 25

4 CONCLUSIONS ................................................................................................................................ 27

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5 AREAS FOR FUTURE INVESTIGATION.............................................................................................. 30

6 ACKNOWLEDGEMENTS .................................................................................................................. 30

7 REFERENCES ................................................................................................................................... 31

8 APPENDICES ................................................................................................................................... 33

APPENDIX 8.1 PREPARATION OF SAMPLES AT CAMPDEN BRI ................................................................................... 33

8.1.1 Preparation of canned rice puddings .......................................................................................... 33

8.1.2 Preparation of a body building powder ..................................................................................... 34

APPENDIX 8.2 TYPICAL RESULTS OBTAINED USING THE PROTEIN 80 LABCHIP .............................................................. 36

APPENDIX 8.3 EXAMPLE CALCULATION OF THE MILK CONTENT OF RICE PUDDING ....................................................... 40

APPENDIX 8.4 CALCULATION OF WHEY PROTEIN IN BODY BUILDING POWDER ............................................................ 41

APPENDIX 8.5 STANDARD OPERATING PROCEDURE ............................................................................................... 42

9. FIGURES ......................................................................................................................................... 64

GLOSSARY

2100 Bioanalyzer: A small-scale capillary electrophoretic system using lab-on-a-chip

technology and microfluidics for the specific separation of DNA fragments or proteins.

Capillary electrophoresis (CE): Electrophoresis in narrow bore capillaries, normally 25-

100µm internal diameter. Electrophoresis may be in free solution, i.e. capillary zone

electrophoresis (CZE); in a sieving matrix, i.e. capillary gel electrophoresis (CGE); or in a

partitioning matrix, i.e. micellar electrokinetic chromatography (MEKC).

Fluorescent Units (FU): A measure of fluorescence intensity used by the 2100

Bioanalyzer.

Electrophoresis: Differential movement or migration of ions by attraction or repulsion in

an electric field.

Gel electrophoresis: Electrophoresis performed in a gel of acrylamide or agar. Proteins

migrate through the gel when an electric current is applied. The gel matrix acts as a

sieve to separate the proteins based on size and charge.

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Protein LabChip: A small (3cm2), disposable, single-use plastic and glass unit containing

etched capillaries attached directly to ten sample loading wells. Two protein LabChips

are available, the Protein 80 LabChip for separating fragments of 5-80kDa and the

Protein 230 LabChip for separating 14-230kDa fragments.

ABBREVIATIONS

AA - amino acid

α-Lg - α-lactoglobulin

β-Lg - β-lactoglobulin

BBP - body building powder

BMR - a solubilisation buffer prepared from phosphate buffered saline (PBS) buffer,

pH7.4, containing 8M urea

CE - capillary electrophoresis

CZE - capillary zone electrophoresis

DL-DTT - dithiothreitol

FSA - Food Standards Agency

PBS - phosphate buffered saline

QUID - quantitative declaration of ingredients

SDS - sodium dodecyl sulphate

TPS buffer - total protein solubilisation buffer

WPC - whey protein concentrate

WPI - whey protein isolate

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1. INTRODUCTION

The ability to quantify the level of ingredients in composite food products provides a

means of monitoring products for consumer protection and regulatory compliance. At

the time of commissioning this work, changes to the European Commission Regulation

covering veterinary checks on imported products of animal origin were expected. It was

anticipated that they would stipulate checks to be carried out on composite foods

imported from third countries that contain 50% or more processed animal product

(other than meat, for which separate controls exist) by weight of the food. The FSA, in

its role of protecting consumers by effective enforcement of food legislation, identified

the need for reliable methods to identify foods that would require veterinary checks, as

there is no existing simple, rapid test to determine the level of animal-based ingredients

in composite foods.

The Food Standards Agency (FSA) has supported the development of easy-to-use

approaches for analysing and quantifying food ingredients. Lab-on-a-chip capillary

electrophoresis (CE), using the Agilent 2100 Bioanalyzer, offers considerable advantages

over standard gel electrophoresis for protein analysis. The Agilent 2100 Bioanalyzer is

available with a choice of two Protein LabChips, the Protein 80 LabChip or the Protein

230 LabChip. The Protein 80 LabChip is designed for sizing and analysis of proteins from

5-80kDa and the Protein 230 LabChip is designed for sizing and analysis of proteins from

14-230kDa. The relatively low cost and ease of use of the Bioanalyzer for DNA analysis

has led to the adoption of this system by a number of government enforcement and

private laboratories. The aim of the current study was to assess the feasibility of

transferring existing CE-based methods for the quantitative determination of milk

proteins to this simple lab-on-a-chip, CE platform and to assess whether it could be used

to determine whether composite food products contain 50% or more of processed

animal product. Milk was chosen as an example of an animal-based ingredient to assess

the feasibility of this approach.

Quantitative analysis of specific analytes in food products relies upon obtaining reliable

standards against which the analyte in the unknowns can be measured. When the

analyte is 'milk' this is complicated by: the number of different types of milk available for

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use in food products (e.g. whole milk, skimmed milk, dried milk, whey powder), the type

of processing applied to the milk (e.g. UHT, pasteurised), and variation in the

composition of milk that occurs due to other factors such as cattle genotype, season or

lactation[1-5]. Milk is an aqueous solution of casein (αs-casein, β-casein, κ-casein, γ-

casein) and whey proteins (lactalbumin, lactoglobulin, immunoglobulins). The

proportions of the different proteins can vary considerably. By using CE to generate a

profile of milk proteins, it was envisaged that the profile would provide an initial screen

to identify significant variations in milk protein composition, for example inclusion of

whey protein, and allow for quantification of 'milk' on the basis of one or more

appropriate proteins.

In this work, the method was initially optimised and then validated in-house using

composite food products containing 25%, 50% and 75% milk prepared at Campden BRI.

These products were analysed using the CE method and traditional methods of

compositional analysis. This allowed the milk content determined from quantities of

ingredients, compositional analysis and CE analysis to be compared.

2. MATERIALS AND METHODS

2.1. CHEMICALS AND REAGENTS All chemicals were obtained from Sigma-Aldrich (Poole, Dorset, UK), unless otherwise

stated. Protein profiles were generated using Protein LabChips and specific reagents

from Protein 80 (P/N 5067-1515) or Protein 230 (P/N 5067-1517) kits and the Agilent

2100 Bioanalyzer (Agilent Technologies UK Ltd, Stockport, Cheshire, UK).

2.2. PREPARATION OF SAMPLES Samples were prepared in-house at Campden BRI (Table 1) using pilot scale equivalents

to normal commercial processing equipment (Appendix 8.1) or were purchased from

local retailers.

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Table 1: Samples prepared at Campden BRI.

Sample Description

Canned rice pudding made with 75%, 50% or 25% whole milk

Canned rice pudding made with 75%, 50% or 25% semi-skimmed milk

Canned rice pudding made with 75%, 50% or 25% skimmed milk

Canned rice pudding made with 75%, 50% or 25% Lactofree semi-skimmed milk

Body building powder (high protein weight gain powder)

2.3 PROTEIN EXTRACTION Prior to extraction, samples were stirred or shaken thoroughly to ensure sample

homogeneity. In order that proteins were reduced and that protein denaturation took

place under reducing conditions, samples were extracted in a total protein solubilisation

buffer (TPS buffer) that contained DL-dithiothreitol (DL-DTT). All samples and standards

were extracted using a total protein extraction protocol.

Samples (up to 1,000mg) were weighed into clean glass bijou bottles. The exact weight

of each sample was noted. The minimum volume of TPS buffer (2M urea, 15 % glycerol,

0.1 M DL-DTT and 0.1 M Tris-HCl, pH 8.8) required to solubilise the sample was added to

the bijou bottle. Liquid samples were mixed thoroughly with TPS buffer. Solid samples

were dissolved in TPS buffer by gentle shaking or agitating the sample using a Spiramix5

mixer (Thermo Fisher Scientific). The samples were then centrifuged in an Eppendorf

centrifuge (5415D) at 2,000g for 15 minutes to remove particulate material from the

supernatant. Aliquots (4µl) of the supernatant were denatured prior to analysis on the

2100 Bioanalyzer.

2.4 PREPARATION OF MILK PROTEIN STANDARDS

2.4.1 PREPARATION OF MILK PROTEIN STOCK SOLUTIONS

Individual milk protein powders (Table 2) were weighed into separate glass bijou bottles.

The exact weight of protein taken was noted. Exact volumes of TPS buffer were added

to achieve milk protein stock solutions of 1,000µg/ml, 5,000µg/ml or 10,000µg/ml (or 1,

5 or 10mg/ml, respectively) (Table 3). Bijou bottles of stock solution were placed on a

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Spiramix5 mixer for 1 hour to ensure that the proteins were fully hydrated, well mixed

and homogeneous.

Table 2: Milk proteins evaluated for use as standards.

Milk Protein Purity by PAGE Source

β-lactoglobulin 94% as b-Lg A and b-Lg B Sigma L0130 Lot 095K7006

α-lactalbumin, from bovine milk Type III, calcium depleted.

98% Sigma L6010 Lot 035K7005

α-casein, from bovine milk >70% as as-casein

90% Sigma C6780 Lot 075K7425

β-casein, from bovine milk >90% as beta-casein

99% Sigma C6905 Lot 105K7410

κ-casein Minimum 70% κ-casein Sigma C0406 Lot 051K7575

After 1 hour, 500µl of the 5,000µg/ml β-casein solution was mixed with 750µl of TPS

buffer in a clean glass bijou bottle to achieve a 2,000µg/ml stock solution of β-casein.

The 2,000µg/ml β-casein stock solution was mixed on a Spiramix5 mixer for 30 minutes

to achieve solution homogeneity.

Table 3: Preparation of milk protein stock solutions.

Milk protein Approximate weight taken

(mg)¹

Approximate volume of TPS buffer (ml)²

Protein stock concentration

(µg/ml)

Protein stock concentration

(mg/ml)

α-casein 20 2 10,000 10

β-casein 20 2 10,000 10

β-casein 10 2 5,000 5

κ-casein 20 2 10,000 10

α-lactalbumin 20 2 10,000 10

α-lactalbumin 5 5 1,000 1

β-lactoglobulin 20 2 10,000 10

¹ The exact weight of protein taken was noted. ² The actual volume of buffer added was dependent on the actual weight of protein taken and the desired final concentration.

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2.4.2 PREPARATION OF F-MILK PROTEIN STANDARDS

A milk protein standard, referred to as F-milk protein standard, containing each of the

five milk proteins at 2,000µg/ml, was prepared from the 10,000µg/ml milk protein stock

solutions. Aliquots (400µl) of the individual milk protein stock solutions were mixed in a

clean glass bijou bottle. The 2,000µg/ml F-milk protein standard solution was mixed on

a Spiramix5 for 30-60 minutes to ensure that solution homogeneity was achieved. A

series of F-milk protein standards at 1,500; 1,000; 500 and 250µg/ml was prepared by

diluting the 2,000µg/ml F-milk protein standard solution using TPS buffer as detailed in

Table 4. The F-milk protein standards were mixed on a Spiramix5 mixer for 30-60

minutes to ensure homogeneity

Table 4: Preparation of the F-milk protein standards.

Final concentration of F-milk protein standard (µg/ml)

Volume of 2,000µg/ml total milk protein standard (µl)

Volume of TPS buffer (µl)

Final volume (µl)

2,000 375 ~ 375

1,500 750 250 1,000

1,000 500 500 1,000

500 250 750 1,000

250 125 875 1,000

2.4.3 PREPARATION OF T-MILK PROTEIN STANDARDS

A series of milk protein standards, referred to as T-milk protein standards, was prepared

in bijou bottles, from the α-lactalbumin and β-casein stock solutions (Table 5). Where

necessary, T-milk protein standards were diluted using TPS buffer. T-milk protein

standards were mixed on a Spiramix5 mixer for 30-60 minutes to ensure that the

standards were homogeneous. The final volume and concentration of each milk protein

in the T-milk protein standards are shown in Table 6.

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Table 5: Volumes of stock milk protein solution used to prepare the T-milk protein standards.

T-milk protein standard

Volume of each stock solution (µl) Final volume (µl)

5,000µg/ml β-casein stock

2,000µg/ml β-casein stock

1,000µg/ml α-lactalbumin stock

TPS buffer

Standard 1 300 - 300 400 1000

Standard 2 - 500 200 300 1000

Standard 3 - 250 150 600 1000

Standard 4 - 125 100 775 1000

Standard 5 - 50 50 900 1000

Table 6: Final concentration of α-lactalbumin and β-casein proteins in the T-milk protein standards.

T-milk protein standard

Final concentration of β-casein (µg/ml)

Final concentration of α-lactalbumin (µg/ml)

Standard 1 1,500 300

Standard 2 1,000 200

Standard 3 500 150

Standard 4 250 100

Standard 5 100 50

2.5 ANALYSIS OF SAMPLES TO DETERMINE MILK CONTENT ON THE 2100

BIOANALYZER

2.5.1 SAMPLE DENATURATION AND DILUTION

All samples and protein standards, used to generate standard curves, were denatured

using denaturing solution supplied in the Agilent Protein 80 reagent kit. An aliquot (4µl)

of the sample or standard was combined with 2μl of denaturing solution in a 0.5ml

microtube. The lids were closed and secured, using a tube cap-lock, to prevent them

opening during heating. The tubes were boiled at 95°C for 5 minutes using a water bath.

