Cgna18890enc 001 PDF

European cooperation
in the field of scientific

and technical research
European Commission
Laboratory manual
for chemical analysis of cheese
Ylva Ardo and Anna Polychroniadou, Editors
COST 95
Improvement of the quality of the production of raw milk cheeses
EUR 18890 EN
EUROPEAN COMMISSION
Edith Cresson, Member of the Commission
s
gv^ responsible for research, innovation, education, training and youth
DG XII/AP2 Political co-ordination and strategy COST
i Contact: Ms F. Serra
Address: European Commission, rue de la Loi 200 (SOME 1/50)
B-1049 Brussels Tel. (32-2) 29-69591; fax (32-2) 29-64289
LABORATORY MANUAL
FOR CHEMICAL ANALYSIS OF CHEESE
Collected and edited by
1 } 2 )
Y L V A ARD and A N N A POLYCHRONIADOU
department of Dairy and Food Science

The Royal Veterinary and Agricultural University
Rolighedsvej 30, DK-1958 Frederiksberg C
laboratory of Food Chemistry and Biochemistry

Faculty of Agriculture, Aristotle University of Thessaloniki
GR-54006 Thessaloniki
Cover page figure. RP-HPLC peptide profile of the water-soluble fraction of
a mature Feta cheese (method 8.1 I). [AP]
A great deal of additional information on the European Union is available on the Internet.
It can be accessed through the Europa server (http://europa.eu.int).
COST home page: http://www.belspo.be/cost
Cataloguing data can be found at the end of this publication.
Luxembourg: Office for Official Publications of the European Communities, 1999
ISBN 92-828-6599-1
European Communities, 1999
Reproduction is authorised provided the source is acknowledged.
Printed in Belgium
PRINTED ON WHITE CHLORINE-FREE PAPER

ARDO & POLYCHRONIADOU, Ed (1998) CHEESE ANALYSIS MANUAL
CONTENTS
Pages
Contributors vi
Preface ix
Abbreviations xii
1. INTRODUCTION 1
2. SAMPLING AND GENERAL SAMPLE PREPARATION 5
3. CHEMICAL COMPOSITION 7
3.1. Fat 7
3.2. Total Nitrogen 8
3.3. Moisture - Total Solids - Dry Matter 9
3.4. Water activity 10
3.5. Sodium chloride 12
3.6. pH 13
3.7. Carbohydrates 13
3.7.1. GLC for analysis of carbohydrates 13
3.7.2. HPLC for analysis of carbohydrates 14
3.8. D-/L-Lactate 18
3.9. Citrate 18
3.10. Carbon dioxide 19
3.11. Ammonia 19
3.11.1. Photometric method 19
3.11.2. Enzymatic method 20
3.12. Calcium 21
3.13. Phosphorus 22
3.14. Other minerals - Na, K, Mg, Zn, Fe, Cu 22
4. MILK ORIGIN 25
5. NITROGEN FRACTIONATION 31
5.1. Fractionation scheme for water soluble N compounds 31
5.2. Fractionation scheme for a citrate dispersion of cheese 32
5.3. WSN - Water soluble nitrogen 33
in
5.4. pH 4.4-SN - pH 4.4 soluble nitrogen 35

5.5. TCA-SN - Trichloroacetic acid soluble nitrogen 37
5.6. PTA-SN - Phosphotungstic acid soluble nitrogen 37
5.7. EtOH-SN - Ethanol soluble nitrogen 39
5.8. Ultrafiltration of the soluble fraction of cheese 40
6. RAPID METHODS FOR MONITORING

PROTEOLYSIS IN CHEESE 41
6.1. Cadmium-ninhydrin method for determination of free amino
groups in cheese 41
6.2. Trinitrobenzenesulphonic acid (TNBS) method for monitoring
proteolysis in cheese 41
7. ANALYSIS OF CASEINS 43
7.1. Reverse phase HPLC of caseins 43
7.2. Ion exchange HPLC of caseins 43
7.3. Urea Polyacrylamide gel electrophoresis of cheese 44
7.4. PAGE of the water insoluble fraction 48
7.5. Capillary electrophoresis 50
8. ANALYSIS OF PEPTIDES 55
8.1. Peptide profiles obtained by RP-HPLC 55
8.2. Isolation of individual peptides 58
8.2.1. Fractionation of peptides 58
8.2.2. Electroblotting of peptides 61
8.3. Techniques for identification of peptides 64
8.3.1. N-Terminal sequence analysis 64
8.3.2. C-Terminal sequence analysis 64
8.3.3. Amino acid composition of peptides 65
9. ANALYSIS OF FREE AMINO ACIDS AND AMINES 67

9.1. Analysis of free amino acids 67
9.1.1. IEC with ninhydrin post-column derivatization 67
9.1.2. RP-HPLC with OPA/FMOC precolumn derivatization 71
9.1.3. IE-HPLC with OPA post-column derivatization 74
9.1.4. Other liquid chromatography methods 75
9.2. Analysis of amines 75
IV
10. ANALYSIS OF FREE FATTY ACIDS 79

10.1. Gas chromatographic methods 79
10.2. Determination of the totalfreefatty acids 89
11. ANALYSIS OF VOLATILE COMPOUNDS 91

11.1. Short carboxylic acids 91
11.2. Volatile compounds in cheese 93
12. ANALYSIS OF ENZYMES IN CHEESE 101

12.1. Alkaline phosphatase 101
12.2. Acid phosphatase 104
12.3. Lactoperoxidase 104
12.4. Chymosin 105
12.5. Plasmin and plasminogen 106
12.5.1. Determination of plasmin and plasminogen by ELISA 106
12.5.2. Activity test of plasmin and plasminogen in cheese 107
12.6. Peptidases 108
12.6.1. Determination of peptidase activities 108
12.6.2. Determination of peptidase concentration by ELISA 109
12.7. Lipases and esterases 111
13. REFERENCES 115

Contributors
[AP] ANNA POLYCHRONIADOU
Laboratory of Food Chemistry and Biochemistry, Faculty of Agriculture
Aristotle University of Thessaloniki, 54006 Thessaloniki, Greece
Fax: +30 31 998789; E-mail: annapoly@agro.auth.gr
[API] ANNAPITOTTI
Dipartamento Scienze degli Alimenti, Via Marangoni 97, 33100 Udine, Italy
Fax: +39 432 501637; E-mail: dsa@hydrus.cc.uniud.it
[DD] DIDIER DUPONT

INRA-SRTAL, B.P. 89, Poligny Cedex, France
Fax: +33 3 84373781; E-mail: dupont@poligny.inra.fr
[ D J ] DORIS JAROS
Department of Dairy Science, Gregor Mendel Str. 33, 1180 Vienna, Austria
Fax: +43 14 789114; E-mail: h610pd@mail.boku.ac.at
PLB] DOMINIQUE L E B A R S
Department of Biocehmiastry and structure of proteins, INRA-SRL
78352 Jouy-en-Josas Cedex, France
Fax: +33 1 34652163; E-mail: dlebars@jouy.inra.fr
[HR] HARALD ROHM

Department of Dairy Science, Gregor Mendel Str. 33, 1180 Vienna, Austria
Fax: +43 14 789114; E-mail: rohm@mail.boku.ac.at
[JB] JACQUES O. B O S S E T
Swiss Federal Research Station, FAM
Schwartzerburgstrasse 161, 3003 Bern, Switzerland
Fax: +41 31 3238227; E-mail: jacques-olivier.bosset@fam.admin.ch
[JH] JOHN HOGENBOOM

Department of Food Science and Technology, University of Milan
Via Celoria 2, 20133 Milano, Italy
Fax: +39 2 2361576; E-mail: john@imiucca.csi.unimi.it
[JLQ] JEAN-LUC L E QUERE

Laboratory of Aroma Research, INRA
17 rue Sully, BP 1540, F-21034 Dijon Cedex, France
Fax: +33 380633227; E-max: lequere@aroma.dijon.inra.fr
vi
ARDO & POLYCHRONIADOU CHEESE ANALYSIS MANUAL
[MJ] MANUELA JUAREZ

CSIC - Instituto del Frio, Ciudad University s/n, E-28040 Madrid, Spain
Fax:+34 91 5493627; E-mail: mjuarez@orgc.csic.es
[MR] M E R T X E S DE RENOBALES
Department of Biochemistry, Euskal Herriko University
Apartado 450, 1080 Vitoria, Spain
Fax: +34 45 130756; E-mail: gbprescm@vc.ehu.es
[PMS] PAULMCSWEENEY
Food Chemistry Department, University College Cork
Western road, Cork, Ireland
Fax: +353 21 270001; E-mail: pmcs@ucc.ie
[RLF] ROSINA LPEZ-FANDIO

CSIC - Instituto de Fermentaciones Industriales
Juan de la Cierva 3,28006 Madrid, Spain
Fax: +34 1 5622900; E-mail: ifil04@fresno.csic.es
[SP] SYLVIE P O C H E T
INRA-SRTAL, B.P. 89, Poligny Cedex, France
Fax: +33 3 84373781; E-mail: pochet@poligny.inra.fr
[UB] UELI BTIKOFER

Swiss Federal Research Station, FAM
Schwartzerburgstrasse 161,3003 Bern, Switzerland
Fax: +41 31 3238227; E-mail: ueli.buetikofer@fam.admin.ch
[YA] YLVA ARDO

Department of Dairy and Food Science
The Royal Veterinary and Agricultural University
Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
Fax +45 3528 3190; E-mail: ya@mli.kvl.dk
In alphabetic order from the initials used in the manual to identify contributors
to each analysis method description.
VII
Preface
The European programmes FLAIR-CA2-COST902, AIR-CA3-CT94-
2039 and COST95 gave the opportunity to many laboratories involved in
cheese analysis to collaborate aiming, among others, at the harmonisation of
analytical methods for the characterisation of cheese ripening. Participants
realised that various methods successfully applied by each laboratory could be
useful for other laboratories and decided to collect them creating a
"Laboratory Manual" for cheese analysis. AIR2039 Concerted Action
accepted to print and distribute a first version of the manual as a work material
in 1996 among the laboratories participating in the Action. We want to thank
the several participants that have contributed with corrections and comments
on the original text and also supplemented the manual with new methods.
For this manual a large number of European laboratories having long
experience in cheese analysis have collaborated. However, the list of methods
presented is far from being exhaustive because there is a dynamic situation and
old and new methods are continuously tested at individual or collaborative
level.
It has not been the intention to cover by this publication all methods used
by cheese analysts, but to bring together methods which are of value in the
collaborating laboratories. No work has been done to evaluate and compare all
alternatives. The effort has been concentrated to analysis methods that are not
available as standard methods, but highly needed and regularly used. Official
methods, such as AOAC and IDF standards are given as references so that
each laboratory may complete their own manual with those.
In the description of some methods, brands of materials and instruments
or suppliers of equipment are mentioned. In most cases they are only indicative
and put as footnotes; other brands may also be used.
In the manual the contribution of members of the "Biochemistry Group"
of EU COST/FLORA programme is followed by initials indicating the
laboratory where the method is applied as described. However, the same
method may be used also in other laboratories.
November 1998 ANNA POLYCHRONIADOU

YLVA ARDO
IX
Introduction to COST Action 95
COST (Cooperation in Science and Technology) is a research programme to foment

scientific and technical cooperation at European level through concerted Actions, which
involve the coordination of national research projects. The Actions focus on specific
themes that are targeted by participating countries according to their research priorities
and helps build larger, more effective scientific communities. At present, COST offers
the possibility to co-operate between scientists from up to 28 member countries (other
countries may be admitted on a case by case basis). The scientific quality of COST
projects is well recognised and contributes to a coherent structure for European
research.
In the field of Food Science and Technology, COST is mainly concerned with improving
food safety, food quality and nutrition. Taking into account these main topics, COST
Action 95 is specifically devoted to "Improvement of the quality of the production of raw
milk cheeses". This Action (1995-1999) is chaired by Dr. Rmy Grappin from 'Station
de Recherches en Technologie et Analyses Laitires' in Poligny (France). At present,
twelve countries are actively participating in this COST Action: Austria, Denmark,
France, Greece, Ireland, Italy, Norway, Portugal, Spain, Sweden, Switzerland, and
United Kingdom. The Action is supported by the European Commission Directorate
General for Science, Research and Development, and in particular by Unit DG Xil/AP,
responsible for COST support and its Scientific Secretariat.
Cheese remains practically the only dairy product made from raw milk. Raw-milk
cheeses are of a prime importance for local economies and consumers appreciate
their large diversities in characteristics and taste. Indigenous microflora of raw milk
leads to significant differences in the proteolysis and fermentation products and is
mainly responsible for the typical sensory characteristics of raw-milk cheeses.
The main objectives of the Action are :
to study the dominant microflora in raw milk and in raw-milk cheese: isolation,
identification, and biochemical and genetic characterisation,
to assess the influence of the non-starter microflora in cheese on the ripening
process: growth and activity of the micro-organisms, biochemical modifications,
to develop and apply standardised methods for microbial, biochemical, Theological
and sensory analysis to traditional raw-milk cheeses,
to study the influence of indigenous milk microflora and heat treatment of milk on
the sensory properties of raw-milk cheeses, and try to relate these properties with
biochemical and Theological data. .
By organising European research co-operation on these issues and exchanging the
results, the Action is promoting harmonisation and standardisation of methodology.
With all these activities the Action wishes to make its contribution to raising the quality
of European research within this field.
Francisca Serra
Scientific Secretary
XI
ARD & POLYCHRONIADOU CHEESE ANALYSIS MANUAL
Abbreviations
a. k. accurately known
CN casein
DM dry matter
FPLC fast protein liquid chromatography
GLC gas-liquid chromatography
HPLC high performance liquid chromatography
i.d. inside diameter
IEC ion exchange chromatography
IEF isoelectric focusing
o.d. outside diameter
PAGE Polyacrylamide gel electrophoresis
SDS. sodium dodecylsulfate
uhqw ultra high quality water
Wc weight of cheese sample
XII
ARDO & POLYCHRONIADOU, Ed. CHEESE ANALYSIS MANUAL
1. INTRODUCTION
The traditional meaning of the chemical composition of cheese (chapter
3) is fat, protein, salt and moisture content and pH. In this manual also
methods for other parameters are included, such as water activity,
carbohydrates, lactate, citrate, phosphorous, calcium and other minerals.
Compounds that are produced during cheese ripening are mainly treated in
later chapters.
Instead of protein content, we use total N content, because protein
content is a parameter that is not really possible to define in a meaningful way
during cheese ripening. Protein content of cheese is of interest as a measure of
its nutritional value, and then it is rather the content of the total amount of
available amino acids that counts. As for the other foods, official methods refer
to N determination. The protein content has to be calculated from the N
content using more or less empirically found conversion factors; which factor
should be used will always be a matter of agreement between people. The N
content is a unique parameter very well defined in all types of cheese fractions.
Many traditional European cheeses - a large number of which have a
Protected Designation of Origin (PDO cheeses) - are made from milk of other
ruminants than cow. Thus, methods were proposed to detect milk origin in
cheese. We found it appropriate to include two of these methods and mention
a third, the official method of the European Union (chapter 4).
A large part of the manual is devoted to the determination of proteolysis
in cheese (chapters 5 - 9), which has been studied for at least a century. Over
time more and more sophisticated techniques have been evaluated for
analysing proteolysis in cheese, and at the same period of time efforts have
been made to simplify existing methods. We have today several methods to use
and our current knowledge about the whole system gives us new exciting
possibilities to analyse proteolysis. A through background is given in a series
of review articles that are published by the IDF (International Dairy
Federation). They cover methods for crudefractionationof N components in
cheese (Christensen et al., 1991), evaluating proteolysis by analysing the N
content of cheesefractions(Ardo, 1999), methods for direct measurement of
peptide bond cleavage in cheese (Ardo & Meisel, 1991), electrophoresis of
cheese (Creamer, 1991), capillary electrophoresis used to measure proteolysis
in cheese (Otte et al, 1999), chromatographic methods to measure proteolysis
in cheese (Ardo fe Gripon, 1991), chromatographic analysis and identification

of peptides in cheese (Singh et al., 1999) and quantitative determination of free
amino acids in cheese (Biitikofer & Ardo, 1999).
Methods for the identification of cheese peptides are currently
developed; some of them, using sequencing of the peptides and amino acid
analysis, are presented in the manual. Mass spectroscopy (MS) is a powerful
tool to determine molecular weights of the peptides and combinations of MS
and sequencing techniques provide useful information. Computerised data
bases comprising the amino acid sequences of all milk proteins in their different
genetic variants is required for a real success in this work.
Three methods for free fatty acids analysis are included in chapter 10
applied by different laboratories. Although all these methods seem to give
satisfactory results, a collaborative test showed some variation among results
of different laboratories, when reference samples were analysed (Biitikofer,
1996). When determination offreefatty acids is performed, precaution must be
taken both to get a high recovery and to avoid co-determination of fatty acids
existing in the milk fatfractionesterified with glycerol.
There is a growing interest in characterizing the flavour and aroma
compounds of cheese and the pathway of their formation. Only as example,
two recently published reviews on compounds involved in the flavour of
cheese are cited (Molinard & Spinnler, 1996; Urbach, 1997). New methods
and equipment are developed aiming to an improved analysis of volatile
compounds. Some of these methods are included in chapter 11.
Finally, methods for analysis of enzymes in cheese are given in chapter
12. These methods have been developed with different objectives and are in
many cases restricted to their specific application. Nevertheless, the
descriptions could be very useful when starting up work with these kinds of
analyses because several things must be taken into consideration. The enzyme
may be present in active or inactive form. It may be in active form bu still not
active in the current cheese environment. It may have contributed significantly
to the ripening even if it is inactivated when analysed, or it may become
activated at a later stage during ripening. Extracting the enzyme from the
cheese matrix must be done with special care since several factors may change
during the process activating or deactivating, stabilising or unstabilizing the
enzyme. Substrates, reaction products, enzyme activators and inhibitors may
be concentrated or diluted during preparation and disturb the measurements.
The analysis procedures have to be adapted to the specific objective of each
investigation. Different factors have to be taken into consideration if the

enzyme activity is determined, for instance, as a measure of heat load to cheese
milk, as an index of bacteria autolysis or as a possible contributor to cheese
ripening. [YA, AP]
2. SAMPLING AND GENERAL SAMPLE PREPARATION

Unless specified, arrangements follow the general recommendations of
IDF Standard 50C: 1995.
According to the size of hard cheese loaves, a piece of cheese (50-100
g) is cut out as a sector or using a trier. A piece is removed for each type of
analysis and in comparable areas within a loaf and from loaf to loaf. The
localisation depends on the purpose of the analysis but one must keep in mind
that a gradient of composition exists from the rind to the centre of the loaf.
Generally 1 cm of rind is removed before packing. The resulting piece is
immediately wrapped up in aluminium foil. Wrapping must be airtight to
prevent oxidation and dehydration.
Samples are analysed immediately, at least for pH, dry matter content
and activity of water, or if necessary after a storage period at -15C or lower
for other parameters. In this case, samples are rapidly frozen under -30C for
one night in a precooled freezer, then gathered in plastic bags and kept at -
15C until analysed. Before analysis, the packet is kept one night at 4C for
water to be reabsorbed then one hour at 20-25C. A thin layer (0.5 cm) is
removed on all the surfaces before subsequent operations.
The cheese is then cut in 1 cm3 cubes which are rapidly mixed up then
totally grated with a manual grater. This sequence allows the best
homogenisation avoiding the risk of aggregation for more adhesive cheese.
Samples are then rapidly weighed for the analysis. [SP]
Soft cheeses are preferentially ground frozen, semi-soft to semi-hard
refrigerated and hard cheeses at room temperature. [YA]
3. CHEMICAL COMPOSITION
3.1. Fat
Official methods
IDF Standard 5B:1986 (Schmidt-Bondzynski-Ratzla)
AOAC Method 933.05 (Rse-Gottlieb)
Routine butyrometric methods

Cheese is digested in a Gerber butyrometer for cheese by heating with a
strong mineral acid. Fat is then separated by centrifiigation forming a clear
layer. Amyl alcohol is eventually used for better fat separation. Percent fat
content is directly read on the butyrometer scale to the nearest 0.05%.
Duplicate determinations should not vary more than 0.10%. [AP]
METHOD I
Fat content is measured using the acido-butyrometric method described
by Heiss (1961) modified by Pien (1976). Grated cheese (3 g + 0.01) is
weighed in a Gerber butyrometer1 for cheese. Then 10 mL of a mixture of
acetic acid / perchloric acid (50/50) are added and the butyrometer is kept in a
water bath at 85C for 15 min until complete digestion (no particles). From
this point, the butyrometer is kept about 30 min more at 85 C. Before
removing the butyrometer from the water bath, hot water (63 C) is added to
allow the upper liquid fat column to be in the measurement zone. The
butyrometer is stopped up and centrifuged at 2000 rpm for 10 min, then
allowed to stand in a water bath at 65C for 5 min before carrying out the
direct reading of the fat content (g/kg cheese). Fat is determined in duplicate
per sample. [SP]
METHOD II (Gerber-van Gulik)

Grated cheese (3.0 g) is weighed in the weighing beaker of a cheese
butyrometer according to van Gulik. Then sulfuric acid (d=1.53 g/mL) is
added until cheese is covered. The butyrometer is kept in a waterbath of 65 C
until all the cheese is digested. Shaking occasionally facilitates digestion. Then,
1 mL amyl alcohol is added and the content of the butyrometer mixed
'Butyrometer: Gerber instruments, Effuetikon, CH.

thoroughly. Finally, more sulfuric acid is added in order to bring the upper fat
layer in the measurement zone.
The butyrometer is stopped, inversed carefully 1-2 times to mix the
content, placed again in the waterbath for 5 min, and centrifuged for 5 min at
1200 rpm and 65 C (thermostatically controlled Gerber centrifuge). The fat
content (% w/w) is read on the butyrometer scale. [AP]
3.2. Total nitrogen

A portion of cheese is digested with concentrated sulfuric acid in the
presence of potassium sulfate and of copper sulfate as a catalyst; organic
nitrogen is converted to ammonium sulfate. When cool the clear solution is
made alkaline with NaOH and ammonia is distilled off, collected in an excess
of boric acid solution and titrated with a standard solution of HCl or H2SO4.
[AP]
Official methods
Nitrogen content: AOAC Method 920.123 (Kjeldahl); IDF Standard 20B:1993
METHOD I
Grated cheese (0.2 g a.k)Wc) is thoroughly weighed on and then
wrapped up in a nitrogen free paper. Nitrogen content is analysed as follows:
Nitrogen is measured by the Kjeldahl method according to the IDF semi-
micro procedure (Standard 20B:1993). Sample is introduced in a special Pyrex
tube with 5 mL concentrated sulfuric acid and 3.2 g catalyser (copper
sulfate/potassium sulfate) and is digested in a heating block2 until colorless.
When cool, the tube is fixed on to the distillation unit3. Nitrogen is then
liberated from ammonium sulfate by the addition of an excess of sodium
hydroxide 10 N (25 mL) which is delivered automatically. Ammonia is steam
distillated out of the digest for 7 min and is recovered in 25 mL boric acid 1%
which is then automatically titrated with sulfuric acid 0.04N (VH2SO4) in the
presence of a colored indicator (mixture of methyl red and bromocresol green).
2
Digestion system 20,1015 digester, Tecator, Upsala, SW.
3
Distillation system : Kjeltec autosampler system, Analyzer 1038. Tecator, Upsala, SW.
Complete mineralisation is regularly checked with tryptophan samples.

