THESIS ON NATURAL AND EXACT SCIENCES B149

A Study of Milk Coagulability

TIINA KRIÐČIUNAITE
 TALLINN UNIVERSITY OF TECHNOLOGY
Faculty of Science
Department of Chemistry

Dissertation was accepted for the defence of the degree of Doctor of

Philosophy (in Chemistry) on March 7, 2013

Supervisors: Professor Raivo Vilu

Chair of Biotechnology
Tallinn University of Technology

Senior Researcher Tiiu-Maie Laht

Department of Chemistry
Tallinn University of Technology

Opponents: Dr. Jes Christian Knudsen

Associate professor, Department of Food Science, University
of Copenhagen

Dr. Ivi Jõudu

Associate Professor, Institute of Veterinary Medicine and
Animal Sciences, Estonian University of Life Sciences
Senior Researcher, Bio-Competence Centre of Healthy Dairy
Products Ltd

Defence of the thesis: April 4, 2013

Declaration:
Hereby I declare that this doctoral thesis, my original investigation and
achievement, submitted for the doctoral degree at Tallinn University of
Technology has not been submitted for any academic degree.

Tiina Kriščiunaite

Copyright: Tiina Kriščiunaite, 2013

ISSN 1406-4723
ISBN 978-9949-23-444-8 (publication)
ISBN 978-9949-23-445-5 (PDF)

 
 
 LOODUS- JA TÄPPISTEADUSED B149

Piima kalgendatavuse uurimine

TIINA KRIÐČIUNAITE
CONTENTS

LIST OF PUBLICATIONS ............................................................................ 8

ABBREVIATIONS......................................................................................... 9
INTRODUCTION......................................................................................... 10
1. LITERATURE REVIEW ................................................................... 12
1.1. Milk composition ........................................................................... 12
1.1.1. Lactose ................................................................................... 12
1.1.2. Milk fat ................................................................................... 12
1.1.3. Milk proteins .......................................................................... 13
1.1.4. Milk salts ................................................................................ 14
1.2. Milk structure ................................................................................. 15
1.2.1. Milk fat globules .................................................................... 15
1.2.2. Casein micelles ....................................................................... 16
1.3. Milk coagulation............................................................................. 18
1.3.1. Rennet-induced coagulation ................................................... 18
1.3.2. Acid-induced coagulation....................................................... 20
1.4. Variation in composition and technological properties of milk ..... 21
1.4.1. Natural variation ..................................................................... 22
1.4.2. Disinfectants ........................................................................... 23
1.4.3. Changes occurring during and after milking .......................... 24
1.5. Methods for monitoring milk coagulation ...................................... 24
1.6. Front-face fluorescence spectroscopy in dairy research ................. 27
2. AIMS OF THIS DISSERTATION .................................................... 30
3. MATERIALS AND METHODS ....................................................... 31
3.1. Milk supply and treatment .............................................................. 31
3.1.1. Milk samples from farms, tank trucks and dairy silos
(Publication I) ............................................................................................. 31
3.1.2. UHT milk samples (Publication II) ........................................ 31
3.1.3. Milk samples for monitoring coagulation by front-face
fluorescence spectroscopy and reference methods (Publication III) .......... 31