In addition to the samples, a 6μl aliquot of the Protein 80 ladder was heated in a 0.5ml

microtube at 95°C for 5 minutes. The ladder was heated with the first batch of samples

and was used for the analysis of all LabChips analysed on the day of heating.

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After boiling for 5 minutes, the tubes were left to cool to room temperature before they

were centrifuged at 16,000g for 15 seconds to recover the solution in the bottom of the

tube. The boiled solutions (samples, standards and ladder) were mixed with 84μl of

ultrapure water, by vortexing, before the tubes were centrifuged at 16,000g for 15

seconds to recover the solution ready for loading onto the Protein 80 LabChip. All

sample extracts, stock solutions, standards and the ladder were used within one day and

were stored on ice when not in use.

2.5.2 SEPARATION OF PROTEIN ON THE LABCHIP AND DATA ANALYSIS

Protein profiles were generated on the 2100 Bioanalyzer according to the instructions in

the Agilent Protein 80 Kit Guide. Denatured sample solution or ladder (6µl) was added

to sample or ladder wells on the LabChip. CE was started within two minutes of

preparing the LabChip. Separation conditions are fixed and pre-determined by the

Protein chip assay for the Bioanalyzer. Data analysis was performed using the 'Protein

80 Series II Assay' of the Agilent 2100 Expert (Version B.02.03) software. The 2100

Expert software was run using the absolute quantification method with manual

integration to ensure that protein peaks were detected and correctly assigned. The

concentration of either α-lactalbumin or β-casein was determined using the appropriate

standard curve.

The Agilent software does not have a facility to allow data analysis to be performed

using two standard curves simultaneously. In order to perform analysis of the sample

data, it was therefore necessary to complete the analysis using the α-lactalbumin

protein standards and then repeat the analysis using the β-casein protein standards.

This required resetting all the values for the protein standard concentrations and in

some cases manually selecting the correct protein peaks in the profiles. In addition,

during the analysis of some samples both the α-casein and β-casein peaks were not fully

resolved. Despite using manual integration to define the two protein peaks, precise

measurement of the β-casein peak was compromised, making a significant contribution

to the overall uncertainty of the analysis.

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2.5.3 CALCULATION OF THE MILK CONTENT

The 2100 Bioanalyzer was used to determine the concentration of specific milk protein,

either α-lactalbumin or β-casein, in the extracted solubilised sample applied to the

LabChip. The following calculations were used to convert this into "milk content":

The concentration (mg/ml) of milk protein (α-lactalbumin or β-casein) in the sample (A)

was calculated using the following formula:

1000

DFPA

where

'A' is the concentration of either α-lactalbumin or β-casein in the sample

'P' is the concentration of protein (α-lactalbumin or β-casein) in the sample going

onto the LabChip (µg/ml) and

'DF' is the dilution factor prior to denaturation, i.e. at extraction and

solubilisation.

The percentage "milk content" of the sample was calculated:

%100CF

A

where

'A' is the concentration of either α-lactalbumin or β-casein in the sample

'CF' is a factor derived from the concentration of either α-lactalbumin or β-casein

in 100% whole milk, i.e. an 'α-lactalbumin factor' or a ' β-casein factor'.

(Note this is the same principle as a nitrogen factor used for determining the protein content

from analysis of organic nitrogen in proteins).

2.6 COMPOSITIONAL ANALYSIS Compositional analysis was performed at Campden BRI on the rice puddings, the body

building powder and the ingredients used for their preparation. All methods used are

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accredited by the United Kingdom Accreditation Service (UKAS) and are summarised in

Table 7.

Table 7: Methods applied to determine the compositional analysis of ingredients and products used in this study.

Method code

Method name

TES-AC-086 Determination of Total Ash in Foodstuffs

TES-AC-087 Determination of Organic Nitrogen (and Protein by Calculation) in Foodstuffs by Automated 'Kjeltec' System

TES-AC-097 Determination of Moisture in Foodstuffs

TES-AC-202 Determination of Fat in Foodstuffs - Rose Gottlieb Extraction

TES-AC-335 Method for the Calculation of the Energy Content of Foods from Nutritional Data

TES-AC-536 Determination of Total Fat Content by Weibull Stoldt (Acid Hydrolysis followed by Soxtec Extraction) and Crude Fat (Soxtec Extraction)

TES-AC-628 Determination of Moisture and Fat Content in Foods using the CEM Smart Trac System

3 RESULTS AND DISCUSSION

3.1 METHOD DEVELOPMENT In order to determine the optimal protein solubilisation buffer for use with milk

products, three different extraction buffers were investigated. Investigations were

initially performed using the purified milk proteins α-lactalbumin, β-lactoglobulin, α-

casein, β-casein and κ-casein. After solubilisation, proteins were separated on the

Agilent 2100 Bioanalyzer using two different protein LabChips, the Protein 230 LabChip

and the Protein 80 LabChip, which resolve proteins of 14-230kDa or 5-80kDa,

respectively. The expected protein sizes (Table 8) indicated that it should be possible to

resolve the proteins on either LabChip; however, protein separation was initially

performed using both LabChips to identify the optimal LabChip for milk protein

quantification.

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Table 8: Comparison of milk protein molecular weight determined from amino acid (AA) composition and the 2100 Bioanalyzer with Protein 80 LabChip.

Protein Molecular weight of proteins determined using

AA composition¹ [kDa] 2100 Bioanalyzer [kDa]

α-lactalbumin 14.1 12.2

β-lactoglobulin 18.3 18.3

αs1-casein 23.6 37.7²

αs2-casein 22.5

β-casein 24 32.4

κ-casein 19 45.1

¹ Data from reference Miralles et al 2000[9]

² The two α-casein sub-units (αs1 & αs2) were not differentiated on the 2100 Bioanalyzer

3.1.1. MODIFIED DE JONG EXTRACTION BUFFER

Milk protein standards were analysed using a solubilisation buffer that was based on a

protocol that had been developed previously to solubilise milk proteins for analysis by

capillary zone electrophoresis (CZE)[6]. This buffer was, therefore, considered potentially

useful for solubilising proteins for analysis on the 2100 Bioanalyzer. Individual milk

protein standards (20mg) were dissolved in 10ml of ‘De Jong’ buffer (6M Urea, 5mM DL-

dithiothreitol (DL-DTT), 5mM disodium phosphate, pH 8.0) to achieve a final

concentration of 2000µg/ml (or 2mg/ml). Equal volumes of the five standards were

mixed and, where necessary, diluted with the buffer to produce mixed standards at 400,

300, 100, 50 and 20µg/ml. Analysis of samples extracted with the De Jong buffer

resulted in poor resolution of the protein profiles with both the Protein 230 and Protein

80 LabChips (Figure 1). Results of analysis with either LabChip produced

electropherograms with lost upper markers, high backgrounds or only a few broad

peaks, suggesting that the De Jong buffer was interfering with the separation of proteins

on the 2100 Bioanalyzer. It was, therefore, concluded that this buffer was not suited to

the extraction and analysis of milk proteins using the Agilent 2100 Bioanalyzer.

3.1.2. TOTAL PROTEIN SOLUBILISATION BUFFER (TPS BUFFER)

Milk protein standards were solubilised using TPS buffer. This contained 0.1M DTT to

ensure that protein extraction took place under reducing conditions. Protein profiles

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were produced using both Protein 80 and Protein 230 LabChips (Figure 2). Profiles

generated on the Protein 80 LabChip showed that good separation of all five milk

proteins was achieved and that all five proteins were detected even when loaded at

250µg/ml. When proteins (at a concentration of 2000µg/ml) were profiled on the

Protein 230 LabChip, only the α-lactalbumin, β-lactoglobulin and κ-casein peaks were

resolved. The α-casein and β-casein proteins were not resolved on this LabChip. When

the proteins were run at a concentration of 250µg/ml, the α-lactalbumin peak was lost

and only small peaks corresponding to the β-lactoglobulin, κ-casein and the combined α-

casein and β-casein peak were observed. Profiles from both LabChips showed low

background noise. System peaks were observed in all profiles generated on the Protein

230 LabChips, but only when low concentrations of protein were analysed on the

Protein 80 LabChip. The relative height of any observed system peaks increased as the

amount of protein loaded was reduced; however, it was possible to manually exclude

system peaks from the analysis to avoid erroneous calculations.

Poor detection of the milk proteins and failure to resolve α-casein and β-casein when

using the Protein 230 LabChip were considered to indicate that this LabChip was less

suitable for the analysis of milk proteins compared to the Protein 80 LabChip. In a

previous study of milk proteins using a Protein 200 LabChip, α-lactalbumin co-migrated

with one of the LabChip markers, whereas successful separation had been achieved

using a Protein 50 LabChip[7]. Both the Protein 50 and Protein 200 LabChips have now

been superseded by the Protein 80 and 230 LabChips. Similar protein separation results

were achieved using the newer LabChips.

3.1.3 BMR EXTRACTION BUFFER

Analysis of the milk proteins was also performed using a solubilisation buffer prepared

from phosphate buffered saline (PBS) buffer, pH 7.4, containing 8M urea (BMR buffer)[7].

The PBS solution contained 0.138M NaCl and 2.7mM KCl. Samples were extracted and

diluted using the BMR buffer as described for the previous buffers. Separation of

samples solubilised in this buffer using either the Protein 80 or Protein 230 LabChips

produced variable results (Figure 3). Whilst proteins were separated on both LabChips,

better resolution was achieved on the Protein 80 LabChip compared with the Protein

230 LabChip. When higher concentrations (2000µg/ml) of protein were run on the

12

LabChip, higher peaks were produced compared to peaks produced when smaller

(250µg/ml) amounts of protein were loaded. However, a greater number of small peaks

were also detected when greater amounts of protein were loaded onto the LabChips.

These small peaks could be removed from the analysis by changing the baseline

threshold level. In contrast, as the amount of protein injected on to the LabChip

decreased, an increase in the height of one or more system peaks was observed. The

height of these system peaks meant they could not be removed by changing the

threshold level, therefore they required manual removal. Overall, the results obtained

using BMR buffer were much better than those produced using the modified De Jong

buffer. When results obtained using the BMR extraction buffer (Figure 3) were

compared with those obtained using the total TPS buffer (Figure 2) a number of

differences were observed. Resolution of the α-casein and β-casein peaks was not as

clear with the BMR buffer as was achieved using the TPS buffer. In addition, the whey

proteins both appear to be suppressed when using the BMR buffer compared to the TPS

buffer. For example, the β-lactoglobulin peak appears to be greater than (or at least

equal to) the α-casein and β-casein peaks when extracted using the TPS buffer; however,

when solubilised with the BMR buffer, the height of the β-lactoglobulin peak was

consistently less than that of the casein peaks. This suggests that the BMR buffer may

be less efficient at solubilising whey proteins or that a preferential extraction of caseins

occurs when using this buffer.

All subsequent work was, therefore, performed using the Protein 80 LabChip and the

TPS buffer.

3.1.4 PROTEIN MOLECULAR WEIGHTS

When using the Protein 80 LabChip in auto-analyse mode, it was observed that there

was a difference between the protein molecular weights as determined by the 2100

Bioanalyzer and the expected molecular weights. The molecular weights of the whey

proteins were close to those expected, but the sizes of the caseins were higher than

expected (Table 8). The difference between the expected and observed size of the κ-

casein is particularly noticeable, as the size determined on the 2100 Bioanalyzer is over

twice the expected size. A difference in protein molecular weight due to the different

polymeric sieving matrices and field strengths of sodium dodecyl sulphate

13

polyacrylamide gel electrophoresis (SDS-PAGE) compared to sodium dodecyl sulphate

capillary electrophoresis (SDS-CE) has been reported previously[9]. It is also known that

the binding capacity of sodium dodecyl sulphate (SDS) differs for the individual caseins

and is to some extent dependent on competition, as caseins either bind with other

casein molecules or with SDS. These previously reported differences could account for

observations made here. In addition, the individual protein sizes did not vary when

separated on different LabChips, suggesting that sizing on the Protein 80 LabChip is

consistent and therefore unlikely to affect the detection or measurement of protein

levels in unknown samples.

3.2 QUANTIFICATION OF MILK PROTEINS

3.2.1 COMPOSITION OF MILK AND MILK PROTEIN

Average values for the composition of milk from McCance & Widdowson[10] show that

the level of total protein in each of the liquid milk types is similar (3.2-3.4%) but the

proportion of fat and water differs (Table 9).

Table 9: Average percentage levels of different components of milk. (McCance & Widdowson[10]).

Type of milk product Water (%) Protein (%) Fat (%)

Whole cow’s milk, pasteurised 87.8 3.2 3.9

Whole cow’s milk, UHT 87.8 3.2 3.9

Semi-skimmed cow’s milk, pasteurised 89.8 3.3 1.6

Semi-skimmed cow’s milk, UHT 89.7 3.3 1.7

Skimmed cow’s milk, pasteurised 91.1 3.3 0.1

Skimmed cow’s milk, UHT 91.1 3.4 0.1

Dried whole milk powder 2.9 26.3 26.3

Dried skimmed milk 3 36.1 0.6

Evaporated milk (whole) 69.1 8.4 9.4

Milk protein is composed of a number of different proteins (Figure 4A and Table 10).