Distillation conditions and concentration of the sulfuric acid is checked by
regular analysis of ammonium sulfate standard solutions.
Blank sample (sucrose) is regularly analysed to assess that reagents are
nitrogen free. If not, the value obtained for blank is subtracted from VH2S04.
[SP]
N in the test portion (mg) = 14007 x Vms04 (mL) x Acid normality

(to the nearest 0.0001)
Alternatively, 10 mL of citrate dispersion of cheese (see 4.2) are

analysed for total nitrogen (TN) by Kjeldahl method. [YA]
3.3. Moisture - Total solids - Dry matter

Moisture in a portion of cheese is removed by heating at 102C in the
presence of sand. The mass remaining after completion of the heating is the
total solids content. Weight loss is calculated as moisture. [AP]
Official methods
Moisture: AOAC Method 926.08
Total solids: IDF Standard 4A:1982
TOTAL SOLIDS
A known weight of grated cheese is dried at a constant temperature
(1022C) to a constant weight. The weight after drying is the weight of total
solids and is expressed as % by weight. [PMS]
DRY MATTER
Dry matter is determined by a simplified procedure of the IDF
gravimetric method (IDF Standard 4A:1982). Grated cheese (2 g a.k)Wc) is
uniformly distributed at the surface of an aluminium dish (i.d. = 50 mm, h = 25
mm) containing sand (20 g) both being dried in an oven at 1022C until
constant weight (e.g. 24 h) (Wi). The dish with the cheese sample is then dried
and weighed in the same conditions (W2).
DM (% cheese)=((W2-W!) / Wc) x 100

Moisture (% cheese) = 100 - DM
Dry matter is determined in triplicate per sample. [SP]
3.4. Water activity
The activity of water (aw) is estimated by the measurement of the

relative humidity at equilibrium (RHE) which does not lead to any variation of
the sample mass (Landrock & Proctor, 1951). Grated cheese (lg 0.010) is
weighed in half of a Petri dish (i.d.=55 mm, h=15 mm). The dish is then placed
via an inox ring over 30 mL of a saturated salt solution of known RHE poured
in an airtight box (i.d.=80 mm, h=30 mm) greased with silicone. Solutions of
three different salts are used in triplicate for each sample :
- potassium dichromate (aw20C=0-98): 150 g dissolved in 500 mL
boiling water, then when cool 100 mL water are added and stirred.
- ammonium dihydrogen phosphate (aw20C=0-935): 200 g dissolved in
500 mL boiling water, then when cool 200 g salt are added and stirred.
- potassium dihydrogen phosphate (aw20C=0-965): 170 g dissolved in
500 mL boiling water, when cool 20 g salt are added and stirred.
These solutions must be kept in a very well controlled water bath at
20C several days before use. Petri dishes are thoroughly weighed after
precisely 24 h at 20C. Variation of the cheese weight is recorded (AW). The
differences are plotted on a graph aw=f(AW) (Figure 3.1). The aw of the
cheese is the value at the intercept of the straight on the aw axis (AW=0). This
value can be also calculated as follows :
awf = - [((SAW.Eaw2)-(Saw. S(AW.aw)) ) / ( (3Z(AW.aw)) - (Saw.EAW) ) ]
where aw = activity of water of each salt solution
2 = sum of the values obtained with the three different salts. [SP]
10
AW i
o,c*
O,o$
O.Ol
aW of the cheese sample f m
0,01
1/
*> Jr tOO
-Ol
Relative Humidity al
.0,01
-,05
-004,
e os
Figure 3.1. Plot for the determination of aw [SP].
11
3.5. Sodium chloride

Official methods
Chloride: IDF Standard 88A:1988; AOAC Method 983.14 (Potentiometrie
titration)
IDF Standard 17A:1972 (titrimetric method)
METHOD I
Grated cheese (2 g) is placed into a 150 mL beaker and 100 mL dilute
nitric acid solution added (1.5 mL cone. HNO3 made up to 1 L). This solution
is then heated at 60C for 1 h and titrated with 0.1 N AgNC3. The end point is
determined potentiometrically and is reached when a difference of +255
mV is obtained between the silver electrode and the reference electrode (Fox,
1963).
Percent NaCl = (mL AgN03 x 0.588) / wt. sample [PMS]
METHOD n
Sodium chloride is estimated by a Potentiometrie method4. Grated
cheese (1.5 g a.k.)(Wc) is blended5 for 1 min in 100 mL water. After allowing
to settle for 30 min, the difference in potential is measured between the
reference electrode and the soluble silver electrode using 0.5 mL of the clear
supernatant. The value is recorded when constant. The chloruremeter is
checked with 0.5 mL standard solution of chloride6. Measurements are done
on two different test portions per sample.
NaCl (%DM) - (read value x 0.016)-0.07 / (Wc x DM) [SP]
4
Chloruremeter Corning 926. Corning limited, Halstead, Essex, GB
^Fltra-Turrax T25 (24 rev/min), KR18G. Janke et Kunkel, IKA Labortechnik, Staufen, DE
6
Chloride Meter Standard (200mg/L). Ciba-Corning. Corning limited, Halstead, Essex, GB
12
3.6. pH
A pH meter equipped with a combined glass/calomel electrode is used to
measure the activity of hydrogen ions in cheese. Measurement may be
performed using grated cheese or a cheese slurry. [AP]
METHOD I
Grated cheese is packed in a cylinder (h=3 cm, o.d.=1.5 cm) and put in
close contact with a combined electrode thoroughly calibrated with two buffer
solutions at pH 4.01 and 7.00. The pH value to the nearest 1/100 unit is
recorded 30 s later. The measurement is repeated consecutively using three
different test portions. Between each measurement, the electrode is wiped to
remove most of the cheese paste, soaked a few seconds in ethanol/ether
(50/50), rinsed with water, then reequilibrated in buffer 4.01 and wiped. [SP]
For some semi-hard and hard cheese varieties addition of a small amount
of water is needed to get proper contact between the cheese and the electrode.
[YA]
METHOD II
Grated cheese (10 g) is thoroughly blended with 10 mL H20 using a
mortar and pestle and the pH of the resultant slurry measured
potentiometrically using a pH meter.
It could be argued that the addition of water results in changes in the salt
balance and hence pH of cheese. It may be preferable, therefore, to measure
the pH of cheese directly by placing the electrodes in contact with grated
cheese (see Method I). [PMS]
3.7. Carbohydrates
Official methods for lactose
IDF Standard 43:1967; 147A:1994
AOAC Method 930.32
3.7.1. GLC method for the analysis of carbohydrates

This gas chromatographic method using a micropacked column was
developed for the analysis of lactose, galactose, lactulose and epilactose in
processed milk (Olano et al, 1986), but was further applied to cows milk
13
cheeses and cheeses made with mixtures of cow's, ewe's and goat's milk with
or without the addition of proteolytic and lipolytic enzymes (Fernandez-
Garca et al, 1988 & 1994).
Preparation of the sample. Carbohydrates are extracted following a
modification of the method of Harvey et al. (1981). A 4-g cheese sample is
homogenized in 20 mL distilled water and 5 mL phenyl--D-glucoside (1
mg/mL), used as internal standard. The mixture is filtered through Whatman
No 1 paper. The resulting extract (5 mL) is diluted to 25 mL using methanol.
The mixture is allowed to stand at room temperature for 1 h, and then filtered
through Whatman No. 42filterpaper. Thefiltrateis vacuum-dried at 38-40C.
Anhydrous pyridine (2 mL) is added, and the mixture refluxed for 1.5 h.
Trimethylsilylimidazole (100 uL) is added to 200 uL of the pyridine solution.
After 30 min at 65-70C, the solution is cooled and 0.1 mL hexane and 0.2 mL
distilled water are added. The hexane solution (2 uL) is injected.
Chromatographic determination. Chromatographic conditions are those
described by Olano et al. (1986). The GLC analysis is performed using a gas
Chromatograph7 equipped with a flame ionization detector, using a 3 m x 1.0
mm stainless steel column8 packed with 2% OV-17 on non-silanized 120140
Volaspher A-29. Nitrogen is used as carrier gas at a flow rate of 10 mL/min.
The temperature of the injector and the detector is 300C. The analysis is
performed using temperature programming from 200 to 270C at a heating
rate of 15C/min with an initial holding at 200C for 2 min.
Figure 3.2 shows a chromatogram of trimethylsilyl derivatives of free
carbohydrates in processed milk. [RLF]
3.7.2. HPLC method for the analysis of carbohydrates

This method specifies a HPLC procedure for the determination of the
lactose, glucose and galactose contents of cheese. Results are expressed as a
percentage by mass (IDF Standard 147A:1994).
All reagents shall be of recognized analytical grade. The water used shall
be double distilled water or of at least equivalent purity.
7
Sigma 3B, Perkin-Elmer.
8
Chrompack.
9
Merck
14
4 e I K> T2
lim (minutes)
Figure 3.2. A chromatogram of trimethylsilyl derivatives of free

carbohydrates in processed milk. 1-3 galactose, 4 phenyl--glucoside (internal
standard), 5 a-epilactose, 6 lactulose and -epilactose, 7 ct-lactose, 8 -lactose
(After Olano et al. 1986, reproduced by courtesy of Chromatographia).
15
Reagent for sample pretreatment. Dissolve 91.0 g of zinc acetate

dihydrate, Zn(CH3COO)2-2H20, 54.6 of phosphotungstic acid 24-hydrate,
H3[P(W3Oio)4]'24H20, and 58.1 mL of glacial acetic acid in water and make
up to 1000 mL.
Eluent. Filter the water HPLC grade through a membrane filter with a
0.45 um pore diameter and, prior to use, boil to remove dissolved air.
Standard samples. Prepare standard solutions containing 5, 10, 50, 100
and 500 mg of lactose or glucose or galactose per 100 mL of water.
HPLC equipment. Magnetic stirrer and heater for keeping the eluent at
90C. Pump capable of delivering a flow of 0.3 mL/min with a pulsation less
than 1% of the pressure drop over the column (about 1.5 MPa). Thermostatic
column oven, regulated at 75C.
An HPX-87 P column (Bio-Rad, 30 x 0.78 cm) or an equivalent column
packed with sulphonic ion exchanger in the lead form, based on a polystyrene
divinylbenzene 8% crosslinked polymer. The pre-column consists of the Bio-
Rad de-ashing system (a cartridge, 3 x 0.46 cm, packed with a cation exchange
resin in the hydrogen form and a cartridge, 3 x 0.46 cm, packed with an anion
exchange resin in the carbonate form) or a system of equivalent effectiveness.
Highly sensitive refractive index detector, with a noise level less than
5 x IO"9 RIU, measured in water. The internal thermostat should be set at a
temperature above room temperature sufficient to get a stable baseline. A
temperature of 35-40C is in most cases advisable. Integrator capable of peak
height measurement.
Preparation of the sample. Prior to analysis, remove the rind or smear or
mouldy surface layer of the cheese, in such a way as to provide a sample
representative of the cheese as it is usually consumed. Grind or grate the
sample by means of an appropriate device; mix the ground or grated mass
quickly, and if possible grind or grate a second time and again mix thoroughly
by intensive stirring and kneading.
Transfer the test sample to an air-tight container to await analysis, which
should be carried out as soon as possible after grinding. If delay is unavoidable,
take all precautions to ensure proper preservation of the sample and to prevent
condensation of moisture on the inside surface of the container.
Clean the device after grinding each sample.
Test portion. Weigh 10 g of the test sample, to the nearest 1 mg, into a
beaker. Dissolve or suspend the test portion in about 50 mL warm water (30-
40C) using a glass rod. Rinse the beaker with 30 mL water and transfer the
16
content of the beaker quantitatively into a 100-mL volumetric flask. Pipette

10 mL of the reagent for the sample pretreatment with a graduate pipette and
swirl. Dilute to 100 mL with water and mix.
Filter in a glass runnel through medium grade filter paper; discard the
first 5 mL of filtrate.
Blank test. Carry out a blank test using the reagents but omitting the test
portion.
Chromatographic determination. Inject 30 uL (accurately measured) of
filtrate into the HPLC apparatus operating at aflowrate of 0.3 mL/min.
Always include calibration samples with every series of samples.
Recalibrate every 10-15 samples.
Calibration. Least square linear regression analysis for the pairs H(5)-
C(5), H(10)-C(10), H(50)-C(50), H(100)-C(100) and H(500)-C(500), with
C(5), C(10), C(50), C(100) and C(500) as the independent variables, gives the
coefficients K(l) and K(2)
H = K(l)xCK(2)
where:
H = peak height of lactose (or glucose, or galactose) of the standard solutions;
C = concentration of lactose (or glucose, or galactose) of the standard
solutions.
Test sample. Calculate the lactose (or glucose, or galactose) content of
the test sample (E):
H(E)K(2)
C(E) = xlOO
K(l)xM(E)
where:
C(E) = lactose (or glucose, or galactose) content of the sample (E), in grams
per 100 grams of cheese;
H(E) = peak height of lactose (or glucose, or galactose) of the sample (E);
K(l) and K(2) = coefficients calculated as described above (Calibration);
M(E) = mass in grams of the test portion.
Repeatability. The difference between two single results found on

identical test material by one analyst using the same apparatus within a short
17
time interval should not exceed 6% (relative) of the arithmetic mean of the
results.
Reproducibility. The difference between two single and independent
results obtained by two operators working in different laboratories on identical
test material should not exceed 20% (relative) of the arithmetic mean of the
results. [JH]
3.8. D-/L-Lactate
Grated cheese (lg a.k) is blended for 1 min with 50 mL water then
centrifuged for 30 min at 1200 g and 4C. The upper solidified fatty layer is
removed with a spatula and the supernatant is filtered on paper. D- and L-
lactic acid concentrations are determined on the filtrate using the Boehringer
enzymatic method kit10. In the presence of D-(L-) lactate dehydrogenase,
D-(L-) lactic acid is oxidised by the nicotinamide-adenine dinucleotide (NAD)
giving pyruvate along with the production of reduced NAD (NADH). The
quantity of NADH is measured by UV absorption and is proportional to the
quantity of lactic acid. In order to shift the equilibrium in a favorable
way, L-glutamate and glutamate-pyruvate transaminase (GPT) are added to
the medium to eliminate L-glutamate as soon as it is produced. Optical density
is read at 340 nm against air. Each sample is extracted twice and eachfiltrateis
analyzed twice. [SP]
Note: If the reaction is applied to half of the quantities of sample and reagents
suggested by Boehringer, semi-micro cuvettes shall be used.
3.9. Citrate
Official methods
IDF Standard 34C:1992 (enzymatic method)
AOAC Method 976.15 (colorimetrie method)
l0
UV method for the determination of D- and L-lactic acid in foods and other materials.
Re 1 112 821. Boehringer Mannheim, Meylan, FR
18
3.10. Carbon dioxide

Samples are emulsified under reduced pressure in an alkaline citrate
medium in an airtight mixer and CO2 is quantitatively displaced into the head
space by addition of an excess of sulfuric acid. A small gas pump ensures gas
circulation to a specific non-dispersive infrared detector mounted in a by-pass
for measurement. The residual depression in the head space is suppressed
shortly before measuring. The equipment is calibrated with standard gases and
standard additions of sodium carbonate. The method is sufficiently simple and
rapid for routine use (about 30 samples/day). The coefficient of variation is
less than 1 % for standard solutions and less than 2 % for cheese. The matrix
has practically no influence on the accuracy of the measurements Bosset et al,
1980). [UB]
3.11. Ammonia
3.11.1. Photometric method
The cheese is suspended in water. Caseins are precipitated by ZnSO-t.
Nessler reagent is added to the filtrate and the yellow product is measured at
425 nm (Mrowetz, 1979).
Reagents. Hydrochloric acid 10 %; ZnSC<4 solution (30 g Z11SO4.7 H2O
in 100 mL water); Nessler reagent11.
Potassium tartrate solution (50 g potassium tartrate in 100 mL of water.
Add 5 mL of Nessler reagent and filter on the following day through a black
ribbonfilterpaper)
Standard solution 1 mg/mL (297 mg dry ammonium chloride is dissolved
in a 100-mL volumetricflaskwith water)
Apparatus. Homogenizer; photometer (425 nm).
Procedure. 1 g of cheese is given to a 50-mL centrifuge tube. 20 mL
of water are added. The mixture is homogenized 1 min at 10 000 rpm. 1 mL
ZnSC4 solution is added and the suspension is homogenized for 20 s. A black
ribbon paper filter is washed with 10 mL hydrochloric acid 10 % and twice
with 10 mL hot water. The suspension is filtered through the black ribbon
paper filter; the first 4-5 mL are eliminated. Then, 5.00 mL water, 1.00 mL
"Merck 9028
19
filtrate solution and 0.100 mL potassium tartrate solution are mixed in a 10-
mL reagent tube. 1.50 mL Nessler reagent is added and well mixed. After
601 s the absorbance is measured at 425 nm against water. [UB]
Blank value. Use 0.5 mL water instead of the cheese sample.
Calculation.
mg NHt/kg cheese = (A-B) x 1000 / SL / V / S
A = absorbance of the cheese sample

B = absorbance of the blank
V = volume used for the reaction (normally 1 mL)
S = sample weight, g
SL = slope of calibration curve
3.11.2. Enzymatic method

The determination of ammonia is based on an enzymatic test method12.
In the presence of glutamate dehydrogenase and reduced NADH ammonia
reacts with 2-oxo-glutarate to L-glutamate, whereby NADH is oxidized. The
amount of NADH oxidized is stoichiometric to the amount of ammonia
present in the sample solution. NADH is determined spectrophotometrically
via its absorbance at 340 nm.
Preparation of the cheese extract. A mixture of 3.00 g grated cheese and
97.00 mL distilled water is homogenized with a T25 Ultra-Turrax5 for 1 min at
9500 rpm, stored in a refrigerator for 1 h and subsequently filtered over a
Schleicher & Schuell 602 Vi h folded filter. Usually, 100 (iL sample solution
can be taken for the measurement.
Measurement. 100 uL sample solution is added to 1.00 mL reaction
mixture 2 (prepared with Solution 1 and NADH tablets of bottle 2 according
to the instructions) in a polystyrol cuvette and gently mixed. After addition of
1.92 mL (2.02 mL for the blank) distilled water, the absorbances of the
solutions after approximately 5 min at 20-25 C are recorded (Al). The
reaction is then initialized by addition of 20 uL of glutamate dehydrogenase
solution (Solution 4). Approximately 20 minutes later, the second
measurement should be done (photometer reading A2).
Calculation. After calculation of the absorbance differences of sample
and blank (Al - A2) the sample-specific absorbance difference AA is equal to
"Reference to Ammonia Testkit 1 112 732. Boeringer Mannheim GmbH, Mannheim, DE
20
AA = ^SAMPLE - AALANK
The concentration of ammonia in the sample solution c is equivalent to
c = (V MW / e v 1000) x AA
where V being the final volume in the cuvette [mL], v the sample volume
[mL], MW the molecular weight of ammonia [g/mol], the light path length
[cm] and e the molar extinction coefficient of NADH (6.3 1 / mmol g at 340
nm). [DJ]
3.12. Calcium
Official method
AOAC Method 991.25
Calcium in cheese can be determined by titration with a chelating agent

(EDTA) at a high pH range (~13). The end point of the reaction is detected by
the colour change of a metallochromic indicator. [AP]
Total calcium is measured by the complexometric method of Pearce

(1977) modified in our laboratory by Jeunet (np). Grated cheese (1.5 g a.k)
(Wc) is blended5 for 1 min in a mixture of 50 mL water and 10 mL
hydrochloric acid 10%. The Turrax rod is repeatedly rinsed working 30 s with
50 mL water (2 mL of sodium hydroxyde 10 N is added the first time) and the
rinsings are added to the suspension up to 400 mL. After 15 min, 8 mL sodium
hydroxyde 10 N is added. Calcium is titrated under stirring using disodium
ethylenediamine-tetraacetate (EDTA) 0.05 M in the presence of 2 drops of
Parton and Reader colored indicator13 until blue (VEDTA). Special care is
needed to stop titration at the same blue color intensity for all the samples,
which is certainly a limitation for the precision of the method. 10 mL of a
solution of calcium chloride, exactly 0.05 M, is used to check that 10 mL of
the solution of EDTA 0.05 M titrates the equivalent of 2 mg of calcium.
Measurements are done in duplicate from two different test portions. [SP]
Ca (%DM) = 2 x VEDTA X 10 / (WC X DM).
13
CalconCarbonSaure (Merck) (0.4 g) + Triethanolamine (30 mL) + Methanol (10 mL)
21
3.12. Phosphorus
Official methods
IDF Standard 33C:1987; AOAC Method 990.24 (photometric method)
Phosphorus is determined by digestion of cheese with sulfiiric acid and

hydrogen peroxide. Molybdenum blue is formed by addition of sodium
molybdate and ascorbic acid. The intensity of the blue colour is measured
spectrophotometrically at 820 nm. [AP]
Phosphorus is measured by a photometric standardised method (IDF

Standard 33C:1987). Grated cheese (0.5-1 g a.k)(Wc) is weighed on a
phophorus free paper which is transferred in a digestion flask. The mixture is
digested (using a burner) with 4 mL sulfuric acid and as soon as foaming stops,
the mixture is cooled to room temperature and 2 mL hydrogen peroxide 30%
are added. The procedure is repeated until completely clear and colorless. The
mixture is cooled, transferred into a 100-mL volumetric flask, adjusted to the
mark and mixed. A test portion (1 mL) is then diluted 25 times with water in a
50-mL volumetric flask and 20 mL of a mixture of 0.5% ascorbic acid and
0.625 % dehydrated sodium molybdate in 0.125 M sulfuric acid are added.
The coloration is measured at 820 nm and is proportional to the molybdenum
blue which is formed. Phopshorus (V) is calculated from a calibration curve
DO=f(P) established from 5 standard solutions of potassium orthophosphate of
known concentration (0 to 50 ug/mL). [SP]
P (%DM) = (V (ug) x 10"2) / (Wc x DM).
3.14. Other minerals - Na, K, Mg, Zn, Fe, Cu

Official methods
Copper: IDF Standard 76A:1980 (photometric method)
Iron: IDF Standard 103A: 1986 (photometric method)
Zinc: IDF Standard 156: 1992 (atomic absorption method)
22
ATOMIC ABSORPTION SPECTROSCOPY

Sample preparation. 3 mL perchloric acid and 5 mL nitric acid are added
to 0.4 - 0.5 g grated cheese weighed accurately into a 50-mL Erlenmeyer flask
(Maurer, 1974). After adding some glass beads, organic matter is oxidized by
heating on an appropriate heating plate (~ 130C, usually 2 h). In case of
coloured digestions more nitric acid is added in aliquots of 0.5 mL until the
solution appears clear. After cooling the flask to room temperature, the
solution is quantitatively transferred into a 50-mL measuring flask. Further
dilutions depend on the element to be analyzed and on instrumental conditions.
Note: In case of determination of Zn and Cu the use of Suprapur reagents and
polypropylene flasks (50 mL) is strongly recommended. All glassware has to
be cleaned with diluted nitric acid and thoroughly rinsed with distilled
water. [HR]
23
4. MILK ORIGIN
REFERENCE METHOD FOR THE DETECTION OF COWS MILK IN
EWE'S MILK
A reference method to detect bovine casein in cheeses made from ewe's milk
has been published in the Official Journal of the European Community (1992). This
method allows the detection of bovine milk in quantities ^ 1 % . It is based on the
different isoelectric point of Y2-CN and Y3-CN of cow's and ewe's milk after
plasminolysis of the casein fraction.
Other screening methods based on the different electrophoretic mobility or
the different isoelectric point of -lactoglobulins (-LG) of milk of different species
can be used (Amigo et al, 1992). The methods based on the analysis of whey
proteins have the advantage that whey proteins are less susceptible to proteolysis
than casein, although they are more affected by the heat treatment. [RLF]
DETECTION OF COWS MILK IN EWE'S MILK

BY POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)
Electrophoresis in Polyacrylamide gels (9.4% T, 4.25 %C, pH 8.9) is
performed using a vertical slab gel unit14 according to the method of Amigo et al
(1989). Gels are stained by Coomassie Blue G-250 (Blakesley & Boezi, 1977) or
R-250 (Wintere/ al, 1977).
Stock solutions
Acrylamide, bisacrylamide solution: 9 g acrylamide and 0.4 g N-N'-
methylene bisacrylamide are dissolved in, and made up to 100 mL with gel buffer
Gel Buffer: 4.6 g tris (hydroxymethyl) methylamine, Tris, and 0.4 mL HCl
35% are dissolved in, and made up to 100 mL with H2O. The pH may
be adjusted to pH 8.9.
Electrode Buffer: 1.2 g Tris and 5.8 g glycin are dissolved in, and made up
to 2 L with H2O, and adjusted to pH 8.3 with HCl.
Ammonium Persulfate: 10%w/vinH2O.
14
Hoefer Scientific Instruments, San Francisco, CA, USA
25
Staining solutions
a. Commassie Brilliant Blue G-250 (Blakesley & Boezi, 1977)
An aqueous solution of Coomassie Brilliant Blue G-250 (0.2% w/v) is
added to an equal volume of IM H 2 S0 4 and held overnight. The solution is then
filtered through Whatman No 1filterpaper and the filtrate mixed 9:1 with 10 M
KOH. Trichloroacetic acid is then added so that the final solution is 12% (w/v)
with respect to TCA. Gels are stained directly by this solution.
b. Commassie Brilliant Blue R-250 (Winter et al, 1977)