5 
 
 3.1.4. RSM from untreated and irradiated LHSMP
(Publication IV) .......................................................................................... 32
3.2. Starter cultures and inoculation rates (Publication II and IV) ........ 32
3.3. Analysis of milk composition (Publication I) ................................ 32
3.3.1. General characteristics of milk ............................................... 32
3.3.2. Analysis of soluble cations and anions in rennet whey .......... 32
3.3.3. Capillary electrophoresis of milk proteins ............................. 33
3.4. Rennet coagulation of milk (Publication I and III)......................... 34
3.5. Dynamic characterisation of bacterial growth and milk
coagulation ..................................................................................................... 34
3.5.1. Microcalorimetry (Publication II) .......................................... 34
3.5.2. Dynamic rheological measurements
(Publication II, III and IV) ......................................................................... 34
3.5.3. Front-face fluorescence spectroscopy
(Publication III and IV) .............................................................................. 35
3.6. Determination of coagulation time parameters and characterisation
of rennet-induced gels .................................................................................... 35
3.6.1. Rennet flocculation time (Publication I and III)..................... 35
3.6.2. Coagulation time parameters determined from dynamic
rheological measurements (Publication III) ............................................... 35
3.6.3. Coagulation time derived from fluorescence measurements
(Publication III) .......................................................................................... 36
3.6.4. Curd firmness (Publication I) ................................................. 36
3.7. Determination of gelation time and characterisation of acid-induced
milk gelation (Publication II and IV) ............................................................. 36
4. RESULTS AND DISCUSSION ........................................................ 37
4.1. Technological quality of raw bulk milk in Estonia (Publication I) 37
4.1.1. Milk renneting properties ....................................................... 37
4.1.2. Seasonal variations in milk general composition and
renneting properties ................................................................................... 39
4.1.3. Stability of the quality of milk produced in Estonia ............... 40
4.1.4. Comparison of raw bulk milk samples from individual farms
with pooled milk from tank trucks and dairy silos ..................................... 41
4.1.5. Correlation between chemical composition and renneting
properties of bulk milk ............................................................................... 42

6 
 
 4.2. Effect of H2O2 on the growth of starter bacteria and structure of acid
gels (Publication II) ........................................................................................ 44
4.2.1. Inhibition of growth of starter bacteria by H2O2 .................... 44
4.2.2. Milk gelation and small deformation rheological properties of
yoghurts prepared from milk treated with H2O2 ......................................... 47
4.3. Front-face fluorescence spectroscopy monitoring rennet-induced
coagulation (Publication III) .......................................................................... 49
4.3.1. Characteristic tryptophan fluorescence intensity profiles during
rennet coagulation ...................................................................................... 49
4.3.2. Determination of coagulation time parameter by fluorescence
measurements ............................................................................................. 53 
4.3.3.  Coagulation time parameters determined by different
methods ...................................................................................................... 54 
4.4. Front-face fluorescence spectroscopy monitoring milk acidification
(Publication IV) .............................................................................................. 55
5. CONCLUSIONS ................................................................................ 58
BIOBLIOGRAPHY ...................................................................................... 60
ACKNOWLEDGMENTS............................................................................. 69
ABSTRACT .................................................................................................. 70
KOKKUVÕTE .............................................................................................. 72
PUBLICATION I .......................................................................................... 75
PUBLICATION II ........................................................................................ 85
PUBLICATION III ....................................................................................... 95
PUBLICATION IV ..................................................................................... 123
CURRICULUM VITAE ............................................................................. 137
ELULOOKIRJELDUS ............................................................................... 138 