Table 10 summarises the proteins and their concentration in milk as published in various

sources[3,11-15]. Reported levels for each protein differ between publications; however,

these differences are relatively small. In general, the protein fraction of whole milk

14

comprises 79-83% casein, most of which is α-casein, and 17-20% whey protein, of which

β-lactoglobulin contributes the major proportion.

The published concentrations of milk proteins were used to calculate an average value

for each protein. The average values were used to calculate a 'factor' to calculate the

'milk' content from the determination of specific proteins.

3.2.2 QUANTIFICATION OF MILK PROTEINS USING THE BIOANALYZER

The separation of milk proteins on the 2100 Bioanalyzer was assessed using a series of

five mixed standards of α-casein, β-casein, κ-casein, α-lactalbumin and β-lactoglobulin at

250µg/ml to 2000µg/ml. Separation of the five milk proteins was achieved on the

LabChip (Figure 2 Protein 80 LabChip). Each protein from the mixed standard was used

in turn to produce a standard curve and calculate the concentration of a single 1,000

µg/ml protein solution loaded onto the same LabChip. Results (except those for κ-

casein) were calculated using the auto-analyse function on the Bioanalyzer software to

define the peak areas and heights. The mean of two runs (except for α-lactalbumin)

made on two separate LabChips is shown in Table 11. Standard curves showed a near

straight line between points with the linear regression line having a fit (R2) of over 95%.

Recovery was between 102 and 113% for α-lactalbumin, β-lactoglobulin, α-casein, β-

casein and κ-casein. Generally the use of the auto-analyse function of the 2100

Bioanalyzer software produced a satisfactory determination of the concentration of

protein in the solutions. Some problems were observed with the automatic

identification of κ-casein and β-lactoglobulin peaks; however, using the manual analysis

mode overcame these problems. Results obtained using the single protein solutions

indicated that, provided no interference due to the sample occurred, it should be

possible to quantify the amount of individual milk proteins in unknown samples.

15

Table 10: Concentrations of specific proteins in milk. Published values are shown along with relevant references. Calculated values (converted to mg protein per ml of whole milk) are shown underlined¹. The average calculated values are shown in bold text in the

last row.

Total protein Total casein Total whey α–casein β–casein κ–casein α–lactalbumin β–lactoglobulin Reference

3.39% 79.60% of milk protein

17.60% of milk protein

16.2mg/ml 8.4mg/ml 2.4mg/ml 1.5mg/ml 4.5mg/ml [3] – Table 1

3.39g/100ml 27g/L 5.9g/L 16.2mg/ml 8.4mg/ml 2.4mg/ml 1.5mg/ml 4.5mg/ml -

3.4g/100ml 25g/L 6g/L 54% of total casein

33% of total casein

13% of total casein

19% of total whey protein

49% of total whey protein

[14] – Tables 12.1 & 12.2

3.4g/100ml 25g/L 6g/L 13.5mg/ml 8.3mg/ml 3.3mg/ml 1.1mg/ml 2.9mg/ml -

nr nr nr 12.6g/ L 9.3g/ L 3.3g/ L 1.2g/ L 3.2g/ L [14] – Table 2.1

- - - 12.6mg/ml 9.3mg/ml 3.3mg/ml 1.2mg/ml 3.2mg/ml -

30-35g/L ~78% nr 50% of total casein

35% of total casein

12% of total casein

1.2g/L 2.7g/L [15]

3.3g/100ml 26g/L - 13mg/ml 9.1mg/ml 3.1mg/ml 1.2mg/ml 2.7mg/ml -

- 80-83% of milk protein

17-20% of milk protein






[11-13]

33.6mg/ml 26mg/ml 6mg/ml 13.8mg/ml 8.8mg/ml 3.0mg/ml 1.3mg/ml 3.3mg/ml

¹ Calculated values were derived from published data as part of this study. nr: no reported data. Missing data were not reported by source.

16

This information, in conjunction with data from the literature about the level of each

specific protein in milk (Table 10), could be used to determine the amount of milk in

products.

Rather than using a standard containing all five proteins to determine the milk content,

two proteins, one representing the whey fraction (α-lactalbumin) and one the casein

fraction (β-casein) were selected for quantification of milk content. These two proteins

correspond to protein peaks that migrate at about 22 and 30 seconds, respectively

(Figure 5). β-lactoglobulin and κ-casein were excluded due to their requirement for

manual analysis and poorer recovery compared to other proteins. Caseins are heat

stable so are considered to be suitable for quantification of milk regardless of heat

processing. The whey protein provides for products to which whey protein concentrates

or isolates have been added or are the sole source of milk protein.

Table 11: Determination of protein concentration of single protein solutions using the auto-analyse function of the 2100 Bioanalyzer software.

Protein Actual

concentration (µg/ml)

Size [kDa] Calculated

concentration (µg/ml)

Percent recovery

α-lactalbumin 1000 12.2 1,045¹ 105%

β-lactoglobulin 1000 18.3 1,131 113%

α-casein 1000 37.7 1,025 102%

β-casein 1000 32.4 1,046 105%

κ-casein 1000 45.1 1,098² 110%

¹ Result of single measurement ² Result obtained using manual integration

3.3 ANALYSIS OF MILK Milk analysed using the TPS buffer and the 2100 Bioanalyzer showed resolution of the

five milk proteins α-lactalbumin, β-lactoglobulin, α-casein, β-casein and κ-casein (Figure

4B). Different types of milk were analysed. Very little difference in the profiles of dried,

whole and skimmed milk and liquid skimmed and semi-skimmed and UHT semi-skimmed

milk were observed (Figure 6, shows some, but not all, of the milk profiles in simulated

gel format for ease of comparison). All five milk proteins were detected in all the milk

types. However, resolution was not as good as that achieved using CZE where α-s1 and

α-s2 forms of α-casein and A1 and A2 forms of β-casein were resolved (Figure 4A). On

17

the LabChip, protein separation is based on sieving in a gel matrix so is dependent on

molecular weight, whereas in CZE, separation is based on the charge on proteins and to

a lesser extent the size. This difference affected the resolution and the order in which

proteins migrated. However, when using the 2100 Bioanalyzer migration was consistent

and, therefore, did not affect the ability to identify the proteins. The lack of resolving

power when using the LabChip compared to CZE did suggest that there may be

difficulties when analysing products containing complex mixtures of proteins, so analysis

of more complex products, i.e. products that contained more than just milk proteins,

was assessed using combinations of dried milk powder and 30% or 60% dried egg

powder or wheat flour (Figure 7). In the presence of additional proteins, the lack of

resolution when using the LabChip meant that milk proteins could not be clearly

identified. In particular, the whey proteins were difficult to detect due to their low level

compared to other proteins. In addition, proteins from some samples migrated with the

upper marker making detection of this marker difficult. This interfered with the

quantification of milk proteins as the upper marker is used by the 2100 Bioanalyzer

software to quantify the amount of protein in each well. These results suggested that

quantification of milk proteins in samples containing high levels of proteins from other

ingredients may not be possible and the method is only suited to the determination of

milk in relatively simple milk products. Therefore, validation of the method was

performed using rice puddings and whey powder based body building powders as

examples of simple milk products.

3.4 IN-HOUSE VALIDATION USING MILK PRODUCTS PREPARED AT CAMPDEN BRI

3.4.1. PRODUCTION OF RICE PUDDINGS

Rice puddings (made at Campden BRI using a recipe based on commercial rice pudding

recipes) were made using 1% dried whey powder, 9% rice, 4.6% sugar and whole,

skimmed or semi-skimmed milk at either 25%, 50% or 75%. Fresh milk was obtained

from local retailers. The total recipe was brought to 100% by the use of an appropriate

amount of water. A similar recipe was used to produce lactose-free rice puddings

(which were made with Lactofree semi-skimmed milk); however, whey protein isolate

was added to these products as the standard dried whey powder contained lactose.

Initially two cans of each rice pudding, except the Lactofree pudding, were produced to

18

confirm that products were suitable for analysis on the 2100 Bioanalyzer. A further

batch of cans of each rice pudding recipe was produced for analytical measurement; one

can was used for compositional analysis and the others were used to determine the milk

content using the Bioanalyzer.

On opening the cans, differences in the colour of the rice puddings were evident

(Figure 8). Some puddings were a pale, slightly off-white colour that is typical of

rice pudding, whilst others were a darker brown colour. These differences generally

corresponded with the amount and type of milk in the cans, suggesting that the

reactions between proteins and non-reducing sugars (Maillard reactions) had

occurred to a greater or lesser extent during heat processing.

3.4.2. CALCULATION OF MILK CONTENT FROM COMPOSITIONAL ANALYSIS OF RICE

PUDDINGS

Results of the compositional analyses carried out on the rice puddings and individual

ingredients are shown in Table 12. The composition of milk used was similar to the

average values reported by McCance and Widdowson[10] (Table 9). The Lactofree semi-

skimmed milk contained a slightly higher proportion of protein, but the removal of

lactose was mainly balanced by an increase in water. Compositional analysis of the rice

puddings showed that as the percentage of milk in the whole milk rice puddings

dropped from 75% to 25%, the protein level dropped from 3.0g/100g to 1.5g/100g, i.e. a

50% reduction. Similar changes were observed in rice puddings made with other milk

types, i.e. the protein levels were not affected by the type of milk added. Because the

protein concentration of the various forms of milk remains unchanged, it should be

feasible to calculate the milk content on the basis of milk protein. Changes in the fat,

carbohydrate or total energy levels of the products also reflected reductions in the milk

content; however, the actual values measured depended on the type of milk. A

relatively large (2.9%) change in the carbohydrate level was noted between the whole

milk rice puddings, while relatively small (<1.9%) changes were observed between the

different semi-skimmed or Lactofree rice puddings.

19

Table 12: Results of compositional analysis of milks, milk puddings, body building

powder and specific ingredients used in the production of milk-based products.

Sample Protein (g/100g)

Fat (g/100g)

Carbohydrate (g/100g)

Moisture (g/100g)

Energy (kcal/100g)

Ingredients

whole milk 3.2 3.5 4.8 87.8 64

semi-skimmed milk 3.2 1.4 5.1 89.6 46

skimmed milk 3.2 0.1 5 91.0 34

Lactofree semi-skimmed milk 3.4 1.6 2.8 91.6 39

Whey powder (dried) 11.9 0.9 76.9 2.7 363

Whey protein concentrate [Protarmor]

74.2 6.9 7.8 8.4 390

Whey protein isolate (dried)

[90 Instant]

81.1 1.6 6.6 8.2 365

Pudding rice (dried) 6.7 0.3 78.7 13.7 344

Finished products

75% whole milk rice pudding 3.0 2.7 16.4 77.2 102



75% semi-skimmed milk rice pudding 3.1 1.2 15.9 79.0 87



75% skimmed milk rice pudding 3.1 0.2 16.1 79.8 79



75% Lactofree rice pudding 3.2 1.3 13.7 81.2 79



Body building powder (dried) 74.9 6.2 8.5 7.6 389

Taking the 75% whole milk rice pudding as an example, if the recipe is unknown and it

has to be assumed that all the protein in the rice pudding is from milk, the milk content

is calculated:

%7.93%2.3

%100*%0.3

20

where

the protein content of whole milk was determined to be 3.2% (Table 12)

the protein content of the rice pudding was 3.0% (Table 12)

However, this overestimate as compared to the known weight of ingredients of 75% is

expected - other ingredients, pudding rice and whey powder, contribute to the protein

content. Where the recipe is unknown, their contribution can not be determined. This

highlights the need for a method that can distinguish and measure milk protein.

Using analytically derived compositional data (Table 12) and information from the

recipe, the milk content of rice puddings was calculated (Appendix 8.3). Results are in

agreement with the milk content based on the known weight of ingredients in all cases,

except for the rice puddings made with 75% whole milk and 75% and 25% Lactofree

milk, (Table 13) where the calculated milk content is 72%, 73% and 26%, respectively. In

all cases, an additional 0.1% protein content of the pudding measured would account for

the 2-3% underestimate in the milk content.

3.4.3. LABCHIP ANALYSIS OF RICE PUDDINGS AND CALCULATION OF MILK CONTENT

Canned rice puddings were opened and the contents stirred with a clean glass rod to

ensure homogeneity. A sample (100-500mg) of the liquor was removed into a clean

bijou bottle using a disposable pipette. Proteins were solubilised and diluted at 1/20,

1/10 or 1/2 using a minimal volume of TPS buffer prior to analysis on the 2100

Bioanalyzer. Dilutions were performed to bring samples within the range of the

standard curve (Figure 9). The concentration of α-lactalbumin and β-casein in the rice

pudding liquor was determined using the Bioanalyzer.

21

Table 13: Calculation of milk content of rice puddings using recipe and compositional analysis data. For details of calculations see Appendix 8.3.