Steps Reagent Amount Storage Time
lJFix TCA 57.5 g Up to 60 min

Sulphosalicylic acid 17.3 g weeks
+water up to 500 mL
2.Destain Ethanol 500 mL Up to 10min
Acetic acid 160 mL weeks
+ water up to 2000 mL
3.Stain Coomassie Brilliant Same Overnight
Blue R-250 0.46 g day
+destain solution up to 400 mL
4.Destain = Step 2
Sample preparation. Whey samples are prepared by acidification of milk (10

mL) to pH 4.6 with 10% (v/v) acetic acid (5 mL) and 1 N sodium acetate (5 mL)
and centrifugation at 3000 g during 10 min. To one volume of the fraction soluble
at pH 4.6 an equal volume of glycerol solution (40%) and bromophenol blue
solution (1/1000) is added.
Preparation and running the gel. The electrophoresis unit is assembled

according to the manufacturer's instructions. Immediately before use, TEMED (50
uL) and ammonium persulfate solution (0.5 mL) are added to 50 mL acrylamide-
bisacrylamide solution. The gel is allowed to polymerize (30 min). The slot-former
is removed and the gels placed into the gel unit which isfilledto the correct level
with electrode buffer.
The gels are pre-run at 220 V for 10 min and the samples are applied (10-20
ViL of fraction soluble at pH 4.6). The system is cooled by recirculating cold water.
?fi
Samples are run at 220 V (60 min) until the tracking dye front is close to the
bottom of the gel slab (3 h).
Gels are stained by immersing into gel stain G-250 or R-250, and destained
in a few changes of distilled water until the background becomes clear.
Interpretation of results. Bovine -lactoglobulin (-LG) A and B exhibit

higher electrophoretic mobilities than do caprine and ovine -LG, and these
proteins can, therefore, be used to identify mixtures. In the case of admixtures of
cow's and goat's milk, the mobility of both bovine -LG and bovine a-
lactoalbumin (a-LA) was higher than that of caprine -LG. In mixtures of ewe's
and goat's milk, it is the caprine -LG that displays a lower electrophoretic
mobility than ovine -LG (Figure 4.1).
Densitometrie measurements are carried out at 600 nm15. Quantification is
based on the measurement of the height of the peaks of -LG and bovine serum
albumin (BSA). Ewe's milk containing 5, 10, 20, 30 and 40% cow's milk is used
for the calibration curve. Linear relationships are calculated for bovine -LG / BSA
on the percentage of cow's milk. [RLF]
DETECTION OF NATIVE AND DENATURED BOVINE

-LACTOGLOBULIN IN CHEESES MADE FROM EWE'S MILK
The reference method is not valid for detecting whey proteins incorporated
into cheeses to increase the yield and nutritional value. Recently, Molina et al.
(1996) have developed a western blotting method to detect adulterations with
pasteurized bovine milk, UHT or heat denatured bovine whey proteins in cheeses
made of milk from other species. The method is based on the separation of whey
proteins isolated from the casein fraction by PAGE and/or IEF followed by
immunoblotting with antibovine -LG antiserum which allows the detection of
bovine heat denatured whey proteins.
Isolation of native and denatured whey proteins. Native whey proteins are
obtained by homogenization of 5 g of cheese with 8 mL of water. Caseins are
precipitated with IN HCl at pH 4.6 and centrifuged at 3,000 g during 20 min. The
denatured (DN) whey proteins are obtained from the casein fraction, previously
obtained following the EU Reference Method (1992). Briefly, 10 mg of casein are
>s
Shimadzu equipment, consisting of a spectrophotometer with an integration and graphic
printing system (DR-2 Date Recorder).
27
homogenized with 100 mL of 0.01 M Tris-glycine buffer, pH 8.3, and 3 mL of

mercapto-ethanol. After incubation for 2 h at room temperature (23 C), the
homogenates are centrifuged at 3000 g. The supematants are analyzed by PAGE
or 1ER
11 10 9 8 7 6 5 4 3 2
*r
^f^m^ iMMr^Sl*i{|||
\ v i*-" jS '-'
v *.; '
SA
SA * n - m * m*m *w> tMf **m- ?r*>* *& i"*1*
<Hg(got)
/Hglew) ^HHkMBfc ^ ^ ^ A^. M I^Mf u d 'ttMg^ W ' ^ f c i * a-la (cow)
, ^ ^ ^ T ^W 4 H V MM* * i^MV J l ^ * ^ ^ ^ ^ ^ ^ ^ ^ -. m^^ _
-lgBfcow)
-i 'M-&-
Figure 4.1. Electrophoretogram of the proteinfractionsoluble at pH = 4.6 for:

1) cow milk; 2) goat milk; 3) ewe milk; 4) 50 % C/ 40 % B/ 10 % O; 5) 50% C/
30%B/ 20% O; 6) 50 % C/ 20 % B/ 30 % O; 7) 50 % C/10 % B/ 40 % O; 8) 50
% B/ 40 % a 10 % O; 9) 50 % B/ 30 % a 20 % O; 10) 50 % B/ 20 % C/ 30 %
O; 11) 50 % B/ 10 % a 40 % O [B = cow milk; C = goat mk; O = ewe milk]
(After Amigo et al. 1989, reproduced by courtesy of Milchwissenschaft).
28
IEF. The IEF is performed in PhastSystem apparaius16using ready cast

Phast Gels IEF at pH 3 to 917. The following conditions are employed: step 1,
prefocusing: 2,000 V, 2.5 mA, 3.5 W at 15C for 75 Vh; step 2, focusing of the
sample: 200 V, 2.5 mA, 3.5 W at 15C for 15Vh; and step 3, final focusing: 2000
V, 2.5 mA, 3.5 W at 15C for 410 Vh. Sample application is down at 0 Vh of step
2 and up at 0 Vh of step 3.
PAGE. PAGE is also performed in a PhastSystem apparatus using ready
homogeneous 20% gels18. The following conditions are employed: step 1,
preelectrophoresis: 600 V, 12.5 mA, 3.5 W at 15C for 80 Vh; step 2, sample
application: 600 V, 12.5 mA, 3.5 W at 15C for 2 Vh; and step 3, electrophoresis:
600 V, 12.5 mA, 3.5 W at 15C for 180 Vh. Sample application is down at 0 Vh
of step 2 and up at 0 Vh of step 3.
Immunoblotting. Following PAGE or IEF, proteins are transferred by a 10-
min diffusion step from the Polyacrylamide gel onto a 0.45 um nitrocellulose (NC)
paper19, previously soaked during 10 min in a blotting medium [20 mM
tris(hydroxymemyl)aminoamethane, 150 mM glycine, and 20% methanol].
Immunodetection is carried out according to the method of Tsang et al. (1983),
but slightly modified as indicated below. Commercial polyclonal antiserum raised in
rabbit against bovine -LG20 is used as primary antibody. The NC paper is dipped
into a solution of 25 uL of primary antibody in 8 mL PBS-gelatin solution [1%
gelatin, 20% PBS (4.5% NaCI and 3.9% NaH2P04, pH 7), and 0.2% Triton X-
100] for 45 min. The NC paper isflushed3 times with water. After 3 rinses, of 10
min each, with 15 mL PBS-gelatin solution, incubation with 25 uL labeled
secondary antibodies in 8 mL PBS-gelatin solution is performed for 45 minutes.
Horseradish peroxidase conjugated anti-rabbit IgG antibody raised in goat21is used
as labeled secondary antibodies. The NC paper is againflushed3 times with water.
Following a 10-min rinse with 15 mL of PBS-gelatin solution, and two 10-min
rinses with 25 mL of PBS diluted with water 1:5, 25 mL of a solution of 0.05%
diaminobenzidine and 6% of 1% NiCl2 in 0.1 M Tris-HCl buffer, pH 7.5,
containing 0.8% of H2O2 are added. Staining is stopped by rinsing with water
when the desired background and band intensity are reached. [RLF]
16
Pharmacia Fine Chemicals, Uppsala, SW
I7
Pharmacia
18
Pharmacia
19
Bio-Rad, Richmond CA, USA
20
Nordic Immunology, Tilburg, NL
21
Bio-Rad, Hercules, CA, USA
29
5. NITROGEN FRACTIONS
The first step used for cheese nitrogenfractionationis the preparation of
a water extract at the pH of the cheese [SP, PMS, AP, UB, HR] or a citrate
dispersion of cheese at pH >7 where the caseins are soluble. Two fractionation
schemes are described below starting with a water extraction or with a citrate
dispersion and any of them can be used to evaluate proteolysis in cheese during
ripening by analysing N content of each fraction by Kjeldahl (Ardo, 1999).
Each scheme has been developed with the intention to facilitate the laboratory
work and the calculations as much as possible and thereby also optimize
analysis parameters such as precision and reproducibility. [YA]
5.1. Fractionation scheme for water soluble N compounds

Cheesefractionationwith water is a widely used method, especially for
mature Cheddar that has a low and rather constant pH. The method is easy to
perform and no extra chemicals are added, which facilitates further actions like
freeze drying of fractions, extraction with organic solvents and
chromatographic analysis. A fractionation scheme to determine cheese
proteolysis should comprise Kjeldahl analysis of cheese for total N (TN),
water soluble N fraction (WSN), 12 % trichloroacetic acid soluble N fraction
(TCA-SN) and phosphotungstic acid soluble N (PTA-SN), respectively, and
analysis of dry matter content of cheese (DM), which is needed for the
calculations (Btikofer et al, 1993; Jarrett et al., 1982; Kuchroo & Fox,
1982).
Ten grams of cheese are mixed with 50 mL deionized water and
homogenised by using a Stomacher (5 min at 40C) or an Ultra Turrax (1 min
at 10 000 rpm, 1 min rest, 1 min at 10 000 rpm). The homogenate is then held
for 1 h at 40C. The samples are centrifiiged at 3000 g for 30 min at 4C or 30
min at 20C and then cooled to 4C. The suspension is finally filtered
preferentially through glasswool before the N content is determined with
Kjeldahl.
31
For preparing the TCA-SN fraction is used 25 mL WSNfractionwhich

is added 25 mL 24 % TCA solution. The suspension is held at room
temperature for 2 h and thenfilteredthrough Whatman No. 40filterpaper.
For the PTA-SN fraction is used 10 mL WSN fraction which is added
7.0 mL of 3.95 mol/L sulphuric acid solution and 3.0 mL 33 % PTA solution.
ThefinalPTA concentration will be 5 %. The mixture is equilibrated overnight
at 4C and thenfilteredthrough Whatman No. 40 filter paper before Kjeldahl
analysis is made.
Analyses recommended are: double analyses of DM, triple analyses of
TN, two fractions of WSN from cheese, one TCA fraction from each WSN-
fraction, one PTA fraction from each WSN-fraction and double Kjeldahl
titration of eachfraction.[YA]
5.2. Fractionation scheme for a citrate dispersion of cheese

Fractionating a citrate dispersion of cheese under pH control makes it
possible to compare proteolysis in cheeses with different pH due to age or
cheese making procedure. There is no need of analysing dry matter and all
results are given as % TN, analysed from the same citrate solution as is
fractionated. This facilitates the calculations and decreases variation due to
differences between the samples of cheese used for WSN, TN, and dry matter,
respectively. It is also an advantage to not be dependent of the analysis of dry
matter in cheese, which is not easy to perform and often gives a large variation
in the results.
In the method for pH4.4-SN described below a constant amount of acid
is added to secure a pH in an interval around pH 4.4. This method can be used
for most cheese varieties, but pH should be checked after acid addition, when a
new cheese variety is analysed for thefirsttime. A citrate dispersion is made of
the cheese, and from this dispersion samples are taken to Kjeldahl analysis of
total N (TN), soluble N compounds at pH 4.4 (pH4.4-SN), and PTA soluble N
compounds (PTA-SN). A sample for TCA-SN is taken from the pH4.4-SN
fraction (Btikofer et al, 1993; Mogensen, 1947; Stadhouders, 1960).
32
Grated cheese (10 g) is mixed with 50 mL warm (40 - 50C) 0.5 M tri-
sodium citrate solution, stirred by a magnet stirrer for 60 min and then cooled
to room temperature and made 200 mL with deionized water.
Of this citrate dispersion, 10 mL are analysed by Kjeldahl for TN. The
pH4.4-SN is fractionated by adding 11.3 ml 1 M HCl to 80 mL of the citrate
dispersion under vigorous shaking at 12C, giving a pH in the interval 4.35 -
4.55. The volume is increased to 100 mL by adding water. It is filtered and for
instance 25 mL of it are analysed by Kjeldahl for the pH4.4-SN.
To analyse PTA-SN, 40 ml of the citrate dispersion are added 25 ml
PTA (20 %) and then the volume is increased to 100 ml with H 2 S0 4 (25%)
giving a final concentration of 2.5 % PTA. The mixture is kept at 4C over
night and filtered. For the Kjeldahl analysis 50 mL of the filtrate are used.
Fractionation with 12 % TCA is made from the pH4.4-SN fraction (50 mL
sample is added 50 mL 24 % TCA). It is kept at 4C overnight and 50 mL
filtered sample is then analysed by Kjeldahl. Analyses recommended are as
follows: double citrate dispersion of cheese, one fraction at pH 4.4 from each
citrate dispersion, one TCA fraction from each SN4.4 fraction, one PTA
fraction from each citrate dispersion and double Kjeldahl titration of each
fractioa [YA]
5.3. WSN - Water soluble nitrogen

Grated cheese is homogenized with water to extract the water soluble
compounds. Cheese to water ratio may vary as well as the extraction
temperature. Centrifugation followed byfiltrationis used for the separation of
the insoluble material. Nitrogen content of the water extract is determined by
the Kjeldahl method. [AP]
METHOD I
Grated cheese (x g) is homogenized with 2x mL H2O for 5 min using a
Colworth Stomacher 400; the resultant homogenate is held in a waterbath at
40C for 1 h. After incubation, the insoluble material is separated by
centrifugation at 3000 g for 30 min in a refrigerated centrifuge at 4C;
centrifugation at 4C allows easy separation of fat. The centrifugal supernatant
33
is filtered through glass wool and Whatman No. 113 filter paper and the N
content determined on an aliquot of thefiltrateby the Kjeldahl method. If more
complete extraction of the soluble material is desired, the insoluble material
may be reextracted with another 2x volumes of H 2 0.
When necessary, WSN is further fractioned by one or more of the
methods described below. Usually, samples of WSN are freeze-dried for
analysis by Polyacrylamide gel electrophoresis or for storage (Kuchroo & Fox,
1982). [PMS]
Alternatively, WSN is prepared by homogenizing lx g cheese with

5x mL FLÔ. The water extract (WSN) is also used for peptide analysis by
HPLC. [AP]
METHOD n (exhaustive extraction)

Water soluble nitrogen is determined following the protocol of Kuchroo
& Fox (1982) slightly modified to be adapted to hard cheeses (Pochet, np).
Grated cheese (20 g r.fc)(Wc) is weighed in a (180x300 mm) polyethylene bag
for Stomacher22 and 50 mL water are precisely added. The bag is held in a
waterbath at 40C for 5 min, taking care the slurry remains in the lower 1/3
portion of the bag and in the water. The bag is then threshed for 5 min, then
put back in the 40C waterbath for 60 min then threshed again for 5 min. The
slurry is then centrifiiged at 1200 g for 30 min at 4C. The upper fatty layer is
thoroughly removed and the supernatant isfiltratedusing a plug of glass wool.
The pellet is then transferred back into its original bag and reextracted under
the same conditions. Nitrogen content is determined by Kjeldahl method using
5 mL of the pooled supernatantsfilteredon paper (WSF).
[WSN] (% DM) = (14 007 x V H 2 S 0 4 x normality) x (2 x 50) / (5 x Wc x DM)
Three extractions are done per sample and nitrogen is determined twice
byfiltrate.The pellet (water insolublefraction,WIF) isfreeze-driedin view of
analysis of casein composition by electrophoresis. [SP]
22
Stomacher Lab-blender 400, Bioblock Scientific, Ollkirch, FR
34
METHOD UI (routine)
Grated cheese (10 g a.k.)(Wc) is weighed in a 150-mL screw-capped
bottle and 50 mL water are added precisely. The mixture is blended for 3 min
then held in a waterbath at 40C for 30 mia The slurry is then centrifuged at
1200 g for 30 min at 4C.The fatty layer is removed and the supernatant is
recovered and filtered on paper (retention 8 urn). Nitrogen is measured by
Kjeldahl method as previously described ( 3.2). [SP]
[WSN] (% DM) = (14 007 x V H 2 S 0 4 X normality) x 50 / (5 x Wc x DM)
METHOD IV
lOx g distilled water of 50C are added to lx g cheese weighed into an
Erlenmeyerflask.The mixture is homogenized with a T25 UltraTurrax5 for 2
min at 9500 rpm, moderately stirred for 1 h at 50C and subsequently
homogenized as above. After centrifugation (25 000 g, 30 min, 4C) the
suspension is filtered through glass wool, and the nitrogen content of the
obtainedfiltrateis determined by the Kjeldahl method. [HR]
5.4. pH 4.4-SN - pH 4.4 soluble nitrogen

The pH 4.4-SN is generally prepared from a citrate dispersion of cheese.
A sample of grated cheese is dispersed in a trisodium citrate solution and the
pH adjusted to 4.4 with IM HCl. The insoluble material is separated by
centrifugation and/or filtration. Nitrogen content of the filtrate is determined
by the Kjeldahl method. [AP]
METHOD I
Grated cheese (12.50 g) is mixed with 50 mL warm (40-50C) 0.5 M tri-
sodium citrate solution using a magnetic stirrer for 60 min, and then cooled to
room temperature and made 250 mL with deionized water
The pH4.4-SN isfractionatedat 12C by adding 22.6 mL 1 M HCl to
160 mL of the citrate dispersion under vigorous shaking, which gives a pH in
the interval 4.35-4.55. The volume is increased to 200 mL by adding water. It
35
is filtered or centrifugea, as described in Method III, and 25 mL of it are

analysed for pH 4.4-SN.
Note: The method can be used for most cheeses, but whenever it is
adapted to a new cheese variety the pH should be controlled and, in some
extreme cases, the amount of acid has to be changed. [YA]
METHOD n
Grated cheese (5 g a.k)(Wc) weighed in a 200-mL beaker is dispersed
with 100 mL citrate buffer 0.5 M pH 7.00,firstlyheld in a waterbath at 40C
for 15 min then stirred magnetically at room temperature for 30 min. When the
cheese is totally dispersed, the pH is adjusted to 4.4 with hydrochloric acid 4N
(VHCI) taking care to add acid at the same speed from sample to sample. The
suspension is then allowed to stand at room temperature for 30 min and
finally centrifuged for 15 min at 1200 g and 4C. The supernatant is then
immediately filtrated on paper (retention 8 um). Nitrogen content is measured
on 10 mL filtrate (see 3.2).
[PH4.4-SN] (% DM) = (14 007 x VH2SO4 X normality) x (100 + VHci)

/(IOXWCXDM)
Extractions are done in triplicate per sample and nitrogen is determined

twice byfiltrate.[SP]
METHOD m
Five g grated cheese are dispersed in about 70 mL 0.1M trisodium
citrate solution, pH 7.0, for 30 min at 30C. Then, IM HCl is added until pH is
4.4. The volume is adjusted to 100 mL with H2O and the suspension is
incubated at 30C for another 30 min. The insoluble part is then separated by
centrifugation at 3000 g for 30 min at 4C followed by filtration through
Whatman No 40 or equivalent filter paper. The N content of the filtrate (25
mL) is determined by the Kjeldahl method and expressed as g per 100 g
cheese. [AP]
pH 4.4-SN (% cheese) = Vwso* x normality x 1.4) /1.25
36
5.5. TCA-SN - Trichloroacetic acid soluble nitrogen

Peptides in WSN or pH4.4-SN are fractionated by 12% trichloroacetic
acid (TCA). The insolublefractionis separated byfiltrationand the N content
of the filtrate determined by the Kjeldahl method. The nature of the extract
used (water extract or pH4.4-solublefraction)has a strong effect on TCA-SN
level (Polychroniadou et al, unpublished results). [AP]
METHOD I (TCA-SNfrompH4.4-SN)
Thefractionationwith 12% TCA is made from the pH4.4-SN fraction
(50 mL sample is added 50 mL TCA 24%). It is kept at 4C overnight and
50 mLfiltratedsample is then analysed by Kjeldahl. [YA]
METHOD n (TCA-SNfrom WSN)

The nitrogen fraction soluble in TCA is prepared from the water soluble
fraction (WSF) as described at 5.1 II or HI. To 25 mL of WSF contained in a
100-mL screw-capped bottle are added 25 mL TCA 24%. The mixture is
mixed, then allowed to stand overnight at 4C, then stirred and immediately
and entirely filtered on paper (retention 8 urn). One precipitation is done by
each of the triplicate WSF and nitrogen is determined twice by filtrate on 10
mL test portions (see 3.2). [SP]
[TCA-SN] (% DM) = (14 007 x V H 2 S 0 4 X 0.05) x 2 / (10 x Wc x DM).
Alternatively, the WSN / 24% TCA mixture is held for 2 h at room

temperature (Btikofer et al, 1993). However, it has been found that time of
incubation of the mixture has a very weak effect on TCA-SN values.
(Polychroniadou et al, unpublished results). [AP]
5.6. PTA-SN - Phosphotungstic acid soluble nitrogen

Phosphotungstic acid (PTA) and sulfuric acid solutions are added to a
water extract or a citrate dispersion of cheese. The mixture is kept overnight at
37
4C, filtrated and the N content of the soluble fraction determined by the
Kjeldahl method. [AP]
METHOD I {PTA-SNfrom a citrate dispersion)

To analyse PTA-SN 40 mL of the citrate solution ( 5.2 I) are added
25 mL PTA (10 %) and then the volume is increased to 100 mL with H 2 S0 4
(25%). The mixture is kept at 4C overnight and filtered (Whatman No 542).
For the Kjeldahl analysis 50 mL of the filtrate is used. The PTA concentration
in the final sample is 2.5% (Mogensen, 1947; Stadhouders, 1960). [YA]
METHOD n {PTA-SNfrom WSN; Jarett et al, 1982)

Protocol 1. To 5 mL of a WSN fraction, prepared as described at 5.11,
are added 3.5 mL of H 2 S0 4 (3.95 M) and 1.5 mL of PTA (33.3% w/v). The
mixture is allowed to stand overnight at 4C and subsequently filtered through
Whatman No. 542 filter paper. The N content of the filtrate is determined by
the Kjeldahl method. [PMS]
Protocol 2. To 20 mL of WSF ( 5.1 II or III) contained in a 100-mL

screw-capped bottle are added 14 mL sulfuric acid 3.95 M and 6 mL
phosphotungstic acid 33.3%. The mixture is left for one night at 4C and then
centrifuged (1200 g, 5 min, 4C) and filtered on the smaller size of filter paper
(retention 2.7 urn). One precipitation is done by each of the triplicate WSF and
nitrogen is determined twice by filtrate on 15 mL test portions (see 3.2).[SP]
[ PTA-SN] (% DM) = (14 007 x VH 2 SO 4 X 0.05) x 2 / (15 x W c x DM).
METHOD HI (PTA-SNfrom WSN; Gripon et al, 1975)

To 20 mL of WSF contained in a 100-mL screw-capped bottle are added
20 mL of a freshly prepared mixture of sulfuric acid 25% / phosphotungstic
acid 10% (v/v). The mixture is left for one night at 4C and then filtered on the
smaller size of filter paper (retention 2.7 urn). One precipitation is done by
each of the triplicate WSF and nitrogen is determined twice by filtrate on
15-mL test portions (see 3.2).[SP]
[ PTA-SN] (% DM) = (14 007 x V H 2 S 0 4 X 0.05) x 2 / (15 x W c x DM).
38
METHOD IV (PTA-SNfrom WSN)

x mL of a 100 g/L PTA solution is added to x mL of the aqueous cheese
extract as prepared according to 5.1 IV (Frister et al., 1989). The mixture is
equilibrated at 4C overnight and filtered through a Schleicher & Schuell 602
1/2 h folded filter. Nitrogen content of the filtrate is determined by the Kjeldahl
method.
Note: This method yields somewhat lower results than precipitation by
combined action of sulfuric acid and phosphotungstic acid (Rohm et al., 1994).
[HR]
5.7. EtOH-SN - Ethanol soluble nitrogen

Absolute ethanol is added to a water extract of cheese in order to
fractionate peptides. The amount of the precipitated nitrogenous compounds
depends on the ethanol concentration; adjustment of the pH is sometimes
necessary. Soluble fraction is then separated by filtration and its N content is
determined by the Kjeldahl method. [AP]
METHOD I
By using Erlenmeyer flasks, x mL aqueous cheese extract prepared
according to 5.1IV are mixed with y mL ethanol (96 %) to achieve the desired
concentration (usually in the range between 30 % and 70 %). The flasks are
tightly closed with parafilm, placed in a refrigerator overnight and
subsequently filtered through a Schleicher & Schuell 602 1/2 h folded filter. An
appropriate amount of the filtrate is used for nitrogen determination by the
Kjeldahl method.
Note: The recovery of nitrogen depends significantly on the
concentration of ethanol in the mixture, which decreases with increasing
ethanol concentration (Rohm et al, 1996). A simple replacement of 12 %
TCA fractionation by ethanol fractionation at a certain concentration level
cannot be recommended. [HR]
39
METHOD H
Absolute ethanol is added to the WSN fraction of cheese to a final
concentration of 70% (v/v). The suspension is held at room temperature for
30 min and centrifuged at 3000 g for 30 min at 20 C. The supernatant is
filtered through Whatman No 1 filter paper and the ethanol is removed by
rotary evaporation at 30 C. After complete removal of the ethanol, the
ethanol-soluble fraction is freeze dried. The pellet (ethanol-insoluble fraction)
is resuspended in distilled water and freeze-dried (Reville & Fox, 1978).
[PMS]
METHOD m
35 mL of water soluble extract is added to a 100-mL beaker. The pH
value is measured and, if necessary (>5.5), adjusted with 1 mol/L hydrochloric
acid solution to 5.5. 15.0 mL 960 g/kg ethanol are then added to the extract.
The suspension is held at 25C for 1 h and thenfilteredthrough Whatman No.
40 filter paper. The nitrogen content is determined using the Kjeldahl method.
[B]
5.8. Ultrafiltration of the soluble fraction of cheese

Ultrafiltration (UF) of water soluble cheese extracts is performed using a
Milipore Minitan (tangential flow) ultrafiltration unit fitted with acrylic
manifolds and four porysulphone (PGTC) membranes having a nominal
molecular weight cut-off of 10 000 Da.
A volume of WSN (50 mL) isfilteredthrough Whatman No. 113 filter
paper. This volume is made up to 250 mL with H2O. This is subjected to UF
until the retentate volume is 40-50 mL. Then another 100 mL H2O are added
and again subjected to UF until the volume of the retentate is 40-50 mL. This
procedure should give a ~99% removal of permeable material. If desired, the
N content of the permeate may be determined by the Kjeldahl method and the
remainderfreezedried for furtherfractionationand/or analysis. [PMS]
40
6. RAPID METHODS FOR MONITORING PROTEOLYSIS

IN CHEESE
6.1. Cadmium-ninhydrin method for determination of free

amino groups in cheese
Although this reagent reacts to some extent with all amino groups, it
reacts very intensely with the amino group of free amino acids and hence
appears to be a simple method for estimating the concentration of free amino
acids in cheese (Folkertsma & Fox, 1992).
Cadmium Ninhydrin reagent. Ninhydrin (0.8 g) is dissolved in a mixture
of ethanol (80 mL) and acetic acid (10 mL). To the resulting solution is added
1 g CdCl2 dissolved in 1 mL H2O.
Procedure. A water soluble extract of cheese is prepared as outlined
above. An aliquot of the WSN extract (10-100 uL depending on the
concentration of free amino acids expected) is diluted to 1 mL with H2O and 2
mL of Cd-ninhydrin reagent added. The mixture is heated at 84C for 5 min,
cooled and the absorbance at 507 nm determined against a reagent blank,
containing no WSN.
Results are expressed as A507 or as mg Leu / g cheese by reference to a
standard curve. [PMS]
6.2. Trinitrobenzenesulphonic acid (TNBS) method for

monitoring proteolysis in cheese
This reagent reacts with a-amino groups, although it reacts also to some
extent with e-amino groups. An accumulation of free amino groups during
cheese ripening is the result of protein/peptide breakdown; thus, the above
reaction is useful for monitoring proteolysis (Polychroniadou, 1988).
Reagents
Borate buffer: 0.1M Na 2 B 4 0 7 in 0.1M NaOH, pH 9.5.
TNBS solution lmg/mL. The reagent can be kept for a week at 4C in
the dark.
41
Phosphate buffer: O.IM NaH2P04, containing 1.5 mM Na2S03.