7 
 
LIST OF PUBLICATIONS
The following publications form the basis of this dissertation:
I. Kriščiunaite, T., Stulova, I., Taivosalo, A., Laht, T.-M., and Vilu, R.
Composition and renneting properties of raw bulk milk in Estonia.
International Dairy Journal, 23(1), 45-52 (2012)
II. Kriščiunaite, T., Stulova, I., Kabanova, N., Laht, T.-M., and Vilu, R. The
effect of hydrogen peroxide on the growth of thermophilic lactic
starter and acid gelation of UHT milk. International Dairy Journal,
21(4), 239-246 (2011)
III. Kriščiunaite, T., Seiman, A., Laht, T.-M., and Vilu, R. Front-face
fluorescence spectroscopy studying rennet coagulation of milk.
Manuscript
IV. Stulova, I., Kabanova, N., Kriščiunaite, T., Taivosalo, A., Laht, T.-M.,
and Vilu, R. Fermentation of reconstituted milk by Streptococcus
thermophilus: effect of irradiation on skim milk powder. International
Dairy Journal (2013), http://dx.doi.org/10.1016/j.idairyj.2013.02.004
SUMMARY OF AUTHOR’S CONTRIBUTION
I. The author participated in planning the study and handling of the milk
samples, was responsible for evaluation of milk coagulation properties,
put together and analysed the data, and wrote the manuscript.
II. The author planned and performed the experiments, analysed the data,
and wrote the manuscript.
III. The author planned and performed the experiments, calculated and
interpreted the data, and wrote the manuscript.
IV. The author participated in planning the experiments, was responsible for
rheological and fluorescence measurements and interpretation of the data,
and participated in writing the manuscript.
ADDITIONAL PUBLICATIONS
Stulova, I., Kabanova, N., Kriščiunaite, T., Laht, T.-M., and Vilu, R. The
effect of milk heat treatment on the growth characteristics of lactic acid
bacteria. Agronomy Research, 9(2), 473-478 (2010)
Stulova, I., Adamberg, S., Kriščiunaite, T., Kampura, M., Blank, L., and Laht
T.-M. Microbiological quality of raw milk produced in Estonia. Letters in
Applied Microbiology, 51(6), 68-690 (2011)
Porõvkina, L., Petruhhina, N., Vilu, R., and Kriščiunaite, T. Meetod piima
kalgendumisaja määramiseks: U200700012 (2007)

8 
 
ABBREVIATIONS
(dG'/dt)max gelation rate
µmax maximum specific growth rate
CCP colloidal calcium phosphate
CE capillary electrophoresis
CN casein
EHF Estonian Holstein-Friesian
ER Estonian Red
FA Finnish Ayrshire
G' elastic or storage modulus
G" viscous or loss modulus
GDL glucono-δ-lactone
GMP glycomacropeptide
HF Holstein-Friesian
irrLHSMP irradiated LHSMP
irrRSM RSM prepared from irrLHSMP
LAB lactic acid bacteria
LHSMP low-heat skimmed milk powder
LPO lactoperoxidase
NIR near infrared
NS not significant
PC1/PC2/PC3 principal component one/two/three
PCA principal component analysis
RFT rennet flocculation time
RSM reconstituted skimmed milk
SCC somatic cell count
SD standard deviation
SFS spectral fluorescence signatures
t2min time to the minimum of the second order derivative
tan δ loss tangent
TBC total bacteria count
tg gelation time
tG" time to the initial viscosity increase
UHT ultra-high temperature treated
WP whey protein
α-LA α-lactalbumin
β-LG β-lactoblobulin
γ0 shear strain
λem emission wavelength
λex excitation wavelength
τ0 shear stress

9
INTRODUCTION
Milk is defined as the secretion of the mammary glands of mammals and its
primary natural function is nutrition of the young (Walstra & Jenness, 1984).
This study focuses on bovine milk, which will be referred to as milk throughout
this dissertation.
Milk is a raw material for production of wide variety of fermented dairy
foods and cheese. Milk has to meet certain requirements to allow the
manufacturing of high quality products without modifying the routine
technological processes. Raw milk has to be of sufficient microbiological quality
and has to have favourable and stable chemical composition.
In 2010 there were approximately 96 thousand dairy cows in Estonia
(Estonian Animal Recording Centre, 2012) producing 700 thousand tons of
milk. Only 9% of all raw milk produced was consumed as drinking milk. Major
part of milk was utilised for manufacturing of dairy products: 35% for cheese,
23% for butter and 33% for other products (Estonian Dairy Association, 2012).
The annual yield of milk per cow doubled from 3666 kg in 1995 to 7613 kg in
2010. The increase in milk production was considered a result of breeding
strategies and increasing prevalence of Estonian Holstein-Friesian (EHF) breed
over Estonian Red (ER). The percentage of ER breed in the Estonian herd
dropped from 38.0% in 1995 to 22.3% in 2010; accordingly, the share of EHF
breed rose from 61.5% to 76.8% during the same time (Estonian Animal
Recording Centre, 2012). The increasing predominance of the Holstein-Friesian
breed in the dairy herds has become a popular trend in Estonian farms, in line
with the situation in other European countries. Changes of the general
composition of milk – protein, fat and lactose content, have been also observed
during the past decades (Estonian Animal Recording Centre, 2012). Hence, as a
result of breeding strategies and changes in the composition of dairy herd, the
alternations in the renneting properties of milk produced in Estonia can also be
expected to have taken place.
Enzymatic coagulation of milk is an essential process in cheesemaking which
peculiarities define in large extent the quality and yield of the final product
(Fagan et al., 2007a; Johnson et al., 2001; Riddell-Lawrence & Hicks, 1989).
Thus, the assessment of milk coagulation characteristics should be of high
importance to the dairy industry due to the economic reasons. Numerous studies
have been performed on the composition of cow’s milk. The relation of milk
coagulation ability to milk composition reported in literature has been based on
the studies performed mainly on the samples collected from individual cows
(Jõudu et al., 2008; Tsioulpas et al., 2007b; Tyrisevä et al., 2004; Wedholm et
al., 2006), which allows to gain insight into the changes of milk composition
throughout the lactation, differences among breeds or the correlations between
different milk characteristics. This provides us with the understanding, which
components determine milk coagulation ability, but does not allow to evaluate
how the pooling of milk affects this technologically important factor in practice.