(1) (2) (3) (4) (5) (6) (7) (8)

Pudding composition Protein content of pudding

(%)

Total protein/ 1400g batch

Non-milk protein

(g)

Protein from milk

(g)

Protein content of milk

(g/100g)

Amount of milk this protein

equates to (g)

Calculated milk content

(%)

75% whole milk 3.0 42 9.84 32.16 3.2 1005 72

50% whole milk 2.3 32.2 9.84 22.36 3.2 699 50

25% whole milk 1.5 21 9.84 11.16 3.2 349 25

75% semi-skimmed milk 3.1 43.4 9.84 33.56 3.2 1049 75

50% semi-skimmed milk 2.3 32.2 9.84 22.36 3.2 699 50

25% semi-skimmed milk 1.5 21 9.84 11.16 3.2 349 25

75% skimmed milk 3.1 43.4 9.84 33.56 3.2 1049 75

50% skimmed milk 2.3 32.2 9.84 22.36 3.2 699 50

25% skimmed milk 1.5 21 9.84 11.16 3.2 349 25

75% Lactofree semi-skimmed milk

3.2 44.8 9.88 34.92 3.4 1027 73


2.4 33.6 9.88 23.72 3.4 698 50


1.6 22.4 9.88 12.52 3.4 368 26

(2) from Table 12 (5) = (3)-(4) (3) = ((2)/100)*1400 (6) from Table 12 (4) = 9.84 (see example) for all except Lactofree* (7) = (5)*100/(6) (8) = (7)/1400*100 *Lactofree pudding 0.13% whey protein isolate Equivalent to 0.13/100*1400g = 1.82g whey protein isolate in can Contributes 1.48g protein (1.82*81.1/100) Protein from rice and whey = 1.48+8.4=9.88g

The α-lactalbumin peak was generally smaller than other milk proteins and not well

resolved. In a number of runs the α-lactalbumin peak was unsuitable for further data

analysis, therefore β-casein was used to quantify the milk content. Results for sample

dilutions of 1/10 for rice puddings made with 75% or 50% milk and 1/2 for rice puddings

made with 25% milk are shown in Table 14. The β-casein concentration measured in the

22

rice pudding liquors was very variable. The quoted reproducibility for quantification of

proteins using the Bioanalyzer and LabChips is ± 30% across different chips, instruments

and users (Agilent application note). For the rice puddings, the variability was higher

than this (Table 14). Quantification is achieved by comparison of the peak area to that

of the upper marker, a protein incorporated into the LabChip kits which is used as an

internal standard in each sample. The upper marker is added to the sample prior to

denaturation so corrects for differences in sample preparation and sample injection into

separation channels. The Bioanalyzer automatically determines the peak area of

unknowns and the upper marker and the software calculates the relative concentration

based on the known concentration of the upper marker. Where standards of a known

calibrant protein are run, in this case β-casein, the standard curve is generated by

plotting the determined relative concentration against the concentration of the

standards (Appendix 8.2). In principle this approach should result in reproducible

quantification; however, the performance of the upper marker was variable and

unpredictable. In some cases this was due to co-migration with sample peaks, but on

other occasions the size of the upper marker peak was adversely affected for no

apparent reason without a similar effect on the size of the sample peaks. On some

occasions the upper marker per se appeared to be a source of variation and when it co-

migrated with sample peaks, the run was invalid (see number of runs in Table 14 and

number of runs used for calculation. This was greater for α-lactalbumin than β-casein,

data not shown).

In addition to variation caused by the upper marker, the poor resolution of α- and β-

casein was another source of variation. The lack of resolution when using the LabChip

compared to CZE was identified as a potential limitation for its application to more

complex milk products (Section 3.3), but it was also a limitation for quantification of milk

proteins in the liquor from rice pudding - a relatively simple milk product. As the

Bioanalyzer and LabChips are designed to be 'a simple-to-use tool', there are very

limited (if any) options for the analyst to adjust the method to improve resolution (the

running conditions and the separation matrix are all fixed). Using an alternative protein

chip, the Protein 230 LabChip, was one option but in initial studies, whilst this gave

23

greater separation between sample peaks and the upper marker (Figure 2), the α- and β-

casein peaks were not resolved, forming a single casein peak.

Table 14: Summary of the milk content of rice puddings prepared with different amounts and types of milk. Calculated milk content was determined using β-

casein standard curves.

Type and amount of milk added

to rice puddings

Dilution Measured conc. of β-

casein in rice pudding (mg/ml)²

Expected conc. of β-casein

in puddings (mg/ml)¹

Average determined milk content of liquor(%)

3

Number of measurements

used for calculation

Number of runs

75% whole 1/10 4.1 ± 2.4 6.7 46.6 ± 26.7 7 7

50% whole 1/10 2.8 ± 0.7 4.5 32.2 ± 7.8 11 14

25% whole 1/2 1.2 ± 0.5 2.2 14.2 ± 5.6 5 6

75% semi-skimmed

1/10 5.1 ± 0.5 6.7 58.0 ± 5.5 3 4

50% semi-skimmed

1/10 3.6 ± 1.3 4.5 40.5 ± 15.4 7 7

25% semi-skimmed

1/2 1.1 & 0.3 2.2 12.7 & 3.1 2 3

75% skimmed

1/10 3.4 ± 0.2 6.7 38.7 ± 2.3 3 3

50% skimmed

1/10 1.93 ± 1.3 4.5 21.9 ± 14.5 7 7

25% skimmed

1/2 0.63 ± 0.3 2.2 7.2 ± 3.2 3 3

75% Lactofree

1/10 2.36 ± 0.9 7.1 26.8 ± 10.1 3 3

50% Lactofree

1/10 2.86 ± 4.5 4.8 32.5 ± 50.9 8 16

25% Lactofree

1/2 0.86 ± 0.6 2.4 9.8 ± 6.5 7 7

¹ Values have been calculated from published data, rice pudding recipe and compositional analysis. Taking the 75% whole milk rice pudding as an example: the milk contained 2.4g protein/100g (75%*3.2g/100g; protein content from Table 12); of this, 80% is casein [19.2mg/ml], and of this 35% is β-casein [6.7mg/ml]. ² Value measured using LabChip. Mean ± standard deviation (SD) 3

Values shown are average calculated milk content ± standard deviation (SD)

The measured β-casein concentration in the rice pudding liquor was compared with the

expected β-casein concentration in rice puddings calculated using compositional analysis

24

data and published values for the proportion of β-casein in whole milk (Table 14). The

expected value assumes that milk proteins are evenly distributed throughout the rice

pudding i.e. the concentration in the liquor is the same as that in the rice.

In all cases the measured β-casein content of the liquor was an underestimate of the

calculated actual β-casein content of the rice pudding. It is unlikely that testing of the

liquor only, rather than homogenised product, contributed to this. Ideally the whole

sample would have been homogenised prior to solubilisation, but concerns about

interference from rice proteins in the separation of milk proteins led to extraction of the

liquor only. Moreover, the concentration of milk proteins in the liquor would be

expected to be higher than in the rice as water is likely to be taken up by the rice,

increasing the concentration of milk proteins in the liquor. A more likely cause of the

underestimate is the integration of the not fully resolved β-casein peak. Where β-casein

peaks are not fully resolved from α-casein, integration parameters define the peak using

a straight line from the valley of the unresolved peaks to the baseline (Figure 9),

resulting in an underestimation of the β-casein peak area.

The percentage of milk in the rice pudding liquor was calculated from the concentration

of β-casein using a conversion factor based on the average of published levels of β-

casein in milk, i.e. 8.8mg/ml (Table 10) derived from whole milk. This value was used

for calculations applied to all milk types as information for other milk types was not

available. As predicted from the comparison of measured and expected β-casein

concentrations, in all cases the measured milk content of the liquor was an

underestimate of the actual milk content in the rice pudding (Table 14). With the

exception of the rice pudding made with 50% Lactofree milk, the measured milk content

did decrease with the actual milk content. However, the measured milk content of the

liquor of rice puddings was only 30% to 80% of the expected values based on the weight

of ingredients used. The greatest underestimates were for skimmed and Lactofree milk,

i.e. those milk types that have the highest water content. Whilst the calculated

expected value assumes that there is an even distribution of milk protein throughout the

can, this is unlikely to be true. Prior to processing, the milk proteins were only in the

liquor (milk and water), so the concentration of milk proteins in the liquor would be

expected to overestimate that of the whole product (for 75% whole milk rice pudding,

25

the expected protein concentration of the liquor would be 87.5%). During processing,

liquid is taken up by the rice (the solids : liquid ratio changed from 15:85 prior to

processing to 44:55 after processing); if water is taken up preferentially to milk proteins,

the concentration of milk proteins in the liquor would have been expected to increase

further.

Overall the conclusion of the study to validate the use of the LabChip method for

estimating the milk content of rice puddings is that the lack of both precision and

accuracy of the method in its current form make it unfit for purpose.

3.4.4. LABCHIP ANALYSIS OF BODY BUILDING POWDER

The number of body building powders on the market is large, as is the range of

ingredients used in their production and their final nutritional composition. The body

building powder recipe selected for the in-house product was developed with the aim of

achieving a protein content of around 75-80% and a fat content of around 5-7%. These

values generally match levels in products available on the market (Table 15). In order to

challenge the method by using different whey protein extracts in body building powders,

the initial aim was to produce two body building powders using the same recipe but with

two different sources of whey protein extract. However, it was only possible to obtain a

single whey protein extract, therefore only one body building powder was produced.

Table 15: Comparison of the composition of an in-house body building powder with three commercial body building powders.

Body building powder Protein (g/100g)

Fat (g/100g)

Carbohydrate (g/100g)

Energy (kcal/100g)

Campden BRI product 74.9 6.2 8.5 389

Body Fortress® Natural whey protein¹

76.2 8.4 4.7 399

Body Fortress™ Super advanced whey protein²

78.8 6.1 9.1 318

¹ Produced for Holland & Barrett, UK. In-house product was based on this recipe. ² Produced by Healthwatchers (DE) Inc, USA. Values converted from 66g portion quoted on label.

The body building powder was solubilised and analysed using the standard method.

Solutions of 2-5mg body building powder per ml solubilisation buffer were appropriate

26

to ensure that α-lactalbumin peaks fell within the range of the standard curve that was

appropriate for the LabChip. Whilst resolution of α-lactalbumin from β-lactoglobulin

was achieved, the whey protein peaks always appeared as doublets with α-lactalbumin

migrating at around 22 seconds (Figure 10). As no other proteins are present in this

product, the doublet suggests the presence of modified forms of the protein in the whey

preparations, perhaps due to the isolation and drying of the proteins.

A small β-casein peak was detected when samples were analysed at high concentrations

of body building powder (100mg/ml), presumably due to carry over in the whey protein

concentrate and/or isolate. The protein profile of the body building powder, i.e. lack of

β-casein peak and a large α-lactalbumin peak, confirmed that 'milk' was not present in

this product, therefore, it was not appropriate to estimate the 'milk content' of this

sample. Protein profiles of this nature, i.e. profiles that differ from those of standard

milks for unknown samples, are useful indicators of samples that do not contain milk or

which have been modified by the addition of selected milk proteins. In such cases it is

not appropriate to calculate a 'milk content', only to estimate the amount of specific

milk proteins present.

The concentration of α-lactalbumin determined in the body building powder was 56mg

α-lactalbumin/g body building powder with a standard deviation of 18mg. This is an

underestimate of the expected α-lactalbumin concentration of 141mg/g based on the

known weight of ingredients and compositional analysis (Appendix 8.4). Even if the peak

area of the doublet is used to determine the level of α-lactalbumin (83mg/g body

building powder with a standard deviation of 34mg), the α-lactalbumin content is still

underestimated.

In terms of whey protein, assuming α-lactalbumin constitutes 19% of whey protein

(Table 10), this equates to 437mg whey protein per g body building powder, i.e. 44%, a

significant underestimate (59%) of the expected whey protein (74%). In terms of the

overall objective of this project it would lead to an incorrect conclusion, i.e. that the

product contained less than 50% protein of milk origin, when in this case we know there

was 74%.

27

The underestimation may be due to the high dilution factor required to bring the α-

lactalbumin in the body building powder within the range of the concentration of the

standard curve that is suitable for use with the Bioanalyzer. In addition, it may be that

the proportion of α-lactalbumin in whey protein preparations varies from that in milk,

depending upon the method of separation and manufacture of the whey protein

concentrate or isolate such that the 'α-lactalbumin factor' is inappropriate. Such

changes in proportions will be a limitation for any method that relies on using the

'natural' proportions of specific components to calculate milk content. 'α-lactalbumin

factors' for isolated whey proteins or concentrates would be required.

The outcome of the study to validate the use of the current LabChip method to

determine the whey protein concentration of body building powder was the same as for

rice puddings, i.e. that the method in its current form is not fit for the purpose of

estimating when there is more than 50% milk protein present.

4. CONCLUSIONS

A lab-on-a-chip protein separation method using the Agilent 2100 Bioanalyzer and

Protein 80 LabChips was suitable for separating milk proteins in milk. The method

separates proteins extracted under reducing conditions, on the basis of their molecular

size. A number of solubilisation buffers were compared: the one that resulted in best

resolution of milk proteins was used throughout this study. Alternative solubilisation

buffers either caused interference with protein separation or appeared to be biased

towards the extraction of caseins, to the detriment of the whey proteins, when used

with milk samples.

Two protein LabChips, the Protein 80 and Protein 230, available for use with the 2100

Bioanalyzer were assessed for their ability to separate milk proteins. Although profiles

could be generated on both LabChips, better resolution and sensitivity was achieved

with the Protein 80 LabChip and there was less interference from system peaks. The

Protein 80 LabChip was used for the analysis of samples in this study.