Phosphate buffer is kept at room temperature without problems, but Na2SC>3 is
added just before use.
Procedure. Grated cheese (1 g) is dispersed in 20 mL borate buffer,
warmed at 45 C for 15 min with stirring and centrifuged at 3000 g for 20 min.
A portion (3-6 mL) of the supernatant is diluted to 100 mL with distilled
water.
From this extract a portion (0.5 mL) is mixed to 0.5 mL borate buffer;
1 mL TNBS reagent is then added and thoroughly mixed. The solution is
incubated at 37C for 60 min. Blanks are prepared with 0.5 mL H2O instead of
cheese extract. The reaction is stopped by adding 2 mL phosphate buffer and
the absorbance is measured at 420 nm.
Results are usually expressed as A420, but, if appropriate, also as mg
Leu/g cheese by reference to a standard curve.
Repeatability of the method is satisfactory (CV=1.19%). Recovery of
glycine added to the cheese sample (0.10-0.50 mM) is 104.92.9%.
Note. TNBS dry powder (but not its solutions) is explosive and shall be
handled with care. [AP]
42
7. ANALYSIS OF CASEINS
7.1. Reverse phase high performance liquid chromatography

(RP-HPLC) of caseins
The cheese sample is dissolved in 0.1 M tri-Na-citrate buffer pH 8.5.
Proteins are precipitated by acidification at pH 4.6 and fat is removed by
centrifugation. The precipitate is washed with demineralized water on glass
filter and solved in 20 mM bis-tris-propane buffer pH 7.0 containing 6 M urea
and 0.24% 2-mercaptoethanol. The chromatographic equipment could be
FPLC or HPLC with the column pro-RPC 5/10 (Pharmacia) or Nucleosil 10
Cis (250 x 4.6 mm) (Christensen et al, 1989). Samples of 25 - 50 uL are
applied onto the column. Elution is performed by using 30-48 % acetonitril in
0.1 % TFA and flow rate is 0.3 mL/min. With this system separation of otsr
and as2-caseins is complete, however - and y-caseins are only partially
separated. [YA]
7.2. Ion exchange HPLC of caseins

The cheese sample is dissolved in 5 mM Tris-HCl buffer pH 8.0
containing 4.5 M urea and 0.12 % dithiothreitol. The chromatography is
performed with FPLC using the column Mono-Q (Pharmacia). Elution is
performed in 5 mM Tris-HCl buffer pH 8.0 containing 4.5 M urea with a linear
gradient of NaCl from 0 to 30% for 35 min at a flow rate of 2 mL/min.This
method separates y-, -, K- and ocs-caseins but not completely the as r and
as2-caseins (Barrefors et al, 1985; Visser et al, 1991; Jaubert & Martin,
1992). [YA]
43
7.3. Urea Polyacrylamide gel electrophoresis of cheese

Electrophoresis in Polyacrylamide gels (12.5% C, 4% T, pH 8.9) is
performed using a vertical slab-gel unit23 according to the method of Andrews
(1983) with modifications. Gels are stained directly by the method of Blakesley
and Boezi (1977).
Stock Solutions
Acrylamide Solution: 40%, w/v, acrylamide in HjO.
Stacking Gel Buffer

4.15 g tris (hydroxymethyl) methylamine
150 g.urea
2.2 mL concentrated HCl
Dissolved in, and made up to 500 mL with, HjO and the pH adjusted to
7.6 with HCl.
Separating Gel Buffer
192.85 g urea
Dissolved in, and made up to 500 mL with, H2O and the pH adjusted to
8.9 with HCL
Electrode Buffer
15 g tris (hydroxymethyl) methylamine
73 g glycine
Dissolved in, and made up to 5 L with, H 2 0. If desired, the pH may
be adjusted to 8.4 with HCl.
Sample Buffer
49 g urea
^Protean II xi, Bio-Rad Laboratories Ltd., Watford, Herts., UK
44
0.7 mL 2-mercaptoethanol
~0.15 g bromophenol blue
Dissolved in, and made up to 100 mL with H 2 0.
Ammonium Persulphate: 10%, w/v, inH 2 0.

1 mL aliquots are placed into Eppendorfs and frozen (-20C). One
Eppendorf is removed for use on each occasion.
Staining Solution
An aqueous solution of Coomassie Briliant Blue G250 (0.2%, w/v) is
added to an equal volume of 1 M H 2 S0 4 and held overnight. The solution is
then filtered through Whatman No. 1 filter paper and the filtrate mixed 9:1
with 10 M KOH. Trichloroacetic acid is then added so that the final solution is
12% (w/v) with respect to TCA.
Gel solutions
They are not prepared until the day of running the gel.
Stacking Gel Solution

5 mL acrylamide solution
45 mL stacking gel buffer
0.1 g N,N,N,N'-methylene bisacrylamide
The above are mixed together and filtered through Whatman No. 113
filter paper. To the filtrate are added 25 uL N,N,N,N-tetramethylethylene
diamine (TEMED).
Separating Gel Solution:

22.5 mL acrylamide solution
52.5 mL separating gel buffer
0.375 g N, N, N , N-methylene bisacrylamide
The above are mixed together and filtered through Whatman No. 113
filter paper. To the filtrate are added 37.5 uL TEMED.
Sample Preparation. Freeze dried samples (10 mg) are dissolved in 1

mL sample buffer. Heating (~50C for a few minutes) may be necessary if
there is difficulty in dissolving the samples.
45
Preparation and Running the Gel. The electrophoresis unit is assembled

according to the manufacturer's instructions. Immediately before use,
ammonium persulphate solution is added (separating gel: 282 \xL) to initiate
the polymerization. Two (1 mm thick) gels are then poured so that the level
of the top of the gel is such to allow about 1 cm of stacking gel below the
level of the bottom of the wells. A layer of water is then gently overlaid on top
of the gel and the gel is allowed to stand until polymerization is complete
(usually 40-60 min). The layer of water is then removed using filter paper and
ammonium persulphate is then added to the stacking gel solution (300 uL) and
the stacking gel is then poured and a slot-former is placed in position. The
stacking gel is then allowed to polymerize (~60 min). The slot-former is
removed and the gels placed in the gel unit which is filled to the correct level
with electrode buffer.
The gels are pre-run at 280 V for 30-40 min and the samples are applied
(Na caseinate, 3.5 uL; cheese, 8 uL; water-soluble extracts, 8-13 uL and
casein hydrolysates [5 mg mL"1] ~15uL). The system is cooled by
recirculating cold water. Samples are run at 280 V through the stacking gel
and at 300 V through the separating gel until the tracking dye front is close to
the bottom of the gel slab (4-5 h).
Gels are stained by immersing in gel stain overnight and destained in a
few changes of distilled water until the background becomes clear. [PMS]
Alternatively, no pre-running of the gels is used. Samples are run at

500V (constant current 60mA) until the tracking dye front is close to the
bottom of the gel slab (less than 2 h). [AP]
Interpretation of Results. Electrophoretograms are usually recorded by

photography. Densitometry24 can be also performed. The majority of the
bands on electrophoretograms of the water-insoluble and water soluble
extracts of cheese have been identified from their N-terminal sequence
(McSweeney et ah, 1993 and 1994). An electrophoretogram of a water soluble
extract of Cheddar cheese is shown in Figure 7.1. [PMS]
24
Bio-Rad model 620 video densitometer
46
P-CN(f 106-209)(y2)
CN WISF P-CN(f29-209)(y,)
-CN(f 108-209)(y3)
-CN(f 1)
p-CN
P-CN(f 1-189/192XP-I)
P-CN(f 1-.)
a s1 -CN
a s1 -CN(f 102-*)
a s1 -CN(f24-199)(ct sl -l)
a s 1 -CN(f33-*)
a . r C N ( f 60-*)
ots1-CN(f 110-*)
a s1 -CN(f24-*)
",-1 !t!Lv*;;:i*
Figure 7.1. Urea Polyacrylamide gel electrophoretograms (12.5% T, 4% C,

pH 8.9) of casein (CN) and water-insoluble (WISF) fraction of 3 month-old
Cheddar cheese made with Lactococcus lactis subsp. cremoris SK 11
indicating the positions of the caseins and the N-terminal of the principal
peptides identified (After McSweeney et al. 1994, reproduced from J. Dairy
Res. by courtesy of Cambridge University Press).
47
7.4. PAGE of the water insoluble fraction

Composition of the water insoluble fraction (WIF)( 5.1 II) is
determined by vertical slab25 Polyacrylamide gel electrophoresis (PAGE) in
denaturating conditions following the method of Andrews (1983).
The lyophilised sample (50 mg a.k.) weighed in a plastic tube is
dissolved in 5 mL Tris(0.062M)-HCl (pH 7.6) buffer containing urea (8 M)
and -mercaptoethanol (0.1M). Samples are first stirred (Vortex) for 30 s then
agitated overnight on a rotating system at 4C then finally placed in a water
bath at 40C for 15 min. When back at ambient temperature, 10 uL are applied
on a discontinuous gel (1 mm thick) composed of a concentrating gel
(length=2 cm, 7=4.2%, C=5%, urea 4.5 M, pH 7.6) then a separating gel
(length=16 cm, 7=12.5%, 0 4 % , urea 4.5 M, pH 8.9). Migration is run for
4.5-5 h under 300 V26 using a Tris(0.025M)-glycine(0.195M) buffer pH 8.4
as electrode buffer.
Bands are fixed and stained overnight under gentle agitation using a
solution of Coomassie blue G250 prepared as follows (Blakesley & Boezi,
1977): sulfuric acid IM (1 L) is added to 1 L of a 0.2 % Coomassie blue
aqueous solution and allowed to stand overnight at room temperature, then
filtered on paper (retention 2.7 um); one volume of potassium hydroxide 10 M
is then added to 9v of the brown mixture and trichloroacetic acid is finally
mixed to a final concentration of 12% TCA. After staining, gels are soaked
repeatedly in a water bath until the background is clear. Gels are then scanned
by transmission between 595-750 nm (red filter) with a 100 urn resolution27
(Figure 7.2). Each profile is manually integrated using the Molecular analyst
programme28. Identification of the band is evaluated referring to standards but
also to different works published on milk (Andrews, 1983; Jakob, 1993) and
cheese (McSweeney et al., 1994). But uncertainty remains for certain
compounds.
Each sample is analysed in two different gels, running on different days.
Two standards (one pH4.6-insoluble casein from milk, one cheese water
"Protean II xi. Bio Rad, Richmond, USA.

26
Gnrateur CONSORT, Bioblock Scientific, Illkirch, FR.
27
GS670. Bio Rad, Richmond, USA.
28
Ver 1.1. Bio Rad, Richmond, USA.
48
asi
OcSld
^ _
WISF Emmental grand cru
WISF Comt
WISF Beaufort
Figure 7.2. Gel pictures and densitograms of the separation of the

nitrogenous water-insoluble-fraction of hard cheeses by urea-PAGE (Pochet,
np).
40
insoluble fraction) are analysed on each gel to facilitate the identification and
to control the reproducibility of the migration from batch to batch. The
extreme wells of the slab are never used because of band deformation. The
surface of each peak is reported as percentage of the total area. We do not
take into account the differences in absorption coefficient of each casein.
Therefore results can not be considered as quantitative but qualitative. [SP]
7.5. Capillary electrophoresis (CE)

METHOD I
This method was developed by de Jong et al. (1993) and has been
recently optimized by Recio & Olieman (1996) in order to provide quantitative
results. Initially, it was developed to determine heat-denatured serum proteins
in the casein fraction and it was applied to the evaluation of the thermal
treatment undergone by milk used for cheesemaking, but it can be applied to
the study of the serum protein/casein ratio in milk or cheese. Besides, this
method has been used to study the main degradation products arising from the
action of different proteolytic agents such as plasmin and chymosin both in
model systems of caseins and in cheese (Recio et al, 1997). Several of the
main casein breakdown products were identified. These included, among
others: y r CN A1, y r CN A2, y r CN B, y,-CN C, y,-CN A, y r CN A3, y2-CN
B and y3-CN A, y3-CN B, as well as proteose-peptones, arising from the
action of plasmin on the different genetic variants of -CN. asi-I-CN, cisi-CN
(fl-23), para-K-CN and caseinomacropeptide produced by chymosin action on
asi-CN and K-CN respectively were also separated. The knowledge of their
migration times provided information on the extent and origin of casein
hydrolysis in cheese.
Solutions and buffers

Before use, buffers arefilteredthrough a 0.22 um filter29.
29
Sterile Acrodisc with HT Tuflryn membrane, Gelman Sciences, Ann Arbor, MI 48106,
USA.
50
Urea solution
120 g urea,
4 g of mixed-bed ion-exchange resin (analytical grade)30
166 mg methylhydroxyethylcellulose31 or other modified cellulose
giving a high viscosity solution (e.g. methyl hydroxypropyl cellulose,
MHPC)
Make up to 200 mL with H2O. Dissolve and stir until the conductivity is
less than 2 uS/cm.
Separation buffer (pH 3.0 0.1)

148 mg trisodium citrate dihydrate (analytical grade)
1.00 g citric acid
Dissolve in a mixture of 10 mL water and 15 mL of the urea solution.
Sample buffer (pH8.6 0.1)

505 mg hydroxymethylaminomethane (Tris)
620 mg ethylenedinitrilo tetraacetic acid disodium salt dihydrate
220 mg 3-morpholino-propanesulphonic acid32
64 mg of DL-dithiothreitol (DTT)
Dissolve in 25 mL of the urea solution.
Lactic acid 18%. It is prepared diluting concentrated lactic acid.
Sample preparation. Lyophilized isoelectric casein, from milk or cheese

(ca. 18 mg) or 300 uL of milk, are dissolved in 1 mL of diluted sample buffer
(sample buffer/water 1.5:1) or in 700 uL of sample buffer in the case of milk.
After incubation for 1 h at room temperature, 20 uL lactic acid 18% are added
(final pH 7.0) and injected in the CE apparatus.
Capillary electrophoresis. CE is carried out using a Beckman P/ACE

System 2050 controlled by a System Gold Software data system version 81033.
30
AG501-X8, Bio-Rad, Richmond, CA, USA.
31
30000, Serva, Heidelberg, Germany, sold also as Tylose, Hoechst, Germany
32
MOPS, BioChemika MicroSelect, Fluka, Buchs, Switzerland.
33
Beckman Instruments Inc., San Ramon, CA 94583-0701, USA.
51
The separations are performed using a hydrophilic-coated fused-silica capillary

column, Supelco CElect PI 34, of 0.47 or 0.57 m x 50 um i.d., with a slit
opening oflOOx 800 um. The distance between detection window and outlet
is 7 cm, resulting in an effective length of 40 or 50 cm. Electrophoresis is run
0.07
ff.1
0A'
flA'
0 04 a*
P-K
0Lg
* 0.02
OB
\
0.01
V 4l
a...
m
20 X O
Tim (min)
Figure 7.3. Electrophoretogram corresponding to casein from a fresh cheese

clotted enzymically (the total length of the capillary was 0.57 m, and the final
voltage 25 kV), a La, a-lactalbumin; Lg, -lactoglobulin; as2, ct-casein
(CN); asi, aSi-CN; otso-, aS0-CN; aSi-I, aSi-I -CN; B, -CN B; Al, -CN
A1; A2, -CN A2; K, K-CN; p-K, para-K-CN; S, peptides soluble at pH 4.6
arising from the action of plasmin on as- and -CN. 1, asP, peptide derived
from the action of plasmin on asi-CN; 2, y2-CN A; 3, yi-CN A2; 4, y3-CN A; 5,
yi-CN A1; 6, y2-CN C; 7, y r CN B ( After Recio et al 1997, reproduced from
J. Dairy Res. by courtesy of Cambridge University Press).
34
Bellefonte, PA 16823, USA.
52
at 45C with a linear voltage gradient from 0 to 20 (25) kV in 3 min, followed

by constant voltage at 20 (25) kV with ground at the detector side. Before
each separation the capillary is flushed in the reverse direction with the
electrophoresis buffer for 3 min. UV-detection is performed at 214 nm. [RLF]
METHOD H
In this method a citrate dispersion of a cheese is analysed for its casein
components. When the method is used in a new application that differ from
common cheese, it is recommended to precipitate the caseins at pH 4.4 - 4.6
and analyse the N compounds that are still in solution and might disturb the
analysis. For all cheeses tested so fr, this has been no problem.
Sample preparation. The citrate dispersion and, if needed, the pH 4.4
soluble fraction are made as described in 5.4 I, and mixed with sample buffer
(1:1) containing 10 M urea, DTT (2.56 mg/mL) and methylhydroxypropyl
cellulose (0.83 mg/mL) and incubated for 1 h at room temperature before
analysis.
Running buffer (pH 3.0) contains 20 mM trisodium citrate dihydrate,
190 mM citric acid monohydrate, 6 M urea and MHPC (1.5 mg/mL) as
described by Recio and Olieman (1996).
Capillary electrophoresis. Analysis is performed at 45C with a Capillary
Electrophoresis System (e.g. G1600AHP3D, Hewlett-Packard A/S, Birkerd,
Denmark) and a hydrophilically coated fused silica capillary column, 50 mm
(Supelco Celect PI, Bellafonte, PA, USA) with an effective length of the
column of 56.0 cm. Migration starts with a linear voltage gradient from 0 to 25
kV for 3 min and then continues at 25kV for 40 min. [YA]
53
8. ANALYSIS OF PEPTIDES
8.1. Peptide profiles by RP-HPLC

METHOD I
Sample preparation for WSN. Grated cheese (10 g) is mixed with water
(50 mL) using Ultra Turrax5 (1 min at 14 500 rpm, 1 min stop, 1 min at 14 500
rpm). The mixture is incubated for 1 h at 40C and centrifuged at 3000 g at
4C for 30 min. The solid fat is removed and the supernatant is filtered (0.42
urn).
Sample preparation for pH4.4-SN. The pH 4.4-SN is prepared from a
citrate dispersion of cheese as described in 5.41.
HPLC. An injection of 50 uL is made onto the column (Nucleosil 5 urn
Cig, 250 x 4.6 mm)35. Gradient elution is performed at 30C with aflowrate of
1 mL/min and the absorbance is measured continuously at 210 nm. Elution
buffer A contains 1 mL/L trifluoroacetic acid (TFA) and buffer B contains 1
mL/L TFA and 600 mL/L acetonitrile in water. The elution scheme has three
steps, starting isocratic with buffer A for 10 min, then a linear increase from 0
to 80 % of buffer B during 80 min and finally 10 min with 100 % buffer B.
Blanks are analysed to exclude carry-over (Ardo & Gripon, 1995) [YA]
Peptide profiles are obtained from a water extract of the cheese (cheese
to H2O ratio 1:5, 5.1 I). Column and gradient used are as in the method
described above, but buffer B contains 0.85 mL/L TFA. Data aquisition is
from 0 to 95 minutes and time between injections is 112 min.[AP]
METHOD n
Peptide profiles of the water-soluble extracts are performed by RP-HPLC36.
Wide-pore Nucleosil-Cg columns (4.6 mm x 25 cm, 300 pore size) are used
and detection is at 214 nm. Successful separation is achieved also using a Cig
column (Ultrasphere ODS, 125 ).
35
Hichrom, Scantec AB, Prtiile, SW.
36
Shimadzu LC-9A solvent delivery system with FCV-9AL flow control valve, DGU-2A
degassing unit and SIL-9A autoinjector, SPD-6A spectrophotometric detector (Shimadzu)
interfaced with a personal computer (Shimadzu Corp., Kyoto, Japan) or a Waters model
626 pump, 717plus autosampler, 600s system controller and 486 detector interfaced with a
personal computer (Waters, Milford, MA, USA).
55
Chromatographic conditions are as follows :

Solvent A: 0.1% trifluoroacetic acid (v/v, TFA37) in deionized, HPLC grade
H 2 0.
Solvent B: 0 . 1 % TFA (v/v) 1CH3CN38.
Samples (4 mg mL"1 of freeze-dried water-soluble extract or freeze-dried
UF permeate of a water-extract) are dissolved in solvent A and filtered
through a 0.45 urn cellulose acetate filter39. Filtrate (20 uL) is applied to the
column and eluted at a flow rate of 0.75 mL min"1. The following gradient is
used for elution:
Time (min) %A %B
0 100 0
5 100 0
60 50 50
66 50 50
70 40 60
73 40 60
75 5 95
77 5 95
78 100 0
101 100 0
Data aquisition is from 0 to 73 min. Time between injections is

101 min.
Examples of typical chromatograms of a UF permeate and retentate and
a water-soluble extract of Cheddar are shown in Figure 8.1; the identity of a
number of peaks is indicated. Chromatograms from individual cheeses can be
quite different and chromatograms also differ markedly between varieties, thus
identification of individual peptides should be based on sequence data only.
[PMS]
37
Sequential grade, Sigma, St. Louis, MO, USA.
38
Far UV HPLC grade: Labscan, Dublin, EI.
39
Sartorius GmbH, Gottingen, DE.
56
Ori-CN n-9
^^-Osi-CNfl-lS
il
0,1-CN fi-?
e-.
m
UFP
S

13 O^ 30 o- ao t'j JTJ
UFR
o o o Vo ~ d " 'T' t'e-
Figure 8.1. HPLC of fractions from water extract of Cheddar (Singh,

unpublished). UFP, 10 kDa ultrafiltration permeate; UFR, 10 kDa
ultrafiltration retentate.
57
METHOD m
Water solublefraction( 5.1 II or III),filteredon 0.45 urn, is injected40
(10-50uL) on a precolumn+column system (length: 15+225 mm, i.d.: 4.6 mm)
filled with Ci8-bonded silica gel (Nucleosil, porosity: 300, partic.diam.:
5um). Nitrogenous compounds are eluted at 37C mainly by increasing order
of degree of hydrophobicity, using alinary gradient (solvente: trifluoroacetic
acid 0.105% and solvent B : trifluoroacetic acid 0.100 % in acetonitrile/water
(60/40)) at a 0.8 mL/min flow rate41: 100% A for 5 min, then linearly up to
100% B in 100 min, then held at 100% B for 5 min, then return to initial
conditions which are reached about 15 min later. Peptides and amino acids are
detected at 214 nm42.
Special attention is taken to control cleanliness of the column (injection
of acetonitrile), reproducibility of the injection (injection of an external
standard) and of the separation (injection of an "home made" standard peptide
mixture). Experimental designs are used to avoid systematic effect.
Maps (Figure 8.2) are then treated in different ways. They can be simply
compared for the occurrence of peaks between samples for further
identification by sequencing. Cumulating the surface of peaks, which are
grouped in hydrophilic or hydrophobic, can permit statistical treatment which
demonstrates differences between cheese samples. [SP]
8.2. Isolation of individual peptides

8.2.1. Fractionation of peptides
The peptide system in cheese is very complex and thus it is not possible
to give details of specific methods for isolating peptides. A fractionation
scheme for cheese peptides is proposed by Singh et al. (1994), which allows
peptides to be grouped into manageablefractions(Figure 8.3).
"^HPLC Autosampler 465. Kontron Instruments, St Quentin en Yvelines, FR.

41
HPLC system Varan LC 5000. Varan, Paris, FR
42
UV 100 detector. Varan, Paris, FR.
58
M \N LI
Figure 8.2. Separation of peptides from water soluble fraction of a hard

cheese (Emmental (E), Comt (C) and Beaufort (B)) by RP-HPLC. (Pochet,
np)
59
I Grated Cheese |
Water : Cheese, 2: I
Homogenize ( Stomacher or similar apparatus )
Centrifuge ( 10 000 g x 30 min )
f
WISN WSN
1Fat
UF. IO kDa membranes

Re-extract as above
f 1
WISN
Retntate Permeate
I pH 6.5
30J5 Ethanol
Precipitate
Cit 3er
Supernatant
I PH 5.5 L 11, ili. JJ, X. VI
I I
I
Precipitate
1 fl
Supernatant Sep-pakcie
and HPLC
Amino acids ?
T
DEAE chromatography
or
IEC -FPLC
or
RP-HPLC
Figure 8.3. Fractionation of cheese peptides. WSN: Water-soluble N; WISN:

Water-insoluble N; UF: Ultrafiltration (Reprinted from Singh et al, 1994 with
permissionfromElsevier Science).
60
ARDO &POLYCHRONIADOU,Ed. CHEESE ANALYSIS MANUAL
Large peptides in the water-insoluble fraction are fractionated by ion-

exchange chromatography on a column of DEAE-cellulose (68.5 x 2 cm)43
using 0.02 M tris (hydroxymethyl) methylamine buffer, pH 8.6, containing
4.5 M urea (Breen, 1992). Prior to application, samples are dissolved in 10 mL
of chromatography buffer and dialyzed against 1 L of the same buffer for 24 h
at 4C. Elution is by a linear, 0 to 0.3 M, NaCl gradient. Eluate is monitored
at 280 run. Fractions (10 mL) are collected and those containing peptides are
pooled, dialyzed overnight against H 2 0 at 4C andfreezedried. Successali
separation is achieved also using a Mono-Q FPLC column44 and similar elution
conditions. Individual peptides from the ion-exchange chromatogram are
isolated by electroblotting (see 8.2.2).
Peptides in the water-soluble fraction are first fractionated by UF.
Peptides in the permeate are further resolved by chromatography on Sephadex
G-25 (distilled water eluent, 214 nm detection) and individual peptides isolated
by HPLC (see above) or further fractionated using Sep-Pak Cs or Cis reverse-
phase cartridges45 eluted using a step-wise acetonitrile gradient (Singh et al,
1994). [PMS]
8.2.2. Electroblotting of peptides

Electroblotting is a simple and rapid technique of isolating large (water
or pH 4.6-insoluble) peptides from cheese or casein hydrolysates. The blotted
peptides may be sequenced directly.
Electrophoresis. Alkaline urea gels are used routinely. SDS gels may
also be used. Reagents for gels should be of the highest quality available and
all solutions (including buffers) should be made upfreshly.Samples should be
dissolved in sample buffer immediately before application onto the gel. It is
better not to use samples which have been stored frozen in electrophoresis
sample buffer.
PAGE gels (1 mm thick) are used. Either large gels (12 x 20 cm) or
mini-gel units may be used. If samples are applied to large gels, then the
region of the gel (of suitable size) containing the band(s) of interest must be
cut out before blotting; mini-gels may be used directly.
The same sample is applied to all the wells on the gel. It is usual to
overload gels for blotting, particularly if relatively minor bands are of interest.
43
Whatman International Ltd., Maidstone, UK.
44
Pharmacia, Uppsala, SW
45
Waters
61
Electrophoresis gels are run as usual but are not stained prior to blotting.
Blotting. Peptides are transferred from the electrophoresis gels onto
polyvinylidene difluoride (PVDF) membranes46 using a Mini Trans-Blot
electrophoretic transfer cell47.
Membranes are treated as follows before transfer:
1. A membrane is removed from its backing material and the package in which
it is supplied using forceps (it is important never to handle the membrane; use
gloves and forceps at all times) and placed in a plastic bag.
2. Two pieces, of suitable size, are cut from the membrane (through the
plastic bag).
3. Pieces are prewetted in 100% methanol,
4. Washed well in water, and
5. Equilibrated in blotting buffer (see below) for at least 15 min.
The electrophoresis gel must also be equilibrated in blotting buffer for
15 min.
Blotting Buffer. 25 mM Tris, 192 mM glycine in 10 % (v/v) methanol.
It is necessary to prepare 2 L buffer. Buffer pH should be -8.3. Buffer must
be at 4C before blotting.
Note: Do not adjust pH; if pH is outside the range 8.1 to 8.4, make up
the buffer again! (Adjusting the pH adds more ions to the solution, which will
cause excessive heat generation during blotting).
Alternative Blotting Buffer. 10 mM cyclohexylaminopropane sulphonic
acid (CAPS), pH 11.0 in 10% methanol. This buffer is prepared as follows:
22.13 g CAPS is dissolved in H 2 0 and made up to 900 mL and adjusted to pH
11.0 (lOx stock solution); 200 mL stock is added to 200 mL methanol and
made up to 2 L with water to yield the working solution.
Transfer Unit Assembly (Bio-Rad unit). Open gel holder cassette in
clean, shallow tray and place dark panel fiat on bottom of tray. Fibre pad is
soaked in blotting buffer and placed on dark panel. A piece of filter paper,
saturated with blotting buffer is placed on top of the fibre pad and its surface is
then flooded with blotting buffer. The gel is placed on top of the filter paper
and aligned in the centre of the unit; make sure there are no bubbles between
the gel andfilterpaper. Flood the surface of the gel with buffer and place the
membrane on top. This is done by holding the membrane by its edges and
lowering the centre onto the gel. Next, roll a Pasteur pipette on top (like a
46
ProBlott, Applied Biosystems Inc.
47
Bio-Rad.
62
ARDO &POLYCHRONIADOU,Ed. CHEESE ANALYSIS MANUAL
rolling pin) to exclude bubbles (there should be no bubbles between the

membrane and the gel). Flood the surface of the membrane with buffer. The
sandwich is completed by placing a piece of saturated filter paper and a fibre
pad on top. The cassette is then closed and placed in the gel holder so that its
dark panel is facing the dark panel on the gel holder.
Blotting (Bio-Rad unit). The gel holder is placed in the tank, as is the
"Bio Ice Unit" (which had been filled with water and frozen). The tank is filled
with blotting buffer until the liquid level is at the level of the top row of holes
on the cassette. A magnetic stir pellet is placed in the tank and the unit is
placed on a magnetic stirring box.
Electrophoretic transfer is done at 100 V for 40 min. The current
increases during blotting, from -180 to -250 mA (90 V/300 mA at room
temperature for 10-15 min using the CAPS buffer).
Staining
Coomassie Blue: Membranes are stained in 0.2% (w/v) Coomassie Blue
in 45% (v/v) methanol, 10% (v/v) acetic acid for <15 min. Membranes are
destained for 15 to 30 min in 45% (v/v) methanol, 7% (v/v) acetic acid,
followed by <2 min in 90% (v/v) methanol, 7% (v/v) acetic acid.
Ponceau S.: Staining solution consists of 0.2% (w/v) Ponceau S48 in 1%
(v/v) acetic acid. Membranes are removed from the cassette and rinsed in
water. Membranes are placed in the stain until bands appear (up to 30 min).
Bands normally appear within -15 min. Membranes are destained by rinsing in
water.
Membranes are dried thoroughly (this is particularly important when the
Ponceau S stain is used) using a hair-dryer. Membranes are placed in a plastic
bag which is then sealed and stored at 4C (or -20C if using the CAPS
buffer). It is a good idea to record the blot at this stage by photocopying it.
Blots can be stored for a couple of months. Do not cut out the bands at this
stage; wait until immediately before sequencing!
Note: Blots generally do not look as nice as the original gel! Provided
the band(s) of interest is/are intense and well separated from other bands, you
should (hopefully) have no trouble getting good sequences! As a general rule,
if the bands can be visualized by photocopying the blot, it is sufficient for
sequencing.
48
Sigma Chemical Co.
63
More information on blotting is available in refe. Gershoni & Plade

(1983), Yuen et al. (1990) and BioRad instruction manual for blotting cell.
[PMS]
8.3. Techniques for identification of peptides

8.3.1. N-Terminal sequence analysis
Peptides are sequenced by Edman degradation, automated on a pulsed
liquid-phase protein sequencer49. Liberated amino acids are detected as their
phenylhydantoin derivatives by means of an HPLC50. The distance to which
peptides may be sequenced depends on amount and purity. Normally, it is
possible to sequence >15 residues for a clean peptide.
Peptides too long to sequence completely are normally identified from
their N-terminal sequence and their mass as determined by mass spectrometry.
[PMS]
8.3.2. C-Terminal sequence analysis

C-Terminal sequencing of peptides is a useful technique for identifying
the length of a peptide when only the N-terminal sequence is available.
The C-terminal sequence of peptides can be determined by a time-course
hydrolysis with carboxypeptidase Y (E.C. 3.4.16.1, protein sequencing
grade51). Isolated peptides (uM quantities) are dissolved in 100 mM citrate
buffer, pH 5.5 (6 M urea or 0.1% SDS may be added for protein analysis;
proteins should be pre-treated in the denaturing buffer at 60C x 20 min before
the addition of enzyme). The ratio of peptide to enzyme should be
approximately 100:1 (w/w). Enzyme (20 uL of 0.1 fig mL"1 solution in buffer)
is added to 50 uL peptide solution and the mixture incubated at 25C.
Aliquots (5 uL) are taken periodically up to 1 h and the reaction stopped by
addition of the 5 uL aliquot to 95 uL 5.26% (v/v) acetic acid (final
concentration ~5%) or to 95 uL amino acid analysis sample diluent52 (pH 2.20,
[Na+] = 0.27 M).
49
Applied Biosystems Inc., Foster City, CA, USA, model 477A.
50
Model 120A, Applied Biosystems Inc.
51
Sigma, St. Louis, MO, USA.
52
Beckman, San Ramon, CA, USA.
64
Liberated amino acids are then quantified by an ion-exchange method.

The C-terminal sequence of the peptide is deduced from the order in which the
amino acids are liberated (e.g. Figure 8.4). [PMS]
8.3.3. Amino acid composition of peptides

Peptides may be identified from their amino acid composition. Purified
peptides are hydrolysed using 6M HCl53 for 24 h at 105 C in specially designed
hydrolysis tubes54. The tubes are repeatedly flushed with N 2 and evacuated
prior to hydrolysis.
HCl is removed by evaporation and the amino acid composition of the
hydrolysate determined. The molar ratio of amino acids is then calculated and
the peptide identified using commercially available software or a simple
BASIC program for this purpose which may be adapted for use on most
computers (Petrilli, 1982). [PMS]
53
Sequanal Grade, Pierce, Rockford, IL, USA
54
Pierce
65
ARDO & P0LYCHR0N7AD0U, Ed. CHEESE ANALYSIS MANUAL
40 60 80 100 140
Hydrolysis time (min)
Figure 8.4. Liberation of amino acids from the C-terminal of the peptide
asl-CN 165-199 by carboxypeptidase Y. Deduced C-terminal sequence
-Met-Pro-Leu-Trp-COOH.
66
9. ANALYSIS OF FREE AMINO ACIDS AND AMINES
9.1. Analysis of free amino acids

Quantitative determination of free amino acids can be done either with
ion exchange chromatography and post column derivatization (ninhydrin or
OPA) or with precolumn derivatization and high performance liquid
chromatography. Ion exchange chromatography is for most amino acids more
precise than HPLC separation of OPA drivtes. Both methods are
recommended for the quantitative determination offreeamino acids in cheese.
A collaborative study has been made to evaluate precision parameters of
the two methods mentioned above (Biitikofer, 1993). The cheese sample was
a Parmigiano-Reggiano cheese with a total amount of 94 g free amino
acids/kg cheese (27 % of total nitrogen). The relative standard deviation of the
repeatability and reproducibility in the cheese sample was dependent on the
amino acid concentration. The median of relative standard deviation of the
repeatability and reproducibility for all amino acids with a concentration >10
mmol/kg for the IEC- and the OPA-method was <5.3 and <10%, respectively.
Results are shown in Table 9.1.
Similar precision parameters were obtained in previous studies with
Fontina, Gruyre de Comt and Parmigiano-Reggiano (Btikofer, 1992a &
b). [UB]
Sampling. If possible a 1-2 cm thick rind of the cheese sample is cut off
and discarded. The hard cheese samples are ground at room temperature,
whilst semi-hard cheese samples are cooled to 4 C and soft cheese are frozen
before grinding.
The water used for reagent preparation is deionised water from Milli-Q
installation55.
9.1.1. Ion exchange chromatography with ninhydrin post-column

derivatization
An amino acid analyser56 with ninhydrin post column derivatisation and
UV detection57 at 570 nm and 440 nm for primary and secondary amino acids
serves for the chromatographic amino acid determination offreeamino acids.
55
Millipore
67
Table 9.1. Precision parameters of free amino acid determination with ion
exchange chromatography and RP-HPLC in cheese [mmol/kg]
Amino IEC HPLC

Acid n mean sr sR n mean sr- sR
phosphoserme 5 6.0 0.49 2.42 3 3.0 0.66 1.08
aspartic acid 11 27.2 1.80 2.01 3 26.5 0.86 1.45
threonine 11 19.7 0.78 1.50 6 18.8 0.91 1.83
serine 10 50.9 1.92 3.83 7 44.7 2.52 5.62
asparagine 8 ' 21.3 1.41 2.41 6 19.7 1.57 6.07
glutamic acid 12 100.7 3.86 19.71 3 96.9 2.16 4.78
glutamine 4 1.2 0.12 0.31 6 .1.5 0.10 0.39
proline 12 75.0 4.25 8.11 3 67.3 2.88 13.65
glycine 11 31.9 1.41 1.89 7 34.8 2.28 8.02
alanine 12 25.6 1.64 2.14 5 24.1 0.95 2.25
citrulline 8 15.6 0.69 1.53 3 15.4 0.36 1.22
a-amino butyric acid - 3 0.6 0.05 0.33
valine 11 54.0 2.47 3.93 6 53.4 1.84 4.57
cystine 3 0.7 0.03 0.59 -
methionine 11 15.0 0.84 1.02 6 16.5 0.68 2.94
isoleucine 13 42.5 2.71 3.75 6 42.2 0.99 2.89
leucine 10 59.2 4.00 4.08 5 57.2 1.28 5.93
tyrosine 13 13.7 2.28 2.76 6 12.3 1.19 2.55
phenylalanine 12 25.3 1.91 2.66 5 26.0 0.53 0.71
-alanine 2 2.1 0.24 1.52 -
-amino isobutyric acid 4 8.2 0.40 8.30 -
y-amino butyric acid 10 2.7 0.33 0.89 4 2.6 0.29 1.12
ornithine 9 5.9 0.31 0.96 3 5.4 0.25 1.00
lysine 10 69.0 3.30 4.69 5 62.5 2.27 8.13
histidine 10 14.1 0.44 2.76 6 13.9 1.64 2.69
tryptophane 3 1.7 0.21 0.33 3 1.4 0.22 0.69
arginine 8 2.3 0.27 1.31 4 3.4 0.19 1.11
Legend: n, number of laboratories; sr, standard deviation of repeatability;

sR, standard deviation of reproducibility
56
Biotronik LC 7000
57
Biotronik Photometer 7025
68
The separation column (140 x 3.2 mm) is filled with BTC 2710 resin58, the
precolumn (30 x 6 mm) with BTC F resin58. The separation takes place with
five lithium citrate buffers (Table 9.2) at a buffer flow of 0.317 rnL/min and a
ninhydrin reagent flow of 0.156 mL/min (column temperature 56C). The
chromatographic separation takes about 110 min.
Citrate buffer. Weigh 19.61 g tri-sodium citrate dihydrate, 0.1 g
pentachlorophenol or another preservative reagent and 200 mg EDTA into a
1-L volumetric flask and dissolve in about 800 mL deionised water. Correct
the pH value with concentrated hydrochloric acid (-360 g/kg) to pH2.20.
Make the solution up to the mark with deionised water.
Lithium dilution buffer pH 2.20. Weigh 141.0 g of trilithium citrate
tetrahydrate into a 5-L volumetric flask, dissolve in ~4 L of deionised water
and add 12.5 mL of 2,2-thiodiethanol and 0.5 mL of octanoic acid. Add
carefully 115 mL of 370 g/kg hydrochloric acid. After cooling adjust the pH to
2.3 by dropwise addition of 370 g/kg hydrochloric acid. Make up to the mark
with deionised water. Leave the buffer for at least 12 h before adjusting the pH
to 2.20 by dropwise addition of 0.1 mol/L hydrochloric acid.
Lithium acetate buffer pH 5.20. Dissolve 964.8 g of lithium hydroxide
p. A. under stirring in 2 L of deionised water in a 10-L volumetric flask. Add
2.500 L of acetic acid p.A. carefully within 5 min (very exothermic reaction).
After dissolution of all the lithium hydroxide and cooling to room temperature
add deionised water up to ~9 L. Correct the pH to 5.2 by dropwise addition
of acetic acid and add deionised water up to about 9.9 L. Finally, adjust the pH
to 5.20 with acetic acid or concentrated lithium hydroxide solution and make
up to the mark with deionised water.
Sulfosalycilic acid solution. Weigh 75 g 5-sulfosalicylic acid dihydrate
into a 1-L volumetric flask and dissolve in about 800 mL citrate buffer.
Correct the pH value with concentrated sodium hydroxide solution to 1.75 and
make up to the mark with citrate buffer.
Brij Solution 300 g/L. Weigh 30.0 g of Brij into a 250-mL beaker and
liquefy in a hot water bath. Hot deionised water is added under permanent
stirring to a volume of 100 mL.
58
Biotronik
69
Table 9.2. Buffer composition for IEC determination

Buffer A B C D E F
pH value 2.70 3.06 3.57 4.00 3.53
Trilithium citrate.
4H20 18.8 22.6 33.8 37.6 10.0 g
Citric acid --
monohydrate p.A. 14.8 12.0 3.6 g
Lithium chloride 17.0 114.0 g
Methanol (for HPLC) 120.0 100.0 mL
Hydrochloric acid,
370g/kg 11.0 12.0 16.0 17.0 4.0 mL
Brij solution, 300 g/L 2.0 2.0 2.0 2.0 2.0 2.0 mL
Titriplex III p.A. 0.8 g
Lithium hydroxide 25.2 g
Ninhydrin reagent. Pour 500 mL ethylene glycol into a 1-L brown
bottle. Dissolve 20.0 g ninhydrin p.A. under stirring. Add 250 mL ethylene
glycol under stirring. Dissolve 0.7 g of hydrindantin dihydrate (for amino acid
determination) in the ultrasonic bath. Add 250 mL of the lithium acetate buffer
pH 5.2.
Standard solution. Give 5.00 mL of standard amino acid solution59
(2.5 umol/mL) and 5.00 mL of the norvaline solution (2.5 umol/mL in lithium
dilution buffer pH 2.20) to a 100-mL graduated flask. Make up to the mark
with lithium dilution buffer pH 2.20.
Internal standard solution 0.5 umol/mL. Weigh 117.1 mg norvaline into
a 2-L volumetric flask, dissolve and make up to the mark with citrate buffer.
Extraction and deproteination. Disperse 1.50 g grated cheese in
40.0 mL citrate buffer (external standard method) or 40.0 mL of the internal
standard solution on a magnetic stirrer for 15 min. Homogenize on Ultra-
Turrax for 5 min at 10 000 rpm. Filter on S&S black ribbon filter paper (or
similar). Pipette 10.0 mL of the filtrate into a 25-mL volumetric flask and add
slowly 10 mL of the sulfosalycilic acid solution. Keep under magnetic stirring
for 5 min. Adjust to the volume with the citrate buffer. Filter on S&S blue
ribbon filter paper (or similar). Pipette 10.0 mL of this solution into a 50-mL
volumetric flask and adjust to the volume by using the buffer recommended by
the column supplier. Filter the solution through 0.2 um membrane before
injection. Due to the presence of preservative reagent, the final sample solution
59
Benson Type ANB
70
ARDO & POLYCHRONIADQU, Ed. CHEESE ANALYSIS MANUAL
may be stored at 4C for 3 days without undergoing alterations; for longer

periods storage in freezer (-20C) is required (no alterations observed up till
6 months).
Chromatographic parameters. Figure 9.1 shows the separation of the
free amino acids from a cheese sample on a BIOTRONIC LC7000 amino acid
analyser. Chromatographic parameters are given in the legend. The big
advantage of this type of analyser are the accurate results for all known sample
types (e.g. food, biologicalfluids,feed, plants). [UB]
9.1.2. RP-HPLC with OPA/FMOC precolumn derivatization

A HPLC system with binary solvent delivery system, temperature
controlled column compartment and a programmable fluorescence or UV
detector is necessary to execute this method (Btikofer et al, 1991). Use Cis
Hypersil ODS column 250 x 4 mm, 5 urn and precolumn 20 x 4.060.
Standard solution. Transfer 5.00 mL of the amino acid standard solution
(1 umol/mL) to a 25-mL volumetric flask. Add 1.00 mL of the norvaline
solution (5 umol/mL) and 1.00 mL piperidine-4-carboxylic acid solution
(5 umol/mL) and make up the mixture to 25 mL with 0.1 mol/L hydrochloric
acid p.A.
Eluent A: Weigh 4.10 g of sodium acetate trihydrate p.A. and 40 mg
Titriplex III p.A. into a 1-L volumetric flask, add deionised water to a volume
of about 950 mL, adjust the pH with 0.1 mol/L NaOH to 7.20 and make up
the solution to the mark with deionised water. Add 2.5 mL of tetrahydrofiiran
puriss. p.A., mix the solution and filter through a 1.2 urn RAWP Millipore
filter.
Eluent B: Weigh 2.72 g of sodium acetate trihydrate p.A. and 40 mg of
Titriplex III p.A. into a 1-L flask and dissolve in 200 mL of deionised water.
Adjust the pH to 7.20 with 0.1 mol/L NaOH. Add 800 mL of acetonitrile for
HPLC under good stirring and filter the solution through a 0.5 um FHUP
Millipore filter.
Extraction and deproteination. Disperse 1.00 g grated cheese in
10.0 mL of 0.1 mol/L hydrochloric acid solution containing 0.4 umol/mL
norvaline and piperidine-4-carboxylic acid as internal standards in a 25-mL
centrifuge tube. Homogenize on Ultra-Turrax for 5 min at 20 000 rpm. Keep
the covered tube (Para-Film) for max. 30 min in an ultrasonic bath. Centrifuge
10 min at 3000 g, take 1 mL of the supernatant and add 1 mL of 400 g/L
60
Hewlett Packard
71
trichloroacetic acid solution. Mix the suspension on a Vortex mixer and keep it
in an ice bath for 10 min or alternatively overnight in a refrigerator. Centrifuge
the suspension 10 min at 20 000 g. The pH of the clear supernatant solution is
adjusted to at least 7.5 withNaOH before derivatisation of the amino acids. An
alternative method is to add TCA with the concentration 40 g/L and watch out
for small peptides that may comigrate with the amino acids. Filter the solution
through 0.45 um membrane61 before injection.
60
retention timt [min]
Figure 9.1. Amino acid chromatogram of cheese extract by BIOTRONIK LC