Quantification of 1000µg/ml milk proteins in mixed protein standards prepared from

commercial α-casein, β-casein and κ-casein, and α-lactalbumin and β-lactoglobulin gave

28

recoveries of 103-113%. Standard curves for either β-casein or α-lactalbumin were

selected for quantification of the amount of milk in milk-based products. Mixed standard

solutions (two proteins) were used to produce standard curves. Manual rather than

automatic peak analysis was required to correctly identify some peaks or to define peaks

which were not fully resolved.

When the method was applied to relatively complex mixtures of proteins (milk powder

mixed with wheat flour or egg powder), the identification of milk proteins was severely

impaired, indicating that the method is only suited to simple milk protein matrices, not

complex protein mixtures.

Validation of the method was performed using canned rice puddings made with

different milk types (whole, semi-skimmed, skimmed or Lactofree milk) present at 75, 50

and 25%, and a body building powder, containing predominantly whey protein. These

were prepared in-house so that the quantities of ingredients used were known.

For rice puddings, the milk content was calculated using the recipe with results of

compositional analysis performed on ingredients and the final products. The calculated

milk content agreed with the actual milk content in all cases except for rice puddings

containing 75% whole milk, and 75% and 25% Lactofree semi-skimmed milk, for which

the calculated milk content was 72%, 73% and 26%, respectively. Compositional analysis

of the rice puddings confirmed that the protein level was unaffected by the type of milk

added - rice puddings made with 75% whole, semi-skimmed, skimmed or Lactofree

semi-skimmed milk had protein levels of 3.0-3.2g/100g, supporting the use of the

protein concentration as a means of determining the milk content.

When using the Bioanalyzer to determine the milk content of rice puddings, only the

liquor was sampled, as inclusion of the rice was considered likely to cause interference

with the milk protein peaks. β-casein was used to quantify milk protein. Variation in

peak areas and therefore the determined milk content was unacceptably high. Variation

was attributed to the performance of the upper marker, also used as an internal

standard, and to the lack of resolution of α- and β-casein peaks in the sample. Whilst

the determined milk content decreased with the expected milk content, it was always

underestimated, being between 30% and 80% of the expected values. The greatest

29

underestimates were for skimmed milk and Lactofree milk, i.e. those milk types with the

highest water content. The lack of accuracy was attributed to integration of the β-casein

peak that was unresolved from α-casein.

In contrast to the rice pudding samples, the use of β-casein for the determination of milk

protein in the body building powder was inappropriate as the product was formulated

using whey protein isolate and whey protein concentrate. The high α-lactalbumin

content of the body building powder required that the sample be tested using 2mg body

building powder/ml solubilisation buffer to ensure that the protein concentration fell

within the range of the standard curve appropriate for the Bioanalyzer. The measured

α-lactalbumin content and total whey protein content calculated from it indicated that

this product contained less than 50% whey protein. However, on the basis of the known

weight of ingredients and their composition, the body building powder contained

74.25% whey protein and, on the basis of compositional analysis, 74.9% protein. The

underestimate when using the LabChip method may be due to a lack of linearity at the

high whey protein concentrations in the body building powder and/or the proportion of

α-lactalbumin in whey isolates and concentrates differing from that found in milk, due to

separation and manufacturing technologies employed.

In conclusion, the validation study showed that the lack of precision and accuracy make

the method in its current form unfit for estimating whether the milk content of even

simple milk-based products exceeds 50%. The Bioanalyzer can be used to give a

satisfactory determination of the protein concentration of protein standards in buffer

solution. A standard operating procedure (SOP) of the method used in this work has

been produced (Appendix 8.5). For very simple milk protein based products, it may be

possible to use the current LabChip technology and the SOP as a relatively simple, rapid

screening method to indicate the presence of a biased peak ratio, i.e. very large α-

lactalbumin peak compared to β-casein peak, for detecting samples that have been

bulked with added whey protein or other milk protein(s), but the LabChip technology in

its current form is unsuitable for estimating the milk content of unknown samples to

determine whether they contain 50% or more milk.

30

Following commissioning of this research, the requirement for vet checks on imported

products with over 50% animal ingredient was not taken forward in the legislation.

However, existing food labelling legislation requires a quantitative declaration of

ingredients (QUID) associated with a product or used in the name of the food. This

project was therefore continued, as the work undertaken could be used to assess the

feasibility of the Bioanalyzer platform to quantify milk ingredients in composite products

and thereby allow enforcement of QUID requirements. The results show that LabChip

technology in its current form is not suitable for this application either.

5. AREAS FOR FUTURE INVESTIGATION

The following were identified as areas for future investigation to improve the method

using protein-based LabChips:

The need for data on the concentration of specific milk proteins (e.g. α-

lactalbumin and β-casein) in milk types in addition to whole milk, e.g. skimmed,

semi-skimmed and Lactofree milks and dried milk powders and whey protein

isolates and concentrates. This would assist with the use of specific milk protein

levels to estimate milk content.

Investigate the use of the Protein 230 LabChip. A number of LabChip runs were

rejected when the upper marker was lost due to co-migration with sample peaks

or the peak area of the upper marker was affected by the sample. This affects

quantification of the samples. Use of the Protein 230 LabChip, where the upper

marker is well resolved from sample peaks, may overcome some of these

problems.

Use a total casein protein standard to overcome issues arising from poor

resolution of specific caseins in combination with Protein 230 LabChips.

6. ACKNOWLEDGEMENTS

Campden BRI gratefully acknowledges the financial support of the Food Standards

Agency for the work described in this report.

31

7. REFERENCES

1. Auldist, M.J., Thomson, N.A., Mackie, T.R. Hill, J.P. & Prosser, C.G. (2000). Effects

of pasture allowance on the yield and composition of milk from cows of

different β-lactoglobulin phenotypes. Journal of Dairy Science, 83(9): 2069-2074.

2. McLean, D.M., Graham, E.R., Ponzoni, R.W. & McKenzie, H.A. (1984). Effects of

milk protein genetic variants on milk yield and composition. Journal of Dairy

Science, 51(4): 531-546.

3. Ng-Kwai-Hang, K.F., Hayes, J.F., Moxley, J.E. & Monardes, H.G. (1987). Variation

in milk protein concentrations associated with genetic polymorphism and

environmental factors. Journal of Dairy Science, 70(3): 563-70.

4. Pollott, G.E. (2004). Deconstructing milk yield and composition during lactation

using biologically based lactation models. Journal of Dairy Science, 87: 2375-

2387.

5. Verdi, R.J., Barbano, D.M., Dellavalle, M.E. & Senyk, G. F. (1987). Variability in

true protein, casein, nonprotein nitrogen, and proteolysis in high and low

somatic cell milks. Journal of Dairy Science, 70: 230-242.

6. De Jong, N., Visser, S. & Olieman, C. (1993). Determination of milk proteins by

capillary electrophoresis. Journal of Chromatography, 652: 207-213.

7. Bütikofer, U., Meyer, J. & Rehberger, B. (2006). Determination of the

percentage of alpha-lactalbumin and beta-lactoglobulin of total milk protein in

raw and heat treated skim milk. Milchwissenschaft, 61(3): 263-266.

8. Recio, I., Amigo, L. & Lopez-Fandino, R. (1997). Assessment of the quality of dairy

products by capillary electrophoresis of milk proteins. Journal of

Chromatography B: Biomedical Sciences and Applications, 697(1): 231-242.

9. Miralles, B., Bartolomé, B., Amigo, L. & Ramos, M. (2000). Comparison of three

methods to determine the whey protein to total protein ratio in milk. Journal of

Dairy Science, 83: 2759-2765.

http://www.ingentaconnect.com/content/els/03784347

http://www.ingentaconnect.com/content/els/03784347

32

10. McCance and Widdowson's The Composition of Foods, 6th edition. (2002).

Published by Royal Society of Chemistry/Food Standards Agency/Institute of

Food Research.

11. Huffman, L.M. & Harper, W.J. (1999). Maximizing the value of milk through

separation technologies. Journal of Dairy Science, 82(10): 2238-2244.

12. Goff, D. Dairy Science and Technology Education. Dairy Chemistry and Physics on-

line course notes. University of Guelph, Canada.

www.foodsci.uoguelph.ca/dairyedu/chem.html. [accessed 25-02-09].

13. Milk proteins. Department of Food Science & Technology. The Ohio State

University. Food Science Degree Programme on-line course notes.

www.class.fst.ohio-state.edu/FST822/lectures/Milk2.htm. [accessed 25-02-09].

14. Hambræus, L. & Lönnerdal, B. (2003) Nutritional aspects of milk proteins. In

Advanced Dairy Chemistry, Volume 1: Proteins. 3rd Edition. Fox, P.F. &

McSweeney, P.L.H. (Eds). Kluwer Academic/Plenum Publishers.

15. Maubois, J.L. & Ollivier, G. (1997) Extraction of milk proteins. In Food Proteins

and Their Application, Damodaran, S. & Paraf, A. (Eds). Marcel Dekker Inc, New

York. 1997.

33

8. APPENDICES

APPENDIX 8.1 PREPARATION OF SAMPLES AT CAMPDEN BRI

8.1.1 PREPARATION OF CANNED RICE PUDDINGS

Ingredients (pudding rice, granulated sugar, sodium bicarbonate, fresh pasteurised

whole, semi-skimmed and skimmed milk, fresh Lactofree semi-skimmed milk1 and whey

protein isolate) for the preparation of rice puddings were obtained from local retailers

or raw ingredient suppliers. Rice puddings were prepared using a recipe based on a

total product weight of 1,400g. The proportion of each ingredient is shown in Appendix

8.1 Table 1. The milk, sugar and bicarbonate were added to a saucepan and heated to

80°C on a domestic hob with stirring to avoid burning. Once heated, 159.25g of the milk

and sugar mix was added to 15.75g of the pudding rice in each small (73mm diameter x

51.5mm) can. The filled cans were immediately seamed with a steam flow closure using

a MB6 can seamer (used in steam flow close mode). The seamed cans were placed in a

retort (John Fraser horizontal steam retort) and processed with end-over-end rotation at

4rpm for 10 minutes at 105°C. After 10 minutes the temperature was increased to

115°C and held for a further 25 minutes. In each batch a total of 24 cans were processed

(8 cans containing 75%, 50% and 25% milk, respectively). Thermocouples were placed at

the geometric centre of three cans and the heat process achieved was monitored using

an Ellab CMC temperature logger and F0 calculator. Each process achieved a minimum

F06 before starting to cool. The remaining cans per recipe were used for analysis.

1 Lactofree semi skimmed milk is prepared by filtering semi-skimmed milk to remove half of the sugars.

The remaining sugars are broken down to simpler forms using a lactase enzyme.

34

Appendix 8.1 Table 1: Proportion of ingredients used to produce rice puddings made with different amounts and types of milk. All recipes were based on a total

weight of 1400g.

Ingredient Whole, semi-skimmed, skimmed milk recipes

Lactofree semi-skimmed milk recipe

Milk 75%¹ 75%²

Pudding rice 9.0% 9.0%

Whey powder 1.0% ~

Whey protein isolate ~ 0.13%

Sugar 4.6% 4.6%

Sodium bicarbonate 0.1% 0.1%

Water 10.3%¹ 11.17%²

¹ Recipe shown contains 75% milk. Other recipes contained 50% milk and 35.3% water or 25% milk and 60.3% water. ² Recipe shown contains 75% Lactofree semi-skimmed milk. Other recipes contained 50% Lactofree semi-skimmed milk and 36.17% water or 25% Lactofree semi-skimmed milk and 61.17% water.

8.1.2 PREPARATION OF A BODY BUILDING POWDER

Dry ingredients for the preparation of a body building powder (weight gain formula)

were obtained from local retailers or specialist ingredients suppliers (Table Appendix 8.1

Table 2).

Appendix 8.1 Table 2: Ingredients and recipe used to prepare the body building powder.

Ingredient Final percentage in the body building powder¹

Supplier/Source

Sucralose 0.01% Tate & Lyle

Cinnamon 0.10% local retailer

Whey protein isolate (90 instant)

5.89% Bacarel & Co. Ltd

Whey protein concentrate

(protarmor 82 sbli)

94.00% Bacarel & Co. Ltd

1Total final weight of product = 200g

All ingredients (making a total weight of 200g) were initially mixed, in the proportions

shown in Appendix 8.1 Table 2, using a domestic Kenwood food processor to form an

homogeneous mixture. Batches of the mixed material were removed and blended in a

domestic Kenwood blender with mill attachment for 1-2 minutes. The 200g bulk

35

mixture was blended in the mill in five smaller batches until each sub-batch formed an

homogenous product. All five sub-batches were then mixed together in the Kenwood

food processor for two minutes to create the final body building powder. Aliquots (45-

50g) of the final body building powder were weighed into foil pouches (19.0 x 12.0 cm)

and sealed under vacuum using a Multivac A300 Pouch Sealer. Samples in sealed

pouches were stored at ambient temperature and humidity and used for the analysis of

milk proteins.