7000 amino acid analyser with UV-detection. Chromatographic conditions
were as follows: Solvent A to E were all lithium citrate buffers with different
methanol content, ionic strength and pH values (Table 9.2); buffer flow rate,
0.317 mL/min, ninhydrin reagent, 0.156 mL/min (at 56C); column
temperature was set at 34C at the start of the chromatogram and changed to
56C at 30 min. Column cleanup with solvent F and reequilibration required
41 min. The detection wavelengths were 570 nm for primary and 440 nm for
secondary amino acids. The separation program was run with the following
buffer solutions: 18 min A, 26 min B, 19 min C, 8 min D, 45 min E, 15 min F
and 35 min A. Temperature was programmed as follows: 34 C during 30 min
and 56C during 80 min.
61
ACRO LC13 0.45 um (Scan)
72
Derivatization reaction. Mix 50 uL of the borate buffer (0.4 mol/L pH

10.4), 10 uL OPA/MPA (o-phtalaldehyde/3-mercaptopropionic acid) reagent
and 10 uL sample solution for 60 s. Add 10 uL FMOC (9-fluorenyl-
methylchlorofonnate) reagent cone, in acetonitrile and mix the solution for 60
s. Because the OPA drivtes are not very stable, inject the solution directly
into the HPLC system.
Chromatographic parameters. Figure 9.2 shows the separation of the
free amino acids from a cheese sample on a HPLC system. Primary amino
acids are derivatized with OPA/MPA, secondary amino acids with FMOC.
Chromatographic parameters are given in the legend. Exitation and emission
wavelengths have to be chosen in relation to the equipment used. In the
example shown (Figure 9.2) the exitation wavelength is 230 nm instead of
optimal 340 irai because of quenching effects from free OPA/MPA reagents.
UJQ.
\\im m L
retention time [min]
Figure 9.2. Amino acid chromatogram of cheese extract by Hewlett-Packard

Type 1090 HPLC equipment with fluorescence detection. Chromatographic
conditions were as follows: solvent A, 30 mmol/L NaOAc pH 7.20 + 0.25 %
tetrahydrofurane + 0.1 mol/L titriplex III; solvent B, 100 mmol/L NaOAc
pH 7.20 + 80 % acetonitrile + 0.1 mol/L titriplex III; flow rate, 1.000 mL/min;
column temperature, 42C. The derivatized amino acids were separated by a
24-min stepwise linear gradient from 3.3 to 40 % B over 20 min and 40 to
100% B over 4 min. Column cleanup with 100% B and reequilibration
required 7 min. The detector parameters were set to detect the OPA drivtes
at the start of the program at Ex: 340 nm and Em: 455 nm, switched to Ex:
230 nm at 3 min and then changed at 17 min to detect the FMOC drivtes Ex:
266 nm and Em: 313 nm.
73
The analysis may also be performed with light absorbance measurements at

340 nm for OPA-derivates and 266 nm for FMOC-derivates.
The separation of free amino acids with OPA/FMOC precolumn
derivatization can be obtained on each HPLC system equipped with binary
gradient system, column oven and UV orfluorescencedetector. The drawback
of this method is the uncontrolled Influence of the matrix. Even in protein
hydrolysates poor precision results were obtained in a feed sample (Btikofer
et al, 1992). [UB]
9.1.3. Ion exchange-HPLC with OPA post-column derivatization

Free amino acids are determined by HPLC62 with one pump ternary
gradient using a Na IEC column63 (length: 25 cm, i.d.: 3 mm). Post-column
derivatization with OPA at 40C (A**: 330 run, A,em: 464 nm).
Buffers (sodium citrate): A: pH 3.15, 0.2 N [Na+]; B: pH 7.40, 1.0 N
[Na+]. Sample buffer: pH 2.20, 0.2 N [Na+]. Rgnrant (Q: 0.1 N NaOH +
0.1 N NaCl. Flow rate: 0.3 mL/rm'n. Column temperature: 50C. Gradient as
follows:
Time %A %B %c
(min)
0 100 0 0
10 100 0 0
36 0 100 0
60 0 100 0
60.1 0 0 100
62.0 0 0 100
62.1 100 0 0
76.2 100 0 0
Sample preparation is performed according to Resmini et al. (1993).

Proline is not determined with this method, except if oxidized with 5%
NaCIO before derivatization. [AP]
62
Scientific Systems, Inc., SSI, USA
63
Pickering Laboratories
74
9.1.4. Other liquid chromatographic methods

Krause et al. (1995) used an automatic precolumn derivatization with
dabsylchloride and RP-HPLC separation of free amino acids and biogenic
amines in cheese.
Kirschbaum et al. (1994) used automated precolumn derivatization with
fluorenylmethyl chloroformate (FMOC) and RP-HPLC for the determination
offreeamino acids in cheese. [UB]
9.2. Analysis of amines

Extraction and dansylation. Amines are extracted from the cheese by the
protocole of Etter et al. (1990) modified by Btikofer et al. (1990). Grated
cheese (5g a.k.)Wc) is blended5 with 50 mL acetonitrile/0.2 M perchloric acid
(50/50) and 500 uL 1,7-diamino heptane (lg/L) as internal standard. A portion
of the slurry (10 mL) is then centrifuged at 1200 g for 10 min at 4C. A
portion of the supernatant (400 uL) is diluted with acetonitrile (1.6 mL) and
uhq water (1.4 mL) and buffered with 400 uL sodium carbonate (0.2g/mL).
Tubes are agitated by vortex for 10 s. Amines are then dansylated by adding
200 uL dansyl chloride (50 mg/mL in acetone), stirred (vortex) for 5 s. The
tubes are then held 30 min in a water-bath at 40C without any light. The tubes
must be stirred by vortex every 5 min. Excess of sodium chloride is then
consumed by addition of 400 uL sodium glutamate (50 mg/mL). Tubes are
kept inthe40C bath for 60 min. Dansylated amines are then extracted by
partition: 2 mL ethyl acetate are added and the tubes are vigorously shaken
for 1 min (vortex). The organic phase (about 2 mL) is recovered by
centrifugation (1200 g, 10 min, 4C) and evaporated under vacuum at 40C.
The dry residue is diluted with 400 uL acetonitrile and sonicated. After a 15-
min stay the mixture is filtrated on 0.45 urn64.
Chromatography. The separation of dansylated amines (5 uL) is
performed at 37C by RP-HPLC65 using a precolumn+column system
(length: 10+250 mm, i.d.: 4.6 mm) filled with silica gel bonded with C)8
(Hypersil ODS, porosity=120 , part, o.d.: 5u). Under our conditions, the
protocol proposed by Etter et al. (1990) was found unsatisfactory and had to
be modified (Pochet, np). The solvents we use are: buffer pH 8 = Tris 0.1M /
64
Minisart polypropylenefilter.Prolabo, Strasbourg, FR.
65
HPLC Autosampler 465. Kontron Instruments, St Quentin en Yvelines, FR.
75
acetic acid O.IM / water (2 / 1 / 2); solvent A = buffer pH 8 (30 mL) /

acetonitrile (550 mL) / water (420 mL); solvent B = buffer pH 8 (2 mL) /
acetonitrile (900 mL) / water(100 mL). The following gradient is used at 0.8
mL/minflowrate :
Time (min) ~~ % solvent A
0 95
15 70
20 37
21 0
31 0
32 95
42 95
Dansylated amines are detected at 254 nm66 and surface of the peak is
estimated with an integrator. Identification of the peaks is done by comparison
of their retention time with standard mixtures of amines. The quantification is
done by referring to calibration curves calculated by the analysis of a mixture
of commercial amines in increasing known concentrations (8 ug to 1 mg/g
equivalent cheese) in solution in 0.02 N sulfuric acid (Figure 9.3). Results are
corrected by the rate of recovery of the internal standard added at the
beginning of the extraction. [SP]
66
HPLC system Varan LC 5000. Varan, Paris, FR
76
e ^
t/3
i/i
o
C/
fe S
H
Mv |U L_^
Figure 9.3. Separation of dansylated amines by RP-HPLC (standard

mixture, 1 mg/mL each): TRY, tryptamine; PHE, phenylamine; ISO,
isopentylamine; PUT, putrescine; CAD, cadaverine; HIS, histamine; DAH,
diaminoheptane (internal standard); TYR, tyramine; SPE, spermidine; SPN,
spermine. (Pochet, np)
77
10. ANALYSIS OF FREE FATTY ACIDS
10.1. Gas chromatographic methods

METHOD I
Standards. Different standard mixtures of the FFAs C4, CO, C8, C10, C)2,
C14, C16, and Cis, >99%, pure by gas-liquid chromatography, can be used to
determine the response factors. Solution of 0.6% (w/v) n-nonanoic acid in
diethyl ether is used as internal standard in the quantitative determinations.
This is added to cheese samples at the start of the analyses.
Preparation of fat extracts from the cheeses and methylation procedure.

Lipid extraction is carried out on an acidified cheese slurry using ethyl ether
according to the procedure described by Martin-Hernandez et al. (1988).
Ten grams of sample are mixed with 5 mL of distilled water and acidified
with H2SO4 to bring the pH down to 1.5 (0.7-1.3 mL). One mL of n-
nonanoic acid 0.6% (w/v) in diethyl ether is added to the cheese as a standard
and the acidified homogenate is mixed with 15 mL of ice-cold diethyl ether,
shaken for 3 min in an electrical homogenizer, then centrifiiged at 3000 rpm
for five minutes, at 0C. The upper organic phase is transferred to a bottle
containing 1 g anhydrous Na2S4 prior to the methylation of the FFA in an
attempt to absorb the water in the cheese slurries.
After 5 min, 3 mL of the ether layer is transferred to a 10-mL screw-
capped vial, 0.2 mL 20% tetramethylammonium hydroxide (TMAH) in
methanol is added and the mixture shaken for 1-2 min. The upper layer
contains the methyl esters from glycerides, while the tetramethylammonium
soaps remain in the bottom layer. For the analysis of the fatty acid composition
of glycerides the upper layer is transferred to another vial and then washed
with 0.5 mL water; an aliquot of the ethereal layer is then injected. For the
FFA analysis the bottom layer is separated, washed three times with 0.5-2
mL ethyl ether and then neutralized to pH 9 using thymol blue as indicator
with 2M and/or 0.5M of methanol HCl before injection (Martinez-Castro et
al, 1986).
79
Gas chromatographic analysis. The methyl esters are analyzed by

programmed gas-liquid chromatography (GLC) as described in Jurez et al.
(1992). Experimental conditions are as follows: Carrier gas: N2 or He, split
flow ratio 1:20 for the entire study. Type of column: a WCOT silica capillary
(50 m x 0.22 mm) with silicone, 50 % phenyl, 50 % cyanopropyl as the
stationary phase (df = 0.22 mm), is suitable for FFA analysis. Column
temperature: initial hold for 3 min at 70C rising to 190C at 13C x min"1; 25
min final hold at 190C. Injector operating conditions: temperature 300C.
Pyrolytic methylation of FFAs in capillary PTV injectors. Pyrolytic

methylation of the FFAs may give rise to unsatisfactory results in some types
of injector. Moreover, split-mode capillary injectors may cause discrimination
in mixtures that span a broad range of volatilities, such as mixtures of FFAs
from milk fat. In the case of the pyrolytic reaction employed in the method
considered herein [TMAH soaps that yield fatty acid methyl esters (FAME)
and TMA], discrimination may impede quantitative determination, since TMA
flash-off is affected by the excess TMAH and sample size.
FFA fraction from a cheese is analyzed using different PTV temperature
programmes and the quantitative determination is performed using n-nonanoic
acid as the internal standard. Samples injected at 300C give the best
quantitative results. A component that has retention time slightly shorter than
that for CO is present in all the chromatograms; it is more intense at lower FFA
contents and has not been identified. Lactic acid is also detected, above all in
the fresh cheese, but it did not interfere with the determinations. Certain
chromatograms presented artifacts that may be caused by the cleavage of
certain bonds produced by reaction of excess, unpyrolyzed catalyst to TMA
with the stationary phase.
Accuracy of the method. Accuracy was tested using two types of cheese,
one a fresh cheese with a low FFA content and the other a cheese that had
undergone moderate lipolysis.
Table 10.1 sets out the mean values and standard deviations for the
major FFAs and the total FFA content in both types of cheese.
The reproducibility values for the total FFA content in the fresh cheese
with the low FFA content [coefficient of variation (CV): 6.3 % for a total FFA
content of 1500 mg/kg] were comparable to those reported by Woo & Lindsay
80
ARD & POLYCHRONIADOU, Ed. CHEESE ANALYSIS MANUAL
Table 10.1. Reproducibility of the analytical method applied to cheese samples

containing different FFA levels (A = fresh cheese; B = cheese aged for two
months).
Cheese A FFA concentration (mg/kg)

Sample 1 2 3 4 5 XSD
Component
c4 41 47 42 31 52 43 8
c6 27 29 26 20 28 264
c8 38 32 23 26 34 31 6
Cio 93 91 81 81 85 866
C,2 74 83 72 61 71 728
CM 172 159 137 141 179 158 19
Cl6 491 455 380 423 504 451 51
C 123 116 105 104 126 11510
Cl8:l 375 468 423 399 394 412 36
Total 1434 1480 1289 1286 1473 1392 97
Cheese B F]T A concentration (mjp&

Sample 1 2 3 4 5 6
Component
c4 453 472 428 437 445 44717
c6 243 266 232 279 277 259+21
c8 258 252 271 346 266 279+38
Cio 699 730 715 743 719 72117
C,2 363 368 360 383 376 37010
CM 967 991 964 1026 1098 100956
Cl6 2092 2290 2077 2166 2141 2153+85
C,8 820 873 843 876 864 855+24
Ci8:l 2800 2492 2779 2516 2476
2613162
Total 8695 8734 8669 8772 8662 870646
X: Mean values; SD: Standard deviation
81
(1982) for Cheddar cheese (CV=5.4 % for a total FFA content of 1500
mg/kg), but they were lower than those recorded by Deeth et al. (1983) and
McNeill & Connolly (1989), also for Cheddar cheese.
In contrast, accuracy improved substantially for the cheese with the
higher FFA content. Analysis of the" cheese studied, with a total FFA content
of nearly 9 000 mg/kg, yielded an overall coefficient of variation of 0.48 %, an
improvement over the CV of 2.2 % for a cheese with a total FFA content of
8000 mg/kg determined with conventional columns, reported by Martin-
Hernandez et al. ( 1988).
Recovery rates. The recovery rates obtained with the method were
examined by adding two standard mixtures of butyric, caproic, caprylic, capric,
lauric, myristic, palmitic, and stearic acids to a sample from a cheese with a
FFA content of approximately 4000 mg/kg prior to analysis.
The first standard added approximately 50 % more of each FFA present,
while the second standard added approximately 100 % more.
Table 10.2 gives the percentage recovery rates for the different FFAs in
the two tests. The percentage recoveries for the different FFAs ranged
between 91 % for C4 and 103 % for Cig. These results were comparable to
those reported by other workers. The results for certain FFAs improved as the
amount of added FFA increased, although the short-chain FFAs presented the
lowest values in both tests, which was ascribed to losses during extraction.
Nevertheless, the error in the determinations for the short and medium-chain
FFAs (C4-C12), which are the FFAs responsible for organoleptic perception of
rancidity, was 5-6 %.
This technique provides a fest and reliable method of FFA analysis for
estimating the degree of lipolysis in cheeses with low FFA contents, and
particularly in those with high FFA contents.
Under the conditions tested chromatograms that were practically devoid
of interference were obtained, providing care was taken to limit the excess
catalyst in the reaction mixture and the mixture was buffered to a pH of around
9 (Martinez-Castro et al, 1986).
With the derivatization technique tested, there was no loss of the volatile
components, because the FFAs were injected in the form of soaps, and
fiirthermore the methyl esters of the glyceridic fraction were also obtained as a
82
Table 10.2. Recovery of FFAs added to a cheese sample (mg/kg)
Compo Initial Amount Total Recovered Recovered

nent amount added a/b calculated a/b a b
XSD XSD XSD (%)

c4 291 22 275 /103 566/394 511 2 4 367 2 3 91/93
c6 159 12 189/ 89 348/ 248 330 1 0 235 9 95/95
c8 183 35 200/ 76 3 8 3 / 259 356 9 236 1 2 93/91
Cio 243 14 222 /114 465 / 357 451 7 343 13 97/96
C12 2 0 5 12 182/101 387/ 306 390 5 297 5 101/97
c 14 584 49 470 / 273 1054/ 857 1055 4 839 2 4 100/ 98
C,6 1536 138 1203/ 748 2739 / 2284 2800 52 2233 41 102/98
Ci8 433 36 384/280 817/713 842 19 704 3 9 103/99
Total 3,634207 3,125/1,784 6,759/5,418 6,735 41 5,25432 99.7/97.0
X: mean values; SD: standard deviation

a: amount added 100 % initial amount; b: amount added 50 % initial amount
separate phase. In addition, carrying out the analysis on a capillary column

using methyl esters following pyrolytic methylation made it possible to quantify
the major and minor FFAs without difficulty, avoiding the adsorption of long-
chain FFAs that takes place when phases like the free fatty acid phase (FFAP) are
used.
Fatty acid composition of the triglyceride and free fatty acid fractions. In
the case of blue cheeses and others with high levels of lipolysis, the fatty acid
composition of the triglyceride fraction can change over the ripening period. In
these types of cheese it is useful to compare the fatty acid profiles of the
triglyceride and FFA fractions. With the proposed method it is possible to achieve
it as mentioned above in a single step (De la Fuente et al, 1993). [MJ]
METHOD II
Analysis of free fatty acid (FFA) content in cheese is done following the
general arrangements of Deeth et al. (1983) for the extraction of fats, and
Needs et al. (1983) for the recovery of free fatty acids. These procedures have
83
been combined and extraction procedure and chromatography in particular

have been adapted to cheese analysis as described here (Pochet, np).
Extraction of fats. Grated cheese (1 g a. k.) is thoroughly weighed in a

10-mL glass screw-capped tube containing 10 Pyrex glass balls (o.d.: 5 mm).
Internal standards are then precisely added (400 uL pentanoic acid 0.06% a.k.
and 400 uL tridecanoic acid 0.12% a.k. in diethyl oxide), followed by 8 mL
cooled diethyl oxide and 100 uL sulfuric acid 4N. The tubes are first
vigorously shaken by hand for 5 min then using a rotary shaker67 for 1 h arid
finally centrifged (1000 g, 5 min, 5C). The supernatant organic phase is
transferred into a 25-mL test tube and filled up to 25 mL with diethyl oxide.
The liquid is then transferred into a 50-mL screw-capped bottle containing
anhydrous sodium sulfate (2.5 g) which is gently shaken68 for 15 min.
Fixation offree fatty acids on resin. The supernatant is then removed

using a Pasteur pipette and transferred into another 50-mL screw-capped
bottle containing about 200 mg of drained strong anionic resin69 (a batch of 20
g resin is prepared before analyses by cleaning it under nitrogen with sodium
hydroxide IM (10 mL/g), rinsing it with uhqw (8x15 mL/g) and methanol
(3x10 mL/g) and stored in methanol). Methanol (5 mL) is then added to the
mixture which is gently shaken for 1 h to allow the whole free fatty acids to be
fixed. At the end, the supernatant containing esterified fats is discarded using a
Pasteur pipette. The resin is then thoroughly rinsed with 5 x 5 mL diethyl
oxide/methanol mixture (2:1 v/v) discarding the supernatant at each rinsing
and taking care not to loose any resin balls. For the last rinsing, the resin is
quantitatively transferred with a shortened Pasteur pipette into a 2-mL screw-
capped vial. The excess of solvent is discarded and an internal standard is
added (400 uL methyl undecanoate 0.07 % a.k. in diethyl oxide.). Solvent is
then evaporated under gentle nitrogen flow70.
67
Agitesi 34050. Bioblock Scientific, Illkirch, FR
68
IKA-Vibrax-VXR horizontal shaker, speed 200. Janke et Kunkel, DCA Labortechnik,
Staufen, DE
Âmberlyst A-26,25-30 m2/g, 4.4 meq/g. Sigma A-5522.
70
Needle evaporator, Meyer N-Evap. Organomation, Berlin, DE .
84
Methylation of free fatty acids. The free fatty acids are then immediately
methylated for one night in a dark place with 300 uL acetyl chloride in
methanol (1:10 v/v)(IDF, 1991). The slurry is then mixed for 10 s with 150 uL
diethyl oxide. The supernatant is recovered in a 5-mL glass disposable tube
and mixed with 2.5 mL sodium chloride solution saturated in salt and diethyl
oxide. The upper organic layer is transferred into another tube containing
about 100 mg anhydrous sodium sulfate, gently mixed, and allowed to dry for
5 min. The upper phase is then recovered with a Pasteur pipette and
transferred into a 2-mL screw-capped vial, diluted with 250 uL pentane and
injected in the gas Chromatograph (0.1-0.3 uL). If necessary, methylated fatty
acids can be kept at -15C but for no more than a few hours.
Special attention is given for the dishes to be very clean. Residual
organic matter is removed by digestion in sulfo-chromic mixture following
soaking in a basic detergent. The dishes are extensively rinsed alternatively
with large quantities of hot tap water and finally uhqw.
Gas chromatography. Methyl esters of FFA are separated on a capillary

gas chromatography system71 fitted with an on column injector and a FID
detector. The column is a fused silica capillary column (length: 30 m, i.d.: 0.32
mm) bonded with a polar phase (D-BWax,filmthickness: 0.5 um)72fittedwith
a naked silica precolumn (length: 1 m). Characteristics of the elution are the
following: carrier gas: 50 kPa helium; temperature gradient: from 40C
(injection) up to 190C (5C/min), then from 190C up to 240C (2C/min)
then 240C for 10 min, then return to initial conditions; F.I.D.: 260C,
flame: hydrogen (50 kPa)-air (100 kPa).
Identity of the peaks (Figures 10.1 and 10.2) is estimated by comparison
with the retention sequences of peaks identified by mass spectrometry in
similar conditions on butter samples (Dorey et al, 1988). But it is necessary to
check the identity of certain peaks in some cheese samples by GC-mass
spectrometry73. Uncertainty could subsist in decanoic acid quantification
because of occasional coelution of a peak identified as succinic acid. Surfaces
71
GC 8000. Carlo Erba Instrumentazione, Milano, IT.
72
J & W, Scientific, Folsom, California, USA.
73
INRA-Laboratoire de Recherches sur les Armes, Dijon, FR.
85
JU
1>
Figure 10.1. Separation of methylated fatty acids of a hard cheese sample by

capillary GC (see text for fiuther details on procedure): \=C4:0, 2=C5:0 (i.s.),
3=C6:0, 4=C7:0, 5=C8:0, 6=C9:0,1=C10:0, %=C10:l(cis-9), 9=C11:0 (i.s.),
10=C12:0, U=C12:l(cis-9), 12=C13:0(iso), 13=C13:0 (i.s.), \4=C14:0 (iso),
15=C14:0, \6=C14:1 (cis9), \1=C15:0 (iso), \%=C15:0 (anteiso), \9=C15:0,
20=CJ5:1, 2l=C16:0 (iso), 22=C16:0, 23=CJ6:1 (?), 24=C16:1 (cis 9),
2S=C16:1 (?), 26=C17:0 (iso), 21=C17:0 (anteiso), 2%=C17:0, 29=C17:1
(cis9), 30=C18:0 (iso), 3\=C18:0, 32=C18:1 (cis9), 33=C18:1 (trans-U),
34=CJ8:1 (?), 35=C18:1 (?), 36=C18:1 (?), 31=C18:1 (?), 3%=C18:2
(cis9,cisl2) 39-C18.-2 (?), 40=C19:0, 4\=C19:1 (?), 42=C18:3
(cis9,cisl2,cisl5), 43=C18:2 (cis9,trans 11), 44=C20:0, 45=C20:1 (9).
(Pochet et al, np)(?= double links non positioned, i.s.=internal standard).
86
1D 11 IS 19 22 24 21 31 J 2 38 42 44
C : .
i >. I . t . 1. u Ik-
u
Figure 10.2. Separation of methylated fatty acids of a standard mixture by

capillary GC (see text for further details on procedure): \=C4:0, 2=C5:0 (i.s.),
3=C6:0, 4=C7:0, 5=C8:0, 6=C9:0, 7=C10:0, &=C10:1 (cis-9), 9=C11:0 (i.s.)
10=C12:0, \\=C12:1 (cis-9), \2=C13:0 (iso), 13=C13:0 (i.s.), \4=C14:0
(iso), \5=C14:0, 16=C14:1 (cis-9), \1=C15:0 (iso), 1S=C15:0 (anteiso),
\9=C15:0, 20=C15:1, 2l=C16:0 (iso), 22=C16:0, 23=C16:1 (?), 24=C16:1
(cis 9), 25=C16:1 (?), 26=C17:0 (iso), 21=C17:0 (anteiso), 2S=C17:0,
29=C17:1 (cis9), 30=C18:0 (iso), 3\=C18:0, 32=C18:1 (cis9), 33=C18:1
(trans-11), 34=C18:1 (?), 35=C18:1 (?), 36=C18:1 (?), 37=CJ8:1 (?),
3S=CJ8:2 (cis9,cisl2) 39=C18:2 (?), 40=C19:0, 4\=C19:1 (?), 42=C18:3
(cis9,cisl2,cisl5), 43=C18:2 (cis9,transll), 44=C20:0, 45=C20:1 (9).
(Pochet et ah, np) (?= double links non positioned, i.s.=internal standard)
87
of the peaks (S) are integrated74 and quantification is done using response
coefficients (K = quantity/surface) previously calculated established by
analysing a mixture of known quantities (Q) of each occurring "dairy" fatty
acid (fa) between C4 and C2o. The coefficient of the fatty acid which could not
be supplied from commercial source are estimated from the available values
(Pochet, np). The ratio is corrected by a similar ratio calculated for the internal
standard (is) i.e. methyl undecanoate.
Kfa (standard) = (Qfa / Sfa) / (Qis / Sfa)