36

APPENDIX 8.2 TYPICAL RESULTS OBTAINED USING THE PROTEIN 80 LABCHIP

40

APPENDIX 8.3 EXAMPLE CALCULATION OF THE MILK CONTENT OF RICE PUDDING

Using compositional analysis and recipe data (example based on rice pudding known

to contain 75% whole milk)

The rice pudding contained 3.0% protein (Table 12)

Therefore the amount of protein per 1400g batch of rice pudding is 42g

(3.0/100%*1400)

Sources of protein other than milk:

9% pudding rice

equivalent to 126g of rice per 1400g batch (9%/100%*1400g)

contributes 8.4g protein (126*6.7/100g)

1% whey protein

equivalent to 14g of whey powder per batch (1%/100% * 1400g)

contributes 1.7g protein (14 * 11.9/100g)

Therefore protein from pudding rice and whey powder is 9.84g (8.4 + 1.7)

The remaining protein, assumed to be from milk, is 32.16g (42 – 9.84)

The protein content of the whole milk used was 3.2% (Table 12).

So the amount of milk used is 1005g (32.16*100/3.2)

If 1005g milk were used per 1400g batch of rice pudding,

the calculated milk content is 71.8% (1005/1400*100)

41

APPENDIX 8.4 CALCULATION OF WHEY PROTEIN IN BODY BUILDING POWDER

The amount of α-lactalbumin in the body building powder was determined using the

following approach. This calculation assumed that no changes in the proportion of the

whey proteins occurred during the manufacture of whey protein concentrate or isolate.

The protein content of the whey protein isolate and whey protein concentrate was

measured by determination of organic nitrogen.

The whey protein products were purified from skimmed milk. The whey protein isolate

(WPI) contained 81.1% protein; the whey protein concentrate (WPC) contained 74%

protein.

The body building powder (BBP) recipe included 5.89% WPI and 94.0% WPC. A total of

200g of BBP was produced.

So...

For WPC

In a 200g batch of BBP there is 200 * 94% = 188g of WPC. The protein content of the BBP due to WPC is therefore 188g * 74% = 139g. For WPI In a 200g batch of BBP there is 200 * 5.89% = 11.78g of WPI. The protein content of the BBP due to WPC is therefore 11.78g * 81.1% = 9.5g. So the total amount of protein in 200g of BBP due to whey is 148.5g (139 + 9.5) protein,

i.e. equivalent to an expected protein content of 74.25g/100g (742mg/g). The measured

protein content was 74.9% (Table 12)

In milk, α-lactalbumin is reported to be around 19% of the total whey protein[14].

Assuming α-lactalbumin to be 19% of the whey protein in the isolates and concentrates,

in 200g of BBP which contains 148.5g whey protein, there is 28.2g α-lactalbumin

(148.5*19/100). This is equivalent to 141mg α-lactalbumin/g BBP.

APPENDIX 8.5 STANDARD OPERATING PROCEDURE

FOOD STANDARDS AGENCY

STANDARD OPERATING PROCEDURE (SOP)

Version 1.0, November, 2009

STANDARD OPERATING PROCEDURE FOR CALCULATING THE MILK CONTENT OF MILK-BASED FOOD PRODUCTS

Prepared by John Dooley & Helen Brown, Campden BRI Date November 2009__

CONTENTS

1. HISTORY / BACKGROUND ........................................................................................................ 44

1.1 BACKGROUND .............................................................................................................................. 44 1.2 CHANGES IN CURRENT VERSION ........................................................................................................ 44

2. PURPOSE................................................................................................................................. 44

3. SCOPE ..................................................................................................................................... 44

4. DEFINITIONS AND ABBREVIATIONS ......................................................................................... 45

5. PRINCIPLE OF THE METHOD .................................................................................................... 45

6. MATERIALS AND EQUIPMENT ................................................................................................. 45

6.1 CHEMICALS .................................................................................................................................. 45 6.2 WATER ....................................................................................................................................... 46 6.3 SOLUTIONS, STANDARDS AND REFERENCE MATERIALS ........................................................................... 47 6.4 COMMERCIAL KITS ......................................................................................................................... 49 6.5 PLASTICWARE ............................................................................................................................... 51 6.6 GLASSWARE ................................................................................................................................. 51 6.7 EQUIPMENT ................................................................................................................................. 51

7. PROCEDURES .......................................................................................................................... 52

7.1 PREPARATION OF SAMPLES .............................................................................................................. 52 7.2 LOADING THE PROTEIN 80 LABCHIP AND STARTING A RUN .................................................................... 53 7.3 ANALYSING THE DATA .................................................................................................................... 56

8. QUALITY ASSURANCE .............................................................................................................. 61

8.1 UPPER AND LOWER MARKER ........................................................................................................... 61 8.2 STANDARD CURVES ....................................................................................................................... 61

9. CALCULATIONS AND DATA ANALYSIS ...................................................................................... 62

9.1 CALCULATE THE PERCENT MILK IN THE SAMPLE .................................................................................... 62

10. REFERENCES ............................................................................................................................ 63

1. HISTORY / BACKGROUND

1.1 Background

A method was developed in response to proposed changes to the European Commission Regulation covering veterinary checks on imported products of animal origin. These stipulated that checks must be carried out on composite foods imported from third countries that contain 50% or more processed animal product (other than meat, such as milk, egg or honey) by weight of the food. Although this legislation was not taken forward, existing food legislation requires a quantitative declaration of ingredients (QUID) associated with a product or used in the name of the food. The ability to quantify the level of ingredients in composite food products therefore provides a means of verifying the QUID declarations in composite food products and consequently monitoring these products for consumer protection and regulatory compliance.

This Standard Operating Procedure (SOP) was developed as part of the FSA commissioned study Q01117, which aimed to transfer a published capillary electrophoresis (CE) method to the Agilent 2100 Bioanalyzer to perform CE using protein LabChips in order to determine the milk content of simple milk-based foods. Results from study Q01117 indicated that it was not possible to use the Agilent 2100 Bioanalyzer to determine the milk content of milk-based products. It was noted, however, that this method (developed as part of study Q01117) had the potential to be used as a screening tool to detect unusual milk protein profiles that could indicate the presence of contaminants or adulterants of milk products.

1.2 Changes in current version This is the original version.

2. PURPOSE

The purpose of this method is to determine if simple milk-based products contain more than 50% milk. The method in its current form is unsuitable for this; however, it could be applied as a screening tool to detect abnormal milk protein profiles resulting from contamination or adulteration of milk products.

3. SCOPE

The original scope of the method was the analysis of proteins in milk and very simple milk-based products to detect the presence of 50% or more milk. Following completion of study Q01117, the method in its current form was deemed unsuitable for this, but has the potential application as an initial screening tool to detect the distortion of a milk protein profile.

4. DEFINITIONS AND ABBREVIATIONS

2100 Bioanalyzer: A small-scale capillary electrophoretic system using lab-on-a-chip technology and microfluidics for the specific separation of DNA fragments or proteins.

Capillary electrophoresis (CE): Electrophoresis in buffer-filled, narrow bore capillaries, normally with 25-100µm internal diameter.

Fluorescence Units (FU): A measure of fluorescence intensity used by the 2100 Bioanalyzer.

Protein LabChip: A small (3cm2), disposable, single-use plastic and glass unit containing etched capillaries attached directly to ten sample loading wells. Two protein LabChips are available, the Protein 80 LabChip for separating fragments of 5-80kDa and the Protein 230 LabChip for separating 14-230kDa fragments.

DMSO: Dimethyl sulphoxide

DS: Destaining solution

DTT: Dithiothreitol

HCl acid: Hydrochloric acid

SDS: Sodium dodecyl sulphate

TPS buffer: Total protein solubilisation buffer

5. PRINCIPLE OF THE METHOD

Proteins are solubilised in a reducing environment and denatured by heating in the presence of SDS. Denatured proteins are separated on the basis of size by capillary electrophoresis using a Protein 80 LabChip on an Agilent 2100 Bioanalyzer. The concentration of α-lactalbumin or β-casein, which are proteins present in milk, in the sample is determined by comparison of peak areas to a standard curve generated using commercial preparations of these proteins. The protein concentration is then converted to “milk” using a factor based on the level of α-lactalbumin or β-casein in whole milk from published literature sources.

6. MATERIALS AND EQUIPMENT

6.1 Chemicals

3M Tris-HCl buffer, pH 8.8

Add 32.3g ± 0.1g Tris-HCl (Sigma, Cat No: T3253, MW=157.6, stored at room temperature) to about 80ml of ultrapure water in a 200ml glass beaker. Adjust

the pH to 8.8 ± 0.1 with HCl acid (AnalaR grade, BDH, Cat No: 101254H, stored at room temperature).

SAFETY NOTE: Handle acids with care and use a fume cupboard. Wear goggles and safety glasses. SLOWLY add acid to water. DO NOT add water to acid.

Once the pH has been adjusted, make up the solution to 100ml in a volumetric flask.

Transfer the Tris solution to a labelled Schott bottle and store cold at +1°C to +6°C for up to 3 months.

Total Protein Solubilisation buffer (TPS buffer) Prepare the total protein solubilisation buffer (0.1M Tris-HCl buffer, pH 8.8 containing 2M urea, 15% glycerol and 0.1M Dithiothreitol [DTT]) (TPS buffer) in a suitable 50ml container, e.g. a 50ml Falcon tube. TPS buffer must be freshly prepared each day.

Weigh 1.8g ± 0.01g of urea (Sigma, Cat No: U6504, MW=60.06, stored at room temperature) into a clean 50ml container.

Add 231mg ± 5mg Dithiothreitol (DTT) (Sigma, Cat No: D5545, MW=154.25, stored at 4°C).

Add 0.5ml 3M Tris-HCl buffer, pH8.8 and 15ml ultrapure water. Shake the tube to dissolve the urea and DTT.

Finally, add 2.81g ± 0.005 glycerol (Sigma, Cat No: G6279, MW=92.09, stored at 4°C).

Invert the tube ten times to mix the glycerol and other reagents. Milk Protein Standards

Alpha-lactalbumin, from bovine milk Type III, calcium depleted. Purity 98%. (Sigma L6010). For traceability purposes note the Lot number of each batch used.

Beta-casein, from bovine milk, >90% as beta-casein. Purity 99%. (Sigma C6905). For traceability purposes note the Lot number of each batch used.

Milk protein standards must be stored dry at -20°C when not in use. A plastic box with a tightly sealed lid and containing silica gel crystals is ideal for storing these standards.

6.2 Water

Unless otherwise stated, ultrapure water (resistance 18.2mΩ·cm) must be used. Suitable water is produced by the Millipore Synergy UV system.

6.3 Solutions, standards and reference materials

Preparation of milk protein standards When preparing the milk protein solutions and standards, handle each solution with care to avoid degradation. Do not vortex any of the standards to hydrate or mix them. Invert the tubes or use the Spiramix rocking roller to mix the reagents.

Stock β-casein standard (5,000µg/ml)

Prepare a fresh 5000µg/ml β-casein solution each day.

In a labelled glass bijou bottle, weigh approximately 10mg of β-casein. Record the exact weight in your notebook. It is also useful to note the weight on the bijou bottle as well.

Use the formula below to calculate the volume of TPS buffer to add to the β-casein protein to achieve a stock of 5,000µg/ml (5mg/ml).

Vol. of TPS buffer (µl) = Actual weight (mg) x Target volume (1000µl)

Target weight (5mg)

Pipette the required volume of TPS buffer into the bijou bottle. Swirl or invert the bijou bottle to dissolve the β-casein powder in the TPS buffer, and then place the bottle on a rocking roller (the Spiramix is suitable) for 1 hour to ensure the β-casein protein is fully hydrated, well mixed and homogeneous.

This is the 5,000µg/ml stock β-casein standard. Stock β-casein standard (2,000µg/ml)

Prepare a fresh 2000µg/ml β-casein solution each day from the 5000µg/ml solution.

Pipette 750µl of TPS buffer into a labelled glass bijou bottle.

Pipette 500µl of the 5000µg/ml β-casein standard stock solution into the bijou bottle. Invert the bijou bottle ten times to mix the solutions and then place the bijou bottle on a rocking roller (the Spiramix is suitable) for 30 minutes to ensure the β-casein protein is well mixed and homogeneous.

This is the 2,000µg/ml stock β-casein standard. Stock α-lactalbumin standard (1,000µg/ml)

Prepare a fresh 1000µg/ml α-lactalbumin solution each day.

In a labelled glass bijou bottle, weigh approximately 5mg of α-lactalbumin. Record the exact weight in your notebook. It is also helpful to note the weight on the bijou bottle.

Use the formula below to calculate the volume of TPS buffer to add to the α-lactalbumin protein to achieve a stock of 1,000µg/ml (1mg/ml).

Vol. of TPS buffer (µl) = Actual weight (mg) x Target volume (1000µl)

Target weight (1mg)

Pipette the required volume of TPS buffer into the bijou bottle. Swirl or invert the bijou bottle to dissolve the α-lactalbumin powder in the TPS buffer, and then place the bijou bottle on a rocking roller (the Spiramix is suitable) for 1 hour to ensure the α-lactalbumin protein is fully hydrated, well mixed and homogeneous.

This is the 1,000µg/ml stock α-lactalbumin standard. Mixed milk protein standard curve (2,000-250µg/ml)

A mixed standard dilution series is produced from the standard solutions prepared above. The dilution series is prepared directly from the standard solutions using the volumes stated in the Table below. All samples are diluted using TPS buffer.

Table of volumes of individual protein standard solutions needed to prepare the mixed Protein Standard Solutions.

Standard

Volume of each solution (µl) Final volume (µl)

5,000µg/ml β-casein standard

2,000µg/ml β-casein standard

1,000µg/ml α-lactalbumin standard

TPS buffer

Standard 1 300 - 300 400 1000

Standard 2 - 500 200 300 1000

Standard 3 - 250 150 600 1000

Standard 4 - 125 100 775 1000

Standard 5 - 50 50 900 1000

Add the correct volumes of protein standard solutions and TPS buffer to a new, clean, labelled bijou bottles. Invert the bijou bottles ten times to mix the solutions and then place the bijou bottles on the rocking roller for 30-60 minutes to ensure the protein standards are thoroughly mixed.

The final volume, and concentration of protein standards, in each of the Standard mixes is shown in the Table below.

Table showing the final concentration of α-lactalbumin and β-casein proteins in the mixed Protein Standard Solutions.

Standard Final concentration of β-casein (µg/ml)

Final concentration of α-lactalbumin (µg/ml)

Final volume (µl)

Standard 1 1500 300 1000

Standard 2 1000 200 1000

Standard 3 500 150 1000

Standard 4 250 100 1000

Standard 5 100 50 1000

6.4 Commercial kits

Protein 80 LabChip kit LabChips and Protein 80 reagents are available from Agilent Technologies (Cat No: 5067-1515). The following reagents, which are required to run LabChips on the 2100 Bioanalyzer, are provided in the kit. Protein 80 sample buffer (white capped tube)

(200 µl per vial, 4 x vials per Part II box). Store at -20°C.

Once a vial has been thawed for use, keep it at 4°C to avoid freeze-thaw cycles.

Remove the Protein 80 sample buffer (white capped tube) from the fridge at least 10 minutes before use and allow it to equilibrate to room temperature.

Protein 80 Size Ladder (yellow capped tube)

To avoid degradation of the ladder due to cycles of freeze-thaw the ladder must be aliquoted into small amounts suitable for daily use.

Upon receipt of the Agilent protein chip kit, or when it is first used, aliquot the ladder into small (6µl) amounts and store frozen as per instructions in the Agilent kit. Each aliquot of ladder is subsequently thawed and used for the analysis of chips on a single day. Spare ladder from each aliquot is discarded at the end of each day.

Completely thaw a tube of ladder (yellow capped tube).

Vortex to mix, then centrifuge at 16,000g for 10 seconds using a microcentrifuge at room temperature to recover the solution in the bottom of the tube.

Aliquot all the ladder solution in 6μl amounts into 0.5ml microtubes. Label the tubes and store at -20°C until required.

Protein 80 Dye Concentrate (blue capped tube)

1 vial in Part I box. Store at 4°C.

Allow 30 minutes to equilibrate to room temperature before use.

This reagent is light sensitive; only remove protective cover when pipetting. SAFETY NOTE: When handling the dye concentrate wear two pairs of gloves. Protein 80 Gel-Matrix (red capped tube).

1. 4 vials in Part I box. Store at 4°C. 2. Allow 30 minutes to equilibrate to room temperature before use. 3. This reagent is light sensitive; only remove protective cover when pipetting.

Protein 80 Gel-Matrix Dye Mix

Using a P1000 pipette, transfer ALL the contents (650µl) of one vial of Protein 80 Gel-Matrix (red capped tube) to a spin filter. To achieve maximum recovery, centrifuge the tube for 10 seconds at 16,000g using a microcentrifuge, then use a pipette to transfer the last of the liquid to the spin filter.

Centrifuge through the spin filter at 2,500g ± 20% for 15 minutes at room temperature.

After centrifugation, discard the filter unit.

Add 25μl Protein 80 dye concentrate (blue capped tube) to the filtered and centrifuged Protein 80 Gel-Matrix.

NOTE: The Protein 80 Dye is light sensitive. Avoid exposing the dye concentrate and the G-Dye solution to light. NOTE: Spin filters are supplied as part of the Agilent Protein 80 Kit. These units are used to filter the Gel-Matrix to ensure it does not contain any lumps which could interfere with protein separation on the LabChip.

Mix thoroughly for 10-20 seconds by vortex mixing or flick the tube to mix the dye into the Gel-Matrix. When mixed correctly the Gel-Dye Mix should have a uniform blue colour.

Label the Gel-Dye mix tube with the preparation date.

Store the Gel-Dye mix in the dark at 2-8°C and use within four weeks of production. This should be sufficient Gel-Dye Mix for running 10 chips.

SAFETY NOTE: The protein-binding dye used in this assay also binds to DNA and is, therefore, a potential mutagen. DMSO in the dye solution facilitates the transfer of compounds through the skin. Handle all solutions containing the dye with care and wear goggles and gloves. When handling the dye concentrate wear two pairs of gloves. Protein 80 destaining solution

Using a P1000 pipette, transfer all the contents (650µl) of one vial of Protein 80 Gel-Matrix (red capped tube) to a spin filter. To achieve maximum recovery, centrifuge the tube for 10 seconds at 16,000g using a microcentrifuge, then use a pipette to transfer the last of the liquid to the spin filter.

Centrifuge through the spin filter at 2,500g ± 20% for 15 minutes at room temperature.

After centrifugation, discard the filter unit.

Label the tube as “DS” (destaining solution) with the preparation date.

Store the DS in the dark at 2-8°C. Use within the expiry date for the kit.

One tube of DS is sufficient for 25 chips.

6.5 Plasticware

General lab plasticware should be obtained from any reputable consumables supplier as detailed below. It is not necessary to use sterile plasticware; however, it should be clean and made from virgin materials. The following plasticware was used:

Filtered pipette tips (various sizes) (Axygen Maxymum Recovery tips from Kinesis. Cat. No.'s: TXLF-10-LRS, TF-100-LRS, TF-200-LRS, TF-1200-CLRS)

Non-filtered tips (Fasttrak Pipette tips refill system from alpha-laboratories. Cat. No.'s: FR1200, FR1250).

Microfuge tubes (1.5ml from alpha-laboratories. Cat. No.: LW2375).

50ml Falcon tubes (alpha-laboratories. Cat. No.: LW1115)

Graduated plastic pastettes (5ml) (alpha-laboratories. Cat. No.: LW4728). 6.6 Glassware

General laboratory glassware (e.g. measuring cylinders, volumetrics, beakers).

Bijou bottles (7ml) (Fisher Scientific, Cat. No.: BTS-160-012X).

Schott bottles (100ml, 500ml) (Fisher Scientific).

6.7 Equipment

Spiramix5 – flatbed, rolling and rocking shaker (Thermo Fisher Scientific).

Agilent 2100 Bioanalyzer (Agilent Technologies).

Single channel manual pipettes (P10, P100 P200, P1000), such as Gilson pipettes (Anachem).

Water bath (Model: Grant Sub14).

Microcentifuge (Model: Eppendorf 5415D).

60-second timer (as supplied with Agilent Bioanalyzer).

7. PROCEDURES

7.1 Preparation of samples Pre-extraction sample preparation

Samples can be used as they are or freeze-dried prior to analysis.

If samples contain <10% water they should be ground to a powder using a sample mill or coffee grinder to produce homogeneous mixtures.

If samples contain >10% water they should be homogenised by blending in a food processor. Alternatively they should be freeze-dried and then milled.

If samples contain >5% fat and or > 10-15% water, they should be defatted using acetone extraction[1]. Use 40ml acetone for every 300mg dry matter.

If freeze drying or defatting, record start and finish weights so that results can be expressed in terms of wet weight or initial product composition.

Sample extraction

Samples must be solubilised, or diluted where necessary, using TPS buffer so that denaturation takes place under reducing conditions. This buffer contains DTT and ensures that proteins are reduced. The ratio of sample to buffer (w/v or v/v) is dependent on the homogeneity of the sample and the protein concentration. The target protein concentration for the samples being pipetted on to the LabChip is 200-300µg protein/ml, which is equivalent to 4,500-6,750µg protein/ml in the sample supernatant prior to denaturation. Where possible, keep the volume of TPS buffer used to a minimum. When analysing unknown samples for the first time, it is advisable to run the sample at more than one protein concentration. This is achieved by making different dilutions in TPS buffer before protein denaturation.

Liquid samples: These should be mixed thoroughly with TPS buffer.

Solid samples: Dissolve samples in TPS buffer.

Ensure homogeneity by mixing samples using the Spiramix5 for 30-60 minutes.

Centrifuge the sample at 10-15,000rpm for 15 minutes.

The supernatant is loaded onto the LabChip after denaturation in accordance with the following steps.

Sample denaturation prior to chip loading The following is applied to all samples and standards that will be loaded onto LabChips. Use supernatant recovered following sample solubilisation.

Before preparing the samples and standards, remove one of the 0.5ml microtubes containing 6μl of Protein 80 ladder and allow to thaw.

Once thawed, briefly vortex the tube to mix the sample and then centrifuge the tube for 15 seconds at 16,000g in a microcentrifuge to recover the solution in the bottom of the tube.

Label a series of 0.5ml microtubes. You will require one tube for each sample and standard.

Use a P10 pipette to combine 4μl of the sample or standard supernatant with 2μl of Protein 80 Sample Buffer (from the Agilent kit) in the respective labelled 0.5ml microtube.

Close the tubes and secure the lids using a tube cap-lock to prevent them opening during heating.

Heat the tubes (including the thawed ladder) at 95-100°C for 5 minutes using a boiling water bath.

NOTE: While the tubes are heating, ensure the lids do not open. If the lids do open,

immediately recover the tube and close the lid to avoid loss of sample through evaporation. Return the tube to the heat.

After boiling for 5 minutes, remove the tubes from the water bath and allow them to cool to room temperature (20-22°C).

Centrifuge the tubes for 15 seconds at 16,000g in a microcentrifuge to recover the solution in the bottom of the tube.

Add 84μl of ultrapure water to each sample, standard or ladder, and vortex to mix.

Centrifuge the tubes for 15 seconds at 16,000g in a microcentrifuge to recover the solution in the bottom of the tube.

This is the final sample, standard or ladder ready for loading onto the Protein 80 LabChip.

NOTE: The samples and ladder are stable for one day. Store the samples and ladder on ice when not in use. NOTE: Once boiled, the ladder can be used for the analysis of all chips that are run on the day of heating. Dispose of any remaining ladder at the end of the day. 7.2 Loading the Protein 80 LabChip and starting a run

Loading the Gel-Dye mix

Prepare the chip priming station by ensuring the station is set to position A and that the syringe clip is set to the middle position (see images below).

Put a new protein LabChip (purple colour) into the chip priming station. The images below show the LabChip (left) and a stylised image of the well layout (right). Well references in the instructions below refer to well numbers as in the stylised image.

Pipette 12μl of the Gel-Dye mix into well 4, marked G (top right).

NOTE: When adding solutions to wells always insert the pipette tip to the bottom of the well when dispensing the liquid. Placing the pipette at the edge of the well may lead to poor results.

Set the syringe plunger at 1ml and close the chip priming station. Press the station shut until a distinct “click” can be heard.

Using a single stroke press the plunger down and slide the top of the plunger under the clip to secure the plunger in the down position.

Wait for 60 seconds (use a timer) and then release the plunger by lifting the clip release catch.

Check that the plunger rapidly returns up to the 0.8ml (between 0.7 and 0.9ml) mark after it is released.

Open the chip priming station by lifting the silver button on the front of the station.

Remove the protein LabChip and invert it and check that all the micro-capillaries are full of Gel Matrix. If there are any bubbles replace the chip in the priming station and re-apply pressure for 60 seconds using the syringe. If the bubbles

1 2 3 4

5 6 7 8

9 10 11 12

16 15 14 13

remain after this second minute of pressure discard the chip and start with a fresh chip.

If the micro-capillaries are full of matrix, replace the chip in the chip priming station and then pipette 12μl of Gel-Dye mix into wells 8, 12 and 15.

Finally, pipette 12μl of destaining solution (DS) into well 16. Loading the ladder & the samples

Pipette 6μl of each sample (or standard) into the ten sample wells. Sample wells are numbered 1-3, 5-7, 9-11 and 13.

Pipette 6μl of the ladder into well 14, which is marked with a ladder symbol.

Ensure that all sample wells contain a sample. If you do not have enough samples to fill the chip, fill the spare wells with 6µl of ladder. Do not leave any wells empty.

Place the protein LabChip in the Agilent 2100 Bioanalyzer and start the analysis within 2 minutes to avoid evaporation from the wells.

Running the LabChip

Open the 2100 Expert software and select the “Protein 80 Series II” Assay type from the drop down menu.

Press the “Start” button to initiate the analysis. Move the mouse cursor away from the start button (which becomes a stop button once the analysis has started).

Watch the software for 1-2 minutes to ensure there are no problems with the chip.

If there are problems with the chip they will be highlighted and the software will stop running. In this case remove the chip and check that all wells contain sample or ladder and that the samples are in the bottom of the wells. If not, correct any obvious problems. Also ensure that there are no bubbles in the wells. If necessary a pipette tip can be used to remove bubbles.

Place the chip back in the 2100 Bioanalyzer and restart the run. If problems persist you will need to discard the chip and start with a fresh chip.