Qfa (in unknown sample) = Sfa x Kfa x (Qis / Sis)
Values are then corrected using the rate of recovery of the two internal
standards added at the beginning of the procedure (C5 for fatty acid chains
between C4 and until d 2 , Cn for fatty acid chains between Cu and C20).
Results are finally expressed on a weight or molecular basis, either in
concentration of the cheese or as a percentage of the mixture of detected FFA.
[SP]
METHOD m
Extraction
Step 1 (to be done the previous afternoon).
Weigh 2.5 g cheese into a 25-mL scintillation screw-cap vial. Add
250 uL of internal standards solution (125 mg 2-methylpentanoic acid + 50
mg pelargonie acid + 100 mg tridecanoic acid + 500 mg heptadecanoic acid +
heptane up to 100 mL; ampoules are stored in deep-freezer at -18C). Add 3
drops 2.5 M H2S04 (25.54 g H2S04 96% + H20 up to 100 mL) by means of a
Pasteur pipette. Add 4.5 g anhydrous Na2SC4. Mix accurately and put into the
refrigerator (4-7 C) overnight.
Step 2.
The following day put a Millipore AP25 prefilter between the body of an
empty Extrelut cartridge (20 mL) and the adaptor & stopcock assembly of
the extraction unit and fill the cartridge with the dried sample. Connect the
extraction unit. Rinse the scintillation vial thrice by means of 5-mL portions of
74
SP 4290. Spectra-Physics, San Jos, CA, USA.
gg
50 mL extraction solvent (diethyl ether/heptane 1:1 v/v) adding the rinsing and
the remaining part of the solvent to the cartridge. Stir gently with a glass stick.
Activate a NH2 Sep-Pak Vac cartridge 6 mL, 500 mg75 by means of 6
mL heptane. Put the Extrelut cartridge on the Sep-Pak Vac cartridge and let
the solution drop at normal pressure (gravity flow). Rinse the Sep-Pak Vac
cartridge by means of 6 mL rinsing solvent (chloroform/isopropanol 2:1 v/v).
Connect a needle to the cartridge barrel and add 3 mL FFA elution solvent
(diethyl ether/formic acid 98:2 v/v). Discharge the first 1.2 mL of the eluate
and save the remaining for the GC analysis.
Gas chromatography. Apparatus: Perkin Elmer Sigma 3B equipped

with a flame ionization detector; Column: FFAP 15 m, 0.53 i.d., 1 um film
thickness; Carrier gas: helium, 5 psi; Injector temperature: 85 C; Detector
temperature: 250 'C; Injection technique: cold on-column injection (1.5
uL); Operating conditions: oven at 80 ' C , then to 235 C at 5C/min,
holding time: 7 min. [JH]
10.2. Determination of the total free fatty acids in cheese

Total free fatty acids in cheese are analyzed by the copper soaps method,
according to Bynum et al (1984).
Reagents
Copper soaps reagent: 100 mL 1.0 M Cu(N03)2-2.5H20 and 50 mL
triethanolamine, diluted to 1 L with saturated NaCl solution and adjusted to
pH8.3withlMNaOH.
Extraction solvent: Chloroform/heptane/methanol (CHM) 49:49:2
Colouring reagent (prepare freshly): Sodium diethyldithiocarbamate
(0.5%, w/v) in 1-butanol
HCl: 0.7 M HCl
Palmitate Standard: 200 mg / 100 mL palmitic acid
75
Waters cat. WAT054560
89
Procedure for cheese samples. Cheese samples (200 mg) are mixed with
0.4 mL 0.7 M HCl (vortex) in glass screw-capped culture tubes (20 x 200
mm); 7.5 mL copper soaps reagent are then added. The tubes are capped,
incubated at 60C for 10 min, cooled in water to room temperature and mixed
vigorously. The Cu soaps of the free fatty acids are then extracted by addition
of 20 mL CHM and gently shaking the tubes for 30 mia The solvent and
aqueous layers are separated by centrifiigation for 10 min using a Gerber
centrifuge. An aliquot (5 mL) of the CHM layer is then transferred to an acid-
washed test tube containing 0.2 mL of the carbamate colouring reagent. The
absorbance of the coloured complex is measured at 440 nm. The concentration
of free fatty acids in the sample is calculated by the equation y = -
0.0459+0.0017x, with y the Auo and x the ug of palmitate or by reference to a
standard curve.
Standard curve andreagent blank. To each of 7 screw-capped glass test

tubes is added 200 mg of fat-free rennet casein. To each tube in turn are added
0, 25, 50, 100, 150, 200 or 300 uL of palmitate standard giving 0, 50,
100, 200, 300, 400 and 600 ug palmitate, respectively. The standards are
subsequently treated as per samples. The absorbance of the standards is
measured at 440 nm, the solution containing 0 ug palmitate acting as the
reagent blank. A plot of AMO against fig palmitate is prepared and the
absorbance values of the samples converted to mg palmitate / 200 mg cheese.
[PMS]
Precision. The copper soap method is simple and rapid and does not
need a GC equipment. However, repeatability is poor and, also, sometimes
technical problems occur. The method is recommended only for a rough
estimation of the FFA in cheese. [AP, SP, PMS]
90
11. ANALYSIS OF VOLATILE COMPOUNDS
11.1. Short carboxylic acids

VOLATILE FATTY ACIDS
Extraction. Grated cheese (15-20 g a.k.) is weighed in a screw-capped
100-mL bottle and 25 mg a. k. pentanoic acid are added as an internal standard
(about 2 drops using a Pasteur pipette). Both are blended with 50 mL sulfuric
acid 10%, the bottle being maintained in a melting ice bath during mixing. The
slurry is then introduced in a Jalade extractor (Figure 11.1) and extracted for 6
h by reflux with 120 mL of a mixture of petroleum spirit (35-60C) and diethyl
ether (50/50). At the end of the extraction, all the clear solvent containing fatty
components is recovered in the bulb glass and transferred into a decant phial.
Ethanol 80% (50 mL) is added and the phial is vigorously shaken 60 times by
hand in order to allow the best partition between free fatty acids in the
alcoholic phase and esterified fats remaining in the ether phase. Fatty acids are
then saponified by adding sodium hydroxide 1% in a slight excess in the
presence of phenolphtaleine and with regular shakings. The mixture is then
allowed to decant for 30 min and the lower phase is then recovered in a 50-mL
beaker. The beaker is allowed to stand in a fume cupboard to eliminate the
remaining traces of diethyl ether. Fatty soaps are then dried using an oven or a
rotaevaporator under vacuum. When completely dry, pellets are transferred
into an airtight 2-mL plastic tube and, if necessary, kept at 4C until analysis
by gas chromatography. Just before chromatography, 1 mL of freshly prepared
trichloroacetic acid 12% in ethanol is added to about 50 mg of dry soap
weighed in a 10-mL glass screw-capped culture tube. The tube is vigorously
shaken (vortex) for 2 min and then allowed to settle for 30 min at 4C. The
upper clear phase is then injected (0.2 to 1 uL).
Gas chromatography. Volatile fatty acids are separated as the acid form
on a gas chromatography system76 fitted with an injector with removable glass
insert and an FID detector. The column is a pure inox column (length: 2 m,
i.d.: 2.1 mm)77filled with Chromosorb G/AW/DMCS (100-120 mesh)
76
GC 8000. Carlo Erba Instrumentazione, Milano, IT.
77
Supelco, L'Isle d' Abeau, FR.
91
impregnated at 10% with a polar phase (Carbowax 20M-acide

78
nitroterephtalic) .
Tap water
J
Reflux
Over filling level
o**-r- sluny + condensed solvents
End of the extraction : procedure to collect clear solvent

tr
Solvents vapor
Solveras (petroleum spirit/diethyloxyde)

+ extracted tats
Heat ine block
Figure 11.1. Scheme of a Jalade extractor.

78
Free Fatty Acid Phase (F.F.A.P.)- Varan, Les Ulis, FR.
92
Characteristics of the elution are the following: carrier gas:

nitrogen 15 mL/min, injector temperature: 190C, oven temperature
gradient: from 145C for 12 min, then up to 180C (5C/min), then 10 min at
180C, then return to the initial conditions. F.I.D.: 200C, flame: hydrogen
(25 mL/min) - air (400 mL/min).
Identity of the peaks is estimated by comparison of the retention time of
pure substances injected in similar conditions (Figure 11.2). Surface of each
peak (S) is integrated79 and quantification is done using response coefficients
(K= quantity/surface) previously established by analysing a mixture of all the
occuring volatile fatty acid (VFA): acetic (C2), n-propanoic (C3), isobutyric
(C4), butyric (C4), isovaleric (C5), isocaproic (iC) and caproic (CO).
The ratio is corrected by a similar ratio calculated for the internal
standard (is) i.e. valeric acid.
Kvfi, (standard) = (Qvfe / Sv&) / (Qis / Sis)

Qvf (in unknown sample) = Sy& x Kv& x(Qis (Q,s/Sis)
Results can be finally expressed on a weight or molecular basis, either in

concentration of the cheese or as a percentage of the mixture of detected VFA.
Separation would be better on a widebore capillary column but the
protocol must be adapted. Because of the degrading effect of the non volatile
fatty acids which accumulate at the beginning, performance of the system tends
to decrease rapidly. Therefore, the inlet of the injector must be regularly
cleaned (each 10 sample injections) and the beginning of the column must be
renewed when necessary. [SP]
11.2. Volatile compounds in cheese
METHOD I (using a dynamic headspace GC/MS-FID)

Reagents and Chemicals. Boil Milli-Q water for approx. 15 min with an
electric heating device to strip off all volatile trace components under a
continuous nitrogen flow.
79
ENICA 31. Delsi-Nermag, Argenteuil, FR.
93
180-
w
170-
o
iso-
us
6 8 10 12 14 16 18 20 22 24
(on)
Figure 11.2. Separation of volatile fatty acids by GC. Standard mixture: C2,
acetic acid; C3, propionic acid; i Q , isobutyric acid; C4 butyric acid; C5,
isovaleric acid; C5, valeric acid (internal standard); iC, isocaproic acid; CO,
caproic acid (Pochet, np).
94
Samples. Freeze sample material. Shortly before the analysis, grind a

representative amount of approximately 20 g of the sample material, weigh it
precisely and disperse it finely in 80 mL water with a high speed homogeniser80
running at 5000 rpm for 5 min. Finally introduce 20 mL of this mixture
carefully in a 25-mL non-fritted sparger of the Purge & Trap extraction
system.
Extraction of the volatiles. The Purge and Trap system LSC 200081
(Figure 11.3) includes a 25-mL non-fritted sparger82, a trap (No 8, containing
a mixture of Carbosieve Sin (0.05 g) and Carbopack B60/80 (0.2g) ) as well
as a cryofocusing unit. The moisture control module should not be used. The
operating conditions are as follows: purge gas: nitrogen; purge flow (vent):
30 rnL/min; prepurge: 1.5 min; water bath: 45C; purge: 10 min; drypurge:
10 min; cap cool-down: -125C; desorb preheat to 210C; desorb: 4 min at
220C; inject: within 1 min from -125 to 200C; bake 5 min at 260C; port
valve: 150C; line: 150C; capillary union heater (= transfer line from purge
and trap to gas Chromatograph): 150C.
Gas chromatography. A Hewlett-Packard (HP) 5890, Series II is used.

The operating conditions are as follows: carrier gas: helium; inlet pressure:
40 kPa; flow: approx. 1.6 mL/min at 45C; transfer line (from GC to MS):
280C; interface: direct inlet; temperature programme: 13 min at 45C,
heating rate: 5C/min up to 240C, and 5 min at 240C; capillary column:
SPB1 (Supelco), 30m x 0.32 mm i.d., film thickness: 4 p.m.
Detectors. Two detectors are mounted in parallel by splitting the flow at

the end of the capillary column (split ratio: approx. 1:1 at 45C), i.e. a
Hewlett-Packardflameionisation detector (FID) and a mass-sensitive detector
(MSD model HP 5972), operating in the scan mode (TIC) from 19 to 250 amu
at 2.9 scan/s, ionisation by EI at 70eV by autotuning; MS-Scan after 3.5 min.
The MSD is used for the identification of the volatile (flavour) compounds, the
FID for their relative quantification. [JB]
80
Polytron PT 3000 used with a PT-DA 6030-6060 cutting system, Kinematica
81
Tekmar, Cincinnati, OH, USA
82
Schmidlin Co, art. no. 14-2333-4SL, CH-6345 Neuheim, DE
95
Note: Details of the method are given in Mariaca & Bosset (1996).
Figure 11.3. "Purge & Trap" System, Teckmar 2000, USA. A. "Purge
modus"; B. "Desorption & injection modus".
1. "Sparger"; 2. Samplefinerydispersed in water; 3. "6 port valve"; 4. "Trap";
5. "Cryofocusing" by liquid N2.
METHOD II (Coldfinger molecular distillation)

This extraction is very suitable for solid sample with maximum 60-70
% of water: cheese, meat... (Forss & Holloway, 1967; Dumont & Adda,
1972).
Step 1: evaporation of water (Figure 11.4).

The frozen cheese is grated and cooled with liquid nitrogen, then crushed
in a coffe-mill.
96
ARDO & POLYCHROMADOU, Ed. CHEESE ANALYSIS MANUAL
Figure 11.4: Evaporation of water. The guard trap for the oil vapors of the
pump is not designed.
The pressure must be decreased in all the distiller with a mechanical

pump (10 Pa), the traps (A,B,C) cooled with liquid nitrogen.
The sample is put in a cone-shaped flask (D). The flask is connected to
the traps. First, the flask is put in ice.
The pressure in the flask (D) is decreased from part to part. Doing that,
at least one of the stopcoks, must be alternatively kept closed to avoid spoilage
of the volatile compounds in the mechanical pump because of a too
considerable flow.
The flask (D) is rotated from time to time slowly to brake the
dehydraded surface of the sample.
Volatile compounds which might come out from the flask (D) are
condensed by traps (A,B,C) held in liquid nitrogen.
When enough water will be evaporated from the sample, the temperature
of the bath can be increased up to 35-40C and the flask (D) can be rotated to
mix the sample without amalgamating it in lump.
97
Step 2: coldfingermolecular distillation (Figure 11.5).

After 2 to 4 h, when the water of the sample is evaporated, the sample is
put in a second flask (E) with finger cooled by liquid nitrogen. The first trap
(C) near theflask(D) and containing the water is taken away.
Figure 11.5: Cold finger molecular distillation. The guard trap for the oil
vapors of the pump is not designed.
The flask (E) with cold finger is connected to the traps (B) and the
pressure is decreased as in thefirststep.
When the pressure reaches 1 Pa, all the stopcocks are open and the oil
difsion pump switched on to reach 0.01 Pa.
After 2 to 4 h, the cold finger is scaped and rinsed with distilled water.
The traps (A, B) are also rinsed except the trap the closest to the pump (guard
trap for the oil vapors of the pump). Waterfromthe rinsing of all the traps and
coldfingerare mixed together.
98
Step 3: solvent extraction

Aqueous solutions from step 1 and step 2 are mixed together and
extracted with a suitable solvent (e.g. dichloromethane). [JLQ]
METHOD m (Distillation under reflux)

This extraction (Figure 11.6) is very suitable for liquid samples : wine,
fruit juice, milk... or solid samples crushed with water: cheese,fruit...(Forss et
al, 1967; Dumont & Adda, 1972; Etivant & Bayonove, 1983).
The pressure must be decreased in all the distiller with a mechanical
pump (lOOPa), the traps (F, G) cooled with liquid nitrogen, and then the
stopcocks closed. The sample is transferred to the first flask (A) by 50 ml
aliquots every ten minutes. The first flask is heated until 35-40C in a
waterbath.
The distillate condensed in the first condenser (C) held at 4C, is
received in the second flask (B) held at 5 or 0C (iced bath). Volatile
compounds which might come out from the second flask (B) are condensed in
traps (F,G) held in liquid nitrogen.
The pressure must be held at 100 Pa. From time to time, particularly at
the beginning of the distillation, the pressure must be decreased in the
apparatus from part to part. Doing that, at least one of the stopcocks, must be
alternatively kept closed to avoid spoilage of the volatile compounds in the
mechanical pump because of a too considerable flow.
When the sample has been completely transferred in the first flask (A),
distilled water is transferred in the first flask (A) by 100ml aliquots, to help to
complete the distillation of the volatiles.
After 2 to 5 hours of distillation, traps (F,G) in liquid nitrogen are rinsed
with distilled water and the aqueous solutions added to the distillate of the
second flask (B), except the trap the closest to the mechanical pump (guard
trap for the oil vapors of the pump). The aqueous distillate is then extracted
with a suitable solvent (e.g. dichloromethane). [JLQ]
Both methods (II and III) give rise to extracts containing acid, neutral
and basic volatiles. To analyse directly these extracts, a suitable GC capillary
column should be choosen (FFAP columns or equivalent, e.g. DB-FFAP from
J&W Scientific, CP-Wax 58CBfromChrompack,...).
99
Figure 11.6. Apparatus for low pressure distillation of volatiles from wine. A,
introduction flask, 6 L; B, condensation flask, 6 L; C and D, condensers, 5 C;
E, to vacuum gauge (1.3 - 1.3 x 104 Pa); F, cooled trap, -80'C; G, cooled
traps, -196 ' C ; H, to mechanical pump; I, tube for wine introduction; J,
magnetic stirrer.
To analyse neutral and basic compounds apart from acids, the pH of the
extracts should be set around 9 (adding dilute NaOH or NaHCOs) and the
neutral fractions extracted from the pH 9-aqueous solutions with a suitable
solvent (e.g. dichloromethane). These fractions could be analysed on any
apolar or polar GC capillary column.
Repeatability of both methods is ca. 20%.
Reproducibility depends greatly on the equipment used for the vacuum.
[JLQ]
100
12. ANALYSIS OF ENZYMES
12.1. Alkaline phosphatase

Official methods
Phosphatase activity: IDF Standard 53:1969 (photometric method)
Phosphatase (residual): AOAC method 946.03 (photometric method)
FLUOROMETRIC METHOD
The fluorometric method (IDF Standard 155:1992) can be used for the
determination of alkaline phosphatase activity in whole milk, semi-skimmed
milk, skimmed milk and in cheese.
Unit of alkaline phosphatase activity is defined as the amount of alkaline
phosphatase enzyme that catalyzes the transformation of 1 micromole of
substrate per minute per litre of sample.
In presence of alkaline phosphatase of the sample, a non-fluorescent
monophosphoric ester (Fluorophos)83 undergoes hydrolysis of its
phosphorate radical, originating a fluorescent (ex. 439 nm; em. 560 ran) phenol
compound (Fluoroyellow)83. Alkaline phosphatase activity is determined by
continuous fluorometric measurement at 38C for 3 min.
Reagents
Substrate: Fluorophos . The freeze-dried substrate is stable for 1 year when
stored in glass vials at 4C.
Substrate diluent: Diethanolamine buffer, 2.4 mol/L, pH 10.0. The buffer
solution is stable for 1 year when stored at 4C.
Working substrate: Solve substrate in substrate diluent at a final concentration
of 1.044 mmol/L, thoroughly mixing.
Calibrator solutions: Different concentrations of Fluoroyellow in
diethanolamine buffer.
Calibrator solution A containing 0 umol/L of Fluoroyellow.
Calibrator solution B, containing 17.24 -10"3 umol/L of Fluoroyellow.
83
Advanced Instruments Inc, Advanced Instruments Inc., 1000 Highland Ave, Needham
Heights, MA02194, USA.
101
Calibrator solution C, containing 34.48 -IO"3 umol/L of Fluoroyellow.

Calibration solutions are stable for 1 year if stored at 4C.
Sample preparation buffer: Cheese extraction buffer83.
Apparatus. Fluorometer equipped with a thermostatted cuvette holder

kept at 38 0.1 C83. Cuvettes, disposable, of diameter 12 mm, length 75 mm.
Incubator block, capable of keeping samples at 38 0.5C. Microsyringe or
pipettor, capacity 0.075 mL83. Fixed-volume dispenser, to dispense 2.0 mL.
Homogeniser. Precision balance. Refrigerated centrifuge, capable of reaching
1500 g. 15-mL centrifuge test-tubes. Blender.
Sample preparation. Cut a triangle of cheesefromthe rind to the core of

the cheese wheel. Remove 0.3 cm of rind and draw the first cm of cheese from
the central area of the triangle just under the rind (Figure 12.1). Thoroughly
grind the sample preventing heating of the cheese. Place 500 mg of the ground
cheese in a test-tube and add 5 ml of Cheese extraction buffer. Homogenise at
3000 rpm for 1 min, centrifuge at 1000 g for 10 min at 4C and recover the
supernatant, discarding fat.
Procedure. Fluorometer must be calibrated for each type of sample (milk

or cheese) to be analysed. Calibration must be run each time a new lot of
reagents is used. Dispense 2.0 mL of calibration solutions, B and C into
cuvettes, each in duplicate. Warm the cuvettes to 38C for 5 min in the
incubator block. Add 0.075 mL of cheese sample supernatant into each
cuvette, to calibrate for cheese. Thoroughly mix the contents of the cuvettes
and perform the following calibration procedure. Insert calibrator solution A
into fluorometer, set fluorescence to zero and readfluorescenceof calibrator
solutions B and C against calibrator solution A.
Dispense 2.0 mL of the substrate solution into a cuvette and place in the
incubator until temperature reaches 38C. Add 0.075 mL of supernatant from
the cheese sample preparation and immediately mix the contents of the cuvette.
Place the cuvette in thefluorometerand after exactly 1 min record the rate of
increase of fluorescence (AF/min) for the next 2 min. Results may be calculated
automatically, by the fluorometer, or manually, according to the procedure
described below.
102
Figure 12.1. Correct drawing of cheese sample
Calculations and expression of results. If an Advanced Instruments Inc.

fluorometer is used, results are automatically calculated by the instrument.
Alternatively results may be calculated manually with the following procedure:
Record the fluorescence values of calibrator solutions B and C reading
against calibrator solution A having zero fluorescence.
Record the rate of increase of fluorescence of the .sample as AF/min.
To calculate alkaline phosphatase activity in mU/L, calculate umoles of
Fluoroyellow formed per minute by 0.075 mL of sample using the
fluorescence value of calibrator solution B which contains 3.448 10" umol of
Fluoroyellow:
umoles Fluoroyellow/min/0.075 mL =
([AF/min/0.075 ml of sample] / [F of calibrator solution B]) x 3.448 x 10"5
To calculate the (imoles of Fluoroyellow formed by 1 litre of sample,

multiply the above obtained result by 13333.3 and then by 1000 to convert the
result to mU/L. Therefore the alkaline phosphatase activity in milli-Units per
litre is:
[AF/min/0.075 mL of sample] / [F of calibrator solution B] x 459.7
103
To calculate alkaline phosphatase activity for cheese samples in mU/kg,

multiply the obtained result by 10. [JH]
12.2. Acid phophatase

Cheese extracts are prepared as follows: 5.0 g of cheese (taken
uniformly along the radius of the piece) are homogenized in a Potter-type
homogenizer on ice with 25.0 mL 0.12 M Tris-HCl buffer pH 8.0 and
centrifuged at 5000 g for 20 minutes at 4C. After removing the fat layer, the
aqueous layer is filtered through Whatman #1 filter paper and the filtrate is
used for lactoperoxidase, alkaline and acid phosphatase determinations.
Acid phosphatase is determined essentially as described by Larsen &
Parada (1988). Milk (80 uL) or cheese extract (0.3 mL) is added to an
appropriate volume of 0.1 M sodium acetate buffer pH 4.4 (total assay volume
is 1.0 mL) containing /7-nitrophenyl phosphate (pNPP; final concentration in
the assay mixture 7.5 mM). Mixtures are incubated at 37C for 1 hour and
reactions are stopped with 1.0 mL 12% (w/v) trichloroacetic acid. After
separating the precipitated proteins by centrifugation at 16 000 g for 10 min,
0.7 mL of the clear supernatant is mixed with 1.5 ml 2M NaOH. Absorbance is
read at 405 ran.
Enzyme units are reported in International Units (U). One U is the
amount of enzyme that catalyzes the production of 1 mmol of product/min
under the described conditions. All determinations are preferably done in
quadruplicate and the results are presented as the mean standard deviation
(Larsen & Parada, 1988; Chvarri et al, 1998). [MR]
12.3. Lactoperoxidase
Cheese extracts were prepared as described above ( 12.2).

Lactoperoxidase is determined as described by Pruitt et al. (1990) in
0.1 M phosphate buffer pH 6.0 and at 20C. The assay concentrations of 2,2'-
azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and H2O2 are 3.88
104
ARDO fe POLYCHRONIADOU, Ed. CHEESE ANALYSIS MANUAL
mM and 0.1 mM, respectively, being 2.5 mL the total assay volume. Reaction
rates are determined at 415 nm and the molar absorptivity coefficient of ABTS
is taken as 32 400 MT1.cm"1
Enzyme units are reported in International Units (U). One U is the
amount of enzyme that catalyzes the production of 1 mmol of product/min
under the described conditions. All determinations are preferably done in
quadruplicate and the results are presented as the mean standard deviation
(Pruitt et al, 1990; Chvarri et al, 1998) [MR]
12.4. Chymosin
Cheese samples (5 g) are finely grated, suspended in 0.4 M trisodium
citrate (25 mL) and mixed using a lab-blender5 for 1 h to allow total
dissolution of the caseins. The suspensions are skimmed by centrifugation at
7000 g and 5C for 15 min. These samples are divided and stored at -20C for
analysis.
Microplates are coated overnight at 4C with a rabbit purified anti-
chymosin antibody solution diluted 1/5000 in 0.05 M carbonate buffer pH 9.6.
Plates are saturated at 37C for 1 h with 0.05 M phosphate buffer pH 7.2, 0.15
M NaCl /0.05 mL/L Tween 20 (PBS-T). Serial dilutions of purified chymosin
solution (0-800 ng/mL) in PBS-T are used as standards. Cheese extracts
diluted in PBS-T (4 dilutions from 1/2 to 1/20, 75 uL), or chymosin standards
are added, in test tubes, to a solution of chymosin-alkaline phosphatase
conjugate (diluted 1/1000 in PBS-T, 75 uL), prepared according to Avrameas
(1969). The mixture is added to the plates that are then incubated at 37C for
1.5 h. The amount of conjugate fixed is detected by addition of/Miitrophenyl
phosphate and the color intensity of the reaction mixture is measured directly
at 405 nm (Boudjellab et al, 1994). [DD]
105
12.5. Plasmin and plasminogen

12.5.1. Determination of plasm in and plasminogen by ELISA.
Cheese (5 g) isfinelygrated and suspended in 20 mL 0.4 M trisodium
citrate buffer pH 8.3. The suspension is homogenized for 1 min in an ice bath
using an ultra-turrax5 and dispersed using a lab-blender22 for 5 min at 22C to
allow total dissolution of the casein. Then, the suspension (10 mL) is incubated
for 2 h at 22C with 50 mM s-amino caproic acid (EACA). Subsequently,
serum containing plasminogen and plasmin is freed of particulate casein by~
ultracentrifugation84 at 100 000 g for lh at 4C and is submitted to assay by
ELISA.
The assay is based on the use of two monoclonal antibodies (Mabs), one
specific for plasminogen and the other specific for plasminogen plus plasmin
(Dupont et al., 1994 & 1997). Plasmin concentration is obtained by
subtracting the first concentration from the second. Flat-bottomed ELISA
plates are coated with Mab anti-plasminogen or Mab anti-
plasmin+plasminogen diluted 1/500 in 0.1 M bicarbonate buffer pH 9.6 (100
uL per well) and incubated for 1.5 h at 37C. Blocking of the remaining
binding sites is performed with 10 g/L gelatin in 0.05 M phosphate buffer pH
7.2, 0.15 M NaCl /0.05 mL/L Tween 20 (PBS-T). Serial dilutions of purified
plasminogen solution (0-100 ng/mL) in PBS-T are used as standards. Cheese
extracts diluted in PBS-T (4 dilutions from 1/25 to 1/200, 100 uL), or
plasminogen standards are added and the plate is incubated 1.5 h at 37C.
Then, rabbit antiserum against plasmin and plasminogen is incubated for 1.5 h
at 37C.
The reaction is revealed by a Goat anti-Rabbit Immunoglobulins-alkaline
phosphatase conjugate85 diluted 1/3000 in PBS-T. /7-Nitrophenyl phosphate85
(pNPP, 100 uL) at 1 mg/mL is used as substrate. Absorbance is measured at
405 nm using a Ceres 900 Microplate Reader86. Plasminogen concentration is
obtained directly whereas plasmin concentration is determined by subtracting
plasminogen from plasminogen+plasmin concentration (Dupont & Grappin,
1998). P D ]
84
Sorvall Ultra Pro 80, Du Pont Company, Newtown CT 06470, USA.
85
Sigma, Saint Louis, MO 63178, USA
86
Bio-Tek Instruments Inc., Winooski, VT 05404, USA
106
12.5.2. Activity test of plasm in and plasminogen in cheese

This method for the determination of the plasmin and plasminogen
activity in hard cheese is based on a photometric assay (Aaltonen et al, 1988;
Ollikainen & Nyberg, 1988; Rollerna et al, 1983). A tripeptide (D-Val-L-Leu-
L-Lys-pNA87), is used as a synthetic substrate. Plasmin is able to cut the
peptide specifically from the p-nitroaniline group showing a yellow colour. In
order to detect the amount of the zymogen, urokinase88 is added immediately
after the determination of plasmin activity. Urokinase transforms plasminogen
into plasmin thus giving some additional activity and, therefore, production of
p-nitroaniline, p N A .
9x g of a suspension buffer containing 100 mM EDTA, 40 mM KCL 50
mM caproic acid, 40 mM Tris, pH adjusted to 7.4, are added to x g grated
cheese. The mixture is homogenized with a T25 Ultra Turrax5 for 2 min at
9500 rpm. The suspension is held for 1 h at 5 C in a refrigerator to allow the
fat phase to separate at the surface. After centrifugation of the liquid phase
(25000 g, 30 min, 4C) the suspension is filtered through a 602 1/2 Schleicher
& Schuell folded filter. Without any further treatment, the filtrate can be used
for the photometric test.
600 uL buffer (see above) and 300 uL tripeptide (diluted with distilled
water to achieve a concentration of 2 mM) are mixed in semi-microcuvettes
placed in a thermostatting device at 37C. After 5 min equilibrium time, 100
uL of the sample solution is added in order to start the enzymatic reaction. At
405 run, absorptions are measured exactly 30 min (A|) and 60 min (A2) after
starting the reaction. Immediately afterwards, 10 uL urokinase (5 units/mL
H2O) are added to the microcuvettes in order to determine the activity of the
plasminogen. Again, absorptions are recorded after 30 min (A3) and 60 min
(A4). Plasmin and plasminogen concentrations are calculated by:
ApLASMIN = 2 x ((E 2 - Ej) - (B 2 - Bj))

A
PLASMINOGEN = 2 x
( ^ 3 ~ E 4 ) ~ 0*3 " B 4 ) ) " EPLASMIN
87
Sigma V-7127
88
Sigma U-5004
107
where B-values represent the corresponding measures of the blank (700 uL

buffer + 300 uL tripeptide solution). The extinction values are converted into
nmolpNA released per g cheese within 1 h. For that purpose, a standard curve
with nmol >NA against absorption has to be used. The variation coefficient of
the plasmin/plasminogen determination (6-fold replication) ranges around 5%.
[DJ]
12.6. Peptidases
The autolysis of starter bacteria in Saint-Paulin type cheese is monitored
by the measure of the activities of intracellular peptidases, the X-prolyl
dipeptidyl peptidase (PepX) and the aminopeptidases with broad specificity
(PepC + PepN) and by the quantification of the intracellular peptidases PepX
and PepC by ELISA in a cheese extract (Chapot Chartier et al, 1994). [DLB]
12.6.1. Determination of the peptidase activities
METHOD I
Two grams of ground cheese are shaken in 8 ml 20 mmol/L bis-Tris
propane buffer pH 7.083 for 2 h at 4C. The samples are then centrifuged for
10 min in a table centrifuge at 4C. The supernatants are filtered (0.2 um) to
remove whole bacterial cells thereby ensuring that any bacterial activity was of
extracellular origin (Ardo et al, 1989).
Enzymatic activities are determined spectrophotometrically by measuring
initial rates using artificial substrates producing /Miitroanilin with absorbance
at 405 nm as a result of aminopeptidolysis. Aminopeptidolytic activities are
typically measured with arginine, leucine, lysine and proline /7-nitroanilides
(arg-NA, leu-NA, lys-NA and pro-NA) and X-prolyl-dipeptidyl
aminopeptidolytic activities with arginyl-proline and glycyl-proline p-
nitroanilides (arg-pro-NA and gly-pro-NA)85.
Assays are performed at 37C in 20 mmol/L bis-Tris propane buffer with
a typical substrate concentration of 0.7 -1.0 mmol/L. [YA]
108
METHOD II
Grated cheese (20 g) is mixed with 80 mL of 50 mM sodium phosphate
buffer pH 7.4 containing 150 mM NaCl which was preheated at 40C. The
mixture is homogenized with an Ultraturrax disperser, twice for 30 s and the
volume then adjusted to 100 mL. The suspension is centrifuged at 8000 g for
20 min at 4C and the upper solid fat layer is removed. The soluble fraction is
recovered and centrifuged again under the same conditions. The supernatant is
filtered and the resulting cheese extract used for determination of peptidase
activities.
The X-prolyl dipeptidyl aminopeptidase (PepX) activity is measured
using Phe-Pro--naphthylamide (Phe-Pro--NA89) as a substrate and the
aminopeptidase (PepC) activity using Lys--naphtylamide (Lys--NA85) as a
substrate. Briefly, 100 uL of cheese extract are incubated at 37C with 0.25
mM substrate in 20 mM Tris-HCl (pH 8 for PepX activity and pH 7 for
aminopeptidase activity) in a final volume of 1 mL. The reaction is stopped by
addition of 500 uL of a mixture containing 1 mg/mL Fast-Garnet GBC85, 10%
Triton XI00, and 1 M sodium acetate, pH 4.0. After incubation for 20 min
at 37C, the samples are centrifuged at 17000 g in Eppendorf tubes for 15 min
to remove the precipitated caseins. The optical density of the supernatant is
read at 550 nra. The unit of activity used is the katal (the quantity of enzyme
releasing one mole of -naphtylamine per second). [DLB]
12.6.2. Determination of peptidases concentration by ELISA

Concentration of cheese extract. The soluble cheese extract is
concentrated as follows. The caseins are first precipitated by addition of IN
HCl to pH 4.6 with stirring. The precipitate is removed by centrifugation at
8000 g for 15 min. The supernatant is recovered and the pH brought to 6.5 by
addition of IN NaOH. The extract is dialyzed against 10 mM sodium
phosphate (pH 7.4), 15 mM NaCl buffer and then against 10 times diluted PBS
(1 mM sodium phosphate (pH 7.4), 15 mM NaCl buffer). The extract is then
lyophilized and is resuspended in 1/10 of the initial volume for the ELISA test.
89
Bachern
109
ELISA for PepX

Preparation of ant-PepX serum. Pep X is purified according to Zevaco
et al. (1990) and antiserum is raised in rabbits. Purified enzyme (80 ug) is
injected intradermally at several points after emulsification with complete
Freund adjuvant. Booster injections of 40 ug of the purified protein in
incomplete Feund adjuvant, are given every 3 weeks after the primary
injection. Rabbits are bled one week after each booster injection. The
specificity is checked by immunoblotting (Tan et al, 1992).
Coupling of immunoglobulins to biotin. Immunoglobulins (Igs) are
purified from rabbit serum by affinity chromatography on a protein A activated
cartridge90 and are then coupled to biotin (Gesdon et al, 1979). Briefly, 1 mL
of purified Igs at 2 mg/mL in 0.1M NaHC03 buffer are incubated with 10 uL
of 0.1 M J-biotm-A/-hydroxysuccinimide ester85. The mixture is incubated for 1
h at room temperature and dialysed extensively against PBS. The conjugate is
stored in 50% glycerol at -20C.
Sandwich ELISA. Microtitration plates91 are coated with rabbit anti-
PepX antiserum (1/1000) in 0.1 M Na2C03, pH 9.5, for 1 h at 37C. After
saturation of the wells with PBS buffer containing 0.1% Tween-20 and 1%
skimmed milk for 1 h at 37C, samples are incubated in serial dilutions in PBS-
Tween 0.1% (PBST) for 1 h at 37C. The enzyme is revealed by successive
incubations at 37C with the specific Igs coupled to biotin and streptavidin-
peroxidase conjugate92 (1/500). After washing with PBS, H202-ABTS
substrate is added and the OD read at 405 nm in a microplate reader93.
The different parameters for ELISA are optimized with the purified
antigen. The detection limit is 4 ng/mL. The concentration of enzyme in cheese
is determined by comparison with a standard curve prepared with purified
PepX.
ELISA for PepC

The concentration of PepC in the cheese extract is determined by the
sandwich ELISA test previously described by Boquien et al (1991) using
90
FMC Byproducts
91
Nunc, Rosklide, DK
Âmersham
93
Titerteck, Flow laboratories, Lugano, CH
110
mouse anti-PepC serum as first antibody for coating the plate and rabbit anti-
PepC serum as second antibody to reveal the presence of antigen, followed by
incubation with goat anti-rabbit antibodies coupled to peroxidase94. The
detection limit determined with purified enzyme is 4 ng/mL. The concentration
of enzyme in cheese is determined by comparison with a standard curve
prepared with purified PepC. [DLB]
12.6. Lipases and esterases

Enzyme extraction. 10 g of cheese are Pofytron homogenized for 3 min
at 3700 rpm and at 20C, with 50 mL of 0.05 M Tris buffer at pH 7.6. The
resulting slurry is centrifiiged at 9000 g for 20 min at 4C. The interlayer was
withdrawn and again centrifiiged at 47 000 g for 30 min at 4C. The
supernatant is filtered using filter paper (Whatman no 40) wet with cold milliQ
water in order to reduce the residual fat content of the extract. The filtrate is
used to determine the lipase activity (Pitotti & Dal Bo, 1996).
Incubation medium and conditions. Substrate emulsion is obtained by

adding 10 mL of p-nitrophenylbutyrate (or other ester) acetonic solution
(lmg/mL) into a stirred solution containing 50 mL of 0.05 M Tris buffer at pH
7.6,10 mL of 25 mM Na-thaurocholate, 10 mL of 70 mM NaCl, and 20 mL of
milliQ water.
Incubation is performed at 30C in 2.5 mL of substrate emulsion to
which 0.5 mL of Tris buffer containing different volumes of cheese extract are
added (Pitotti & Dal Bo, 1996).
a) Continuous spectrophotometry method. Reaction is monitored at 400

nm using UVTKON 860 (Kontron) spectrophotometer.
Different volumes of cheese extract are tested and specific activity (A
A.U./min/mL of extract) is calculated from the slope of linear regression of
velocity vs. extract quantity. The y intercept corresponds to the spontaneous
hydrolysis in the presence of denatured enzymatic extract. This value
corresponds to the value obtained adding two volumes of an "inactivating
94
Diagnostic Pasteur Marnes la Coquette, FR
111
solution" (Humbert et al., 1997) (three volumes 0.06 M EDTA water solution,
adjusted to pH 7.6 with 2 M NaOH, plus one volume of 0.06 M
phenylmethanesulfonylfluoride in dimethylformamide) to five volumes of the
enzymatic extract and using this inactivated enzyme as a reference "blank".
Other inactivation methods tested did not give good results for different
reasons.
Lipase activity unit is expressed as quantity of enzyme that determines
the increase of absorbance of 0.001 A.U./min. n moles of /?-nitrophenol
hydrolyzed can also be obtained through a calibration curve.
This method is useful only when using p-nitrophenylbutyrate as substrate
(esterase activity) because longer fatty acid esters used as substrate cause light
scattering problems. Determinations performed on six extracts obtained from
the same cheese showed that specific activity (activity/mL) was 50.72 3.9 %
(Pitotti & Dal Bo, 1996).
b) Blocked reaction and spectrophotometry determination. After 20

min incubation (or more if activity is feeble) in the medium described, reaction
can be stopped by adding 0.2 mL of the "inactivating solution". In the "zero
time" this solution is added to the enzyme before the substrate solution. After
10 min at 0C and centrifugation, the clear samples are read at 400 nm.
Activity is calculated as described.
Blocked reaction and HPLC determination. Incubation is performed in

the same medium. After 20 min (or more) at 37C reaction is stopped by
adding 0.1 mL of 5 M HCl. At this point 0.1 mL of internal standard solution
(0.15 mg/mL of 2,4-dinitroaniline) is also added. The mixture is mixed, left 10
min at 0C and centrifuged.
After centrifugation, 20 uL of clear samples are analyzed on a Cis
reverse phase column, using a 55:45 v/v water/acetonitrile mobile phase at 1.5
mL/min flow-rate. Detector wavelength is 300 nm.
To obtain a "zero time" HCl is added before the substrate solution. A
reference standard solution (3 (xg/mL /7-nitrophenol plus 15 |Ag/mL internal
standard) is also alternatively injected.
p-Nitrophenol hydrolyzed from the different substrate used is obtained as
follows:
112
h
i ^ 2
ug/?NP = x
\ m
where:
hj = peak height ofpNP in the sample
t^ = peak height of internal standard in the sample
(j.g = internal standard in the sample in ug
h', t
m=
h'2 Mg\
where :
h'j = peak height ofpNP in the standard solution
h'2 = peak height of internal standard in the standard solution
jag'j =pNP in the standard solution
)j.g'2 = internal standard in the standard solution
Peaks are eluted in the order: aceton (used for substrate solubilization),
/7-nitrophenoL 2,4-dinitroaniline. The lower limit of detection was 0.75 um/mL
with a CV of 4% (Maurich et al, 1991). Different volumes of cheese extract
are tested and velocity (n moles/min/mL extract) is obtained from the slope of
the corresponding linear regression.
This method can also be used with every fatty acid ester of p-
nitrophenol. HPLC has some other advantages: no toxic reactants are needed
to inactivate the enzyme, and it is also easier to test activity at different pHs
because of final acidification. HPLC separation of substrate and product is
necessary because at acid pH both absorb at 300 nm. At acidic pHs the
absorbance coefficient of p-nitrophenol is also less influenced by small pH
variation. [API]
113
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Amigo, L., Ramos, M., Calhau, L. & Barbosa, M. 1992. Comparison of
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Btikofer, U., Fuchs, D., Bosset, J.O. & Gmur, W. 1991. Automated HPLC-
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klassischen Ionenstausch-Aminosurenanalysatoren und HPLC-
Systemen mit OPA/FMOC-Vorsulenderivatisierung. Mitt. Gebiete
Lebensm. Hyg. 83 457-466
Btikofer, U., Regg, M. & Ard, Y. 1993. Determination of nitrogen
fractions in cheese: evaluation of a collaborative study. Lebensm.-Wiss.
u. Technol. 26 271-275
Bynum, D.G., Senyk, G.F. & Barbano, D.M. 1984. Determination of free fatty
acids content of Cheddar cheese by a copper soap method. J. Dairy
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123
European Commission
EUR 18890 COST 95 Laboratory manual for chemical analysis of cheese
Edited by Ylva Ardo and Anna Polychroniadou
Luxembourg: Office for Official Publications of the European Communities
1999 X I I , 123 pp. 1 7 x 2 4 . 0 cm
ISBN 92-828-6599-1
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The European programmes COST Action 95, FLAIRCA2COST902 and AIRCA3

CT94203 gave the opportunity to many laboratories involved in cheese analysis to col
laborate aiming, among other things, at the harmonisation of analytical methods for the
characterisation of cheese ripening. Participants realised that various methods suc
cessfully applied by each laboratory could be useful for other laboratories and decided
to collect them, creating a "Laboratory Manual" for cheese analysis. The intention of this
publication is to bring together methods which are of value in the collaborating labora
tories. The effort has been concentrated on analysis methods that are not available as
standard methods, but highly needed and regularly used. Official methods, such as
AOAC and IDF standards are given as references so that each laboratory may com
plete their own manual using them.
Price (excluding VAT) in Luxembourg: EUR 22

ISBN 9282865991
* * OFFICE FOR OFFICIAL PUBLICATIONS
a. * OF THE EUROPEAN COMMUNITIES
op * 9 "789282 865996">
k + + L2985 Luxembourg

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European cooperation

in the field of scientific

Ylva Ardo and Anna Polychroniadou, Editors

Edith Cresson, Member of the Commission

DG XII/AP2 Political co-ordination and strategy COST

Collected and edited by

department of Dairy and Food Science

laboratory of Food Chemistry and Biochemistry

COST home page: http://www.belspo.be/cost

Cataloguing data can be found at the end of this publication.

Luxembourg: Office for Official Publications of the European Communities, 1999

European Communities, 1999

Reproduction is authorised provided the source is acknowledged.

PRINTED ON WHITE CHLORINE-FREE PAPER

2. SAMPLING AND GENERAL SAMPLE PREPARATION 5

5.4. pH 4.4-SN - pH 4.4 soluble nitrogen 35

6. RAPID METHODS FOR MONITORING

9. ANALYSIS OF FREE AMINO ACIDS AND AMINES 67

10. ANALYSIS OF FREE FATTY ACIDS 79

11. ANALYSIS OF VOLATILE COMPOUNDS 91

12. ANALYSIS OF ENZYMES IN CHEESE 101

13. REFERENCES 115

[DD] DIDIER DUPONT

[HR] HARALD ROHM

[JH] JOHN HOGENBOOM

[JLQ] JEAN-LUC L E QUERE

[MJ] MANUELA JUAREZ

[RLF] ROSINA LPEZ-FANDIO

[UB] UELI BTIKOFER

[YA] YLVA ARDO

November 1998 ANNA POLYCHRONIADOU

Introduction to COST Action 95

COST (Cooperation in Science and Technology) is a research programme to foment

in cheese (Ardo fe Gripon, 1991), chromatographic analysis and identification

investigation. Different factors have to be taken into consideration if the

2. SAMPLING AND GENERAL SAMPLE PREPARATION

Routine butyrometric methods

METHOD II (Gerber-van Gulik)

'Butyrometer: Gerber instruments, Effuetikon, CH.

3.2. Total nitrogen

Complete mineralisation is regularly checked with tryptophan samples.

N in the test portion (mg) = 14007 x Vms04 (mL) x Acid normality

Alternatively, 10 mL of citrate dispersion of cheese (see 4.2) are

3.3. Moisture - Total solids - Dry matter

DM (% cheese)=((W2-W!) / Wc) x 100

Dry matter is determined in triplicate per sample. [SP]

3.4. Water activity

The activity of water (aw) is estimated by the measurement of the

Figure 3.1. Plot for the determination of aw [SP].

3.5. Sodium chloride

Percent NaCl = (mL AgN03 x 0.588) / wt. sample [PMS]

NaCl (%DM) - (read value x 0.016)-0.07 / (Wc x DM) [SP]

3.7.1. GLC method for the analysis of carbohydrates

3.7.2. HPLC method for the analysis of carbohydrates

Figure 3.2. A chromatogram of trimethylsilyl derivatives of free

Reagent for sample pretreatment. Dissolve 91.0 g of zinc acetate

content of the beaker quantitatively into a 100-mL volumetric flask. Pipette

Repeatability. The difference between two single results found on

3.10. Carbon dioxide

A = absorbance of the cheese sample

3.11.2. Enzymatic method

Calcium in cheese can be determined by titration with a chelating agent

Total calcium is measured by the complexometric method of Pearce