If there are no problems with the chip, leave the 2100 Bioanalyzer to perform the analysis. Do not touch the 2100 Bioanalyzer during the analysis otherwise spurious peaks will be detected or data will be lost.

Once the analysis has started, enter sample information. In the “Contexts” window (on the left hand side of the screen) select the “Data” tab then select the “Chip Summary” tab to open the sample information page. Complete the fields headed “Sample name”, “Sample comment”, “Use for Calibration” and “Conc *µg/ml+”.

Also complete fields for chip and reagent lot numbers. If required, additional comments can be added in the “Chip comments” box.

Once the chip run has completed a notification window appears. The data is automatically saved to the default or selected file location. Print the results to a colour printer using the instructions below.

Open the lid of the 2100 Bioanalyzer and remove and discard the LabChip.

The 2100 Bioanalyzer electrodes must be cleaned after each LabChip run. Cleaning the 2100 Bioanalyzer electrodes

After each protein chip has been run the 2100 Bioanalyzer electrodes must be cleaned using a cleaning chip (clear plastic chip).

Slowly fill one of the wells on a cleaning chip with 350µl of ultrapure water. Ensure that there are no bubbles then place the chip into the 2100 Bioanalyzer.

Close the lid and leave for 30-60 seconds. Open the lid and leave open for 1 minute to allow the electrodes to air dry.

Remove and discard the water from the cleaning chip.

If further protein chips are to be analysed repeat the steps in the “Loading the Protein LabChip” section above.

If no further protein chips are to be analysed ensure that the 2100 Bioanalyzer electrodes have been cleaned and dried, then shut the lid of the 2100 Bioanalyzer.

7.3 Analysing the data Data is analysed using the two milk proteins α-lactalbumin and β-casein, which correspond to protein peaks that migrate at about 22 and 30 seconds, respectively. It is not possible to analyse the data using both standards at once using the 2100 Bioanalyzer software. Analysis is, therefore, first performed using the α-lactalbumin protein standards and then using the β-casein protein standards.

Before starting the calculations select the “Chip Summary” tab and then the Sample Information (bottom row) tab.

Tick the “Use for Calibration” box next to each of the wells that contain calibration proteins. In the “Conc *µg/ml+” box enter the amount of protein in each of the calibration wells.

When the standards have been identified to the software, select the “Electropherogram” tab.

Double-click on one of the standards to open the electropherogram into full page view.

Click the expansion button located on the right-hand side of the screen to reveal the general assay parameters.

Select the “Global” tab to show the global parameters. Change the “Height threshold” settings from 2FU to 10FU and then press the “RETURN” key to initiate the changes.

When the height threshold has been changed to 10FU, continue with the next steps.

Defining the standards

For each standard, check that the upper and lower peaks have been correctly identified. If not, right-mouse click on the correct peaks and select the option to “Manually set upper/lower peak”, as appropriate.

Right-mouse click on the electropherogram image and select the “Manual Integration” option.

NOTE: Once you have switched to the Manual Integration mode do not revert back to Automatic Integration, otherwise changes you have made and data analysis calculations will be lost.

Once in Manual Integration mode, right-mouse click on one of the blue discs, which define the limits of the peaks, corresponding to each system peak. The system peaks can be identified from the Table of peaks at the bottom of the screen.

Once the peak has been selected, an arrow appears at the maximum point of the peak, the selected blue disc turns green and a drop-down menu appears. From the menu select the “Remove peak” option to remove the peak from the analysis.

Repeat the peak selection and removal process to remove all the small peaks that do not correspond to the α-lactalbumin or β-casein standard milk protein peaks.

Repeat the peak editing process for all five wells containing milk protein standards.

Analysis using the α-lactalbumin standards

When all extraneous peaks have been removed from the standards, right-mouse click on the α-lactalbumin (first) protein standard peak in each of the standard wells and select “Manually Set Calib. Protein”. Ensure that the same protein is selected in each of the standard wells.

Select the “Chip Summary” tab and enter the actual protein concentration of the α-lactalbumin protein standards (300, 200, 150, 100, 50µg/ml), loaded into each well, into the corresponding “Conc *µg/ml+” box.

Once the α-lactalbumin protein standard has been selected in all wells containing standards select “File” from the top toolbar and then select “Save” from the drop-down menu to save changes. Alternatively use the “Save” icon on the toolbar.

NOTE: The file must be saved after protein amendments have been made so that changes are implemented and calculations are updated. The 2100 software does not perform these calculations and updates automatically.

Samples

Select the sample name in the tree view and highlight the “Peak Table” sub-tab (at the bottom of the page) and the “Electropherogram” viewing tab (just under the top toolbar).

Ensure that there are two marker peaks in each sample and that they have been correctly identified.

The marker peaks should be well resolved from the sample peaks; however, this can depend on the sample type. The lower marker peak appears at about 12 seconds and the upper marker at about 45 seconds.

NOTE: Sample peaks should be between the lower and upper marker peaks. Sample peaks outside of the range of the markers may affect correct marker identification, which will affect the analysis. If this occurs the marker peaks must be assigned manually. NOTE: If necessary the actual markers can be identified by switching off the “Automatic analysis”. To do this select the analysis stop button (a round blue button with a white square) from the top toolbar.

To manually assign marker peaks select the correct peak by right-mouse clicking the correct peak in the Peak Table and then selecting “Manually Set Upper Marker” or “Manually Set Lower Marker”. Alternatively right-mouse click on the correct peak on the electropherogram image and then select the correct marker from the drop-down menu options.

When all the markers have been correctly identified, save the file to implement any changes.

Save the file by printing it to a PDF file using an appropriate extension to the filename so that it is obvious that the data has been analysed using the α-lactalbumin standards, e.g. use “...-A-lac.pdf”.

Once analysis has been completed using the α-lactalbumin standards, the analysis must be repeated using the β-casein standards.

Analysis using the β-casein standards

When the analysis using the α-lactalbumin standards has been completed it is necessary to repeat the process using the β-casein standards.

Right-mouse click on the β-casein (second) protein standard peak in each of the standard wells and select “Manually Set Calib. Protein”. Ensure that the same protein is selected in each of the five wells containing standards.

Select the “Chip Summary” tab and enter the actual protein concentration of the β-casein protein standards (1500, 1000, 500, 250, 100µg/ml), loaded into each well, into the corresponding “Conc *µg/ml+” box.

Once the β-casein protein standard has been selected in all wells containing standards select “File” from the top toolbar and then select “Save” from the drop-down menu to save changes. Alternatively use the “Save” icon on the toolbar.

NOTE: The file must be saved after changing the protein standard to β-casein so that changes are implemented and calculations are updated.

Save the file by printing it to a PDF file using an appropriate extension to the filename so that it is obvious that the data has been analysed using the β-casein standards, e.g. use “...-B-CN.pdf”.

Printing results

To print results, click the print icon or select print from the “File” drop-down menu.

NOTE: There is a bug in the 2100 Expert Software (version B.02.05.SI360) and printing directly from the file causes the electropherogram data to be omitted from the printed hard-copy. Printing the results to a PDF file and then printing the PDF file overcomes this problem.

Select the options marked in the Print menu (see image below). These options are:

“Print item” box: tick all options except “Gel like” and “Run logbook”.

“Wells” box: tick “All wells” option.

“Options” box: select 2 per page or 4 per page from the drop-down menu. Tick the “Include ladder” option.

“Save to File” box: tick the “PDF” option. Click the “...” button and select a location for the file to be saved. Select a suitable file name for the file.

When all options are correct press the “Save” button to save the file. Save the PDF file along with the project file using the same file name, except for the following two modifications:

1: change the file extension from <<filename.xad>> to <<filename.pdf>>

2: insert the “-alpha ” after the filename, but before the extension, when the analysis has been performed using the α-lactalbumin standards, e.g. filename -alpha.pdf. When the analysis is performed using the β-casein standards insert the extension “-beta”, e.g. filename -beta.pdf.

NOTE: To review the file prior to printing select the “Preview” button.

To produce a hard-copy of the results, open the PDF file and print to a colour printer.

8. QUALITY ASSURANCE

Protein Ladder Well

When separated, the Protein 80 ladder should feature six (excluding the markers) well-resolved peaks, as shown above.

The baseline should be flat and peak heights/values should be at least 25 fluorescence units (y-axis) higher than the baseline readings.

NOTE: In some runs, there may be double system peaks. If both peaks are identified as ladder peaks, exclude the first system peak by placing the cursor over the second peak in the peak table (which is the first system peak) and right-mouse click. Select “Exclude peak” to remove this peak.

8.1 Upper and lower marker

The lower marker peak should appear between 10.5 and 15.5 seconds.

The upper marker peak should appear between 38 and 48 seconds.

Both the upper and lower marker peaks should be well resolved from the sample peaks; however, this depends on the sample.

NOTE: For easy identification of the upper and lower markers, click “Don’t analyze” and

use the gel-like image to compare the markers in sample and ladder wells to identify and manually assign markers.

8.2 Standard curves

Select the “Chip Summary” tab (top of screen) and then the “Calibration Curve” tab (bottom of screen) to examine the standard curve.

The r2 value of the calibration curve should be >90% and all the points should lie on or very close to the straight line produced.

9. CALCULATIONS AND DATA ANALYSIS

9.1 Calculate the percent milk in the sample

Starting with the α-lactalbumin protein, use the 2100 Bioanalyzer printout (see image below) to find the amount of calibrated protein in the sample. The values in ng/µl can be found under the heading “Rel. Conc. *ng/µl+” (last column). Only the value for the “Calibrated Protein”, which is identified under the “Observations” (second last column) heading, is required.

Use the value for the amount of calibrated protein (from the print-out) to calculate the concentration of milk (α-lactalbumin or β-casein) in mg/ml in the sample (A) using the following formula:

mlmgDFP

A /1000

…where “P” is the amount of α-lactalbumin or β-casein in the sample going onto the LabChip (ng/µl) and “DF” is the dilution factor (prior to denaturation steps).

NOTE: Results from the 2100 Bioanalyzer are expressed in ng/µl. Values are converted from ng/µl to mg/ml by dividing the result by 1000.

The percentage milk in the sample is then calculated using the following formula:

%100CF

A

…where “CF” is a conversion factor derived from the expected amount of each milk protein in 100% whole milk.

CF has a value of 1.3mg/ml for α-lactalbumin and 8.8mg/ml for β-casein (Dooley et al., 2009. Determining the Milk Content of Milk-Based Food Products. FSA Final Report: Q01117).

NOTE: The conversion factor values have been derived from 100% whole milk. There are no reported values for other milk types, e.g. skimmed milk, therefore, these values are used for all sample types.

10. REFERENCES

[1]: Castro-Rubio et al., (2005). J. Agric. Food Chem. 53(2): 220-226.

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9. FIGURES

Figure 1: Separation of mixed milk protein standards at 1000µg/ml or 500µg/ml extracted using the modified DeJong Extraction Buffer and analysed using the Protein 80 or Protein 230 LabChips.

Protein 80 LabChip, 1000µg/ml protein


Figure 2: Separation of mixed milk protein standards at 2000µg/ml or 250µg/ml extracted using the TPS buffer and analysed using the Protein 80 or Protein 230 LabChips.





Figure 3: Separation of mixed milk protein standards at 2000µg/ml or 250µg/ml extracted using the BMR extraction buffer and analysed using the Protein 80 or Protein 230 LabChips.





Figure 4: Separation of proteins in skimmed milk using:

A - a Beckman PACE capillary electrophoresis system

B - a Protein 80 LabChip

Figure 5: Resolution of milk proteins standards on a Protein 80 LabChip. Migration times of the α-lactalbumin and β-casein proteins are about 22 and 30 seconds, respectively.

Figure 6: Artificial gel image generated from separating:

A- dried skimmed milk (hydrated at 34mg/ml TPS buffer) B- liquid semi-skimmed milk (diluted 1/10 in TPS buffer) C- UHT semi skimmed milk (diluted 1/10 in TPS buffer) D- five mixed milk proteins at 1000µg/ml TPS buffer:

κ-casein (1); α-casein (2); β-casein (3); β-lactoglobulin (4) α-lactalbumin (5)

M - upper and lower size markers SP - LabChip system peak

Figure 7: Artificial gel image generated from separating: A- α-casein B- β-casein C- κ-casein D- β-lactoglobulin E- α-lactalbumin F- mixed standards G- dried milk in 30% wheat flour H- dried milk in 60% wheat flour I- dried milk in 30% egg powder J- dried milk in 60% egg powder UM- Upper size marker LM- Lower size markers SP- LabChip system peaks

Figure 8: Example of browning observed in cans of rice pudding made with:

Full milk 25% 50% 75%

Semi-skimmed milk 25% 50% 75%

Skimmed milk 25% 50% 75%

Lacto-free milk 25% 50% 75%

Figure 9: Superimposed protein profiles from rice pudding made with 50% milk (red line) and milk protein standards. The green line shows the lowest concentration of

standards and the blue line a mid-concentration standard.

β-casein peak is within range of standards but α-lactalbumin is below lowest standard.

Figure 10: Resolution of proteins in the body building powder on a Protein 80 LabChip (2mg body building powder/ml). The α-lactalbumin peak at 22 seconds always appeared as a doublet.

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