10
The recent large-scale studies of pooled milk from dairies in Sweden (Lindmark-
Månsson et al., 2003) and the Netherlands (Heck et al., 2009) provide detailed
information on the average milk composition in these countries, but one can only
tentatively make conclusions about the appropriateness of that milk for
cheesemaking. To the best of our knowledge there have been no publications on
the composition and rennetability of raw milk, characterising the actual quality
of commercial pooled milk utilized for processing with a country-wide coverage.
Hence, a part of this dissertation focuses on the evaluation of the renneting
properties of raw bulk milk produced at the farms throughout Estonia by direct
determination of rennet flocculation time (RFT) by classical Berridge method
(Berridge, 1952) and measurement of curd firmness. Further, we developed a
novel method based on front-face fluorescence spectroscopy for instrumental
determination of coagulation time and monitoring the process of milk rennet
coagulation in detail.
The activity of starter cultures in milk is another aspect essential in the
manufacturing of fermented dairy foods. Disinfectant residues getting
incidentally into milk after milking or added on purpose by unfair producers are
potential inhibitors of starter bacteria (Tamime & Robinson, 1999). Starter
cultures containing lactic acid bacteria (LAB) produce lactic acid lowering the
pH for controlling rate and extent of syneresis, retention and activity of
proteolytic enzymes and further proteolysis in cheese mass. For example in case
of yoghurt production, lowering the pH by LAB initiates acid-induced gelation
of milk, which determines the body of the product. Furthermore, LAB may
contribute to aroma, flavour, texture and mouthfeel of the end products (Leroy &
de Vuyst, 2004). The growth and activity of starter bacteria in milk in case of
occurrence of low concentrations of disinfectants or structural modifications of
milk constituents caused by gamma-irradiation were monitored in the present
study applying microcalorimetry and front-face fluorescence spectroscopy
techniques.

11 
 
1. LITERATURE REVIEW
1.1. Milk composition

The composition of milk determines its nutritional value and its properties as
raw material for manufacturing food products.
The average content of milk principal constituents and approximate range of
variation are presented in Table 1. The most abundant component of milk is
water, which comprises about 87% of its content. Lactose comprises
approximately 4.6%, fat 4.0% and protein 3.3% of milk composition. Milk also
contains vitamins, enzymes, minerals (primarily K, Na, Ca, Mg, Cl and
phosphate), organic acids and miscellaneous components (Walstra et al., 2006).
Table 1. Approximate composition and range of variation of milk (adapted from
Walstra et al., 2006).
Component Average content in milk (% w/w) Range (% w/w)
Water 87.1 85.3-88.7
Lactose 4.6 3.8-5.3
Fat 4.0 2.5-5.5
Protein 3.3 2.3-4.4
casein 2.6 1.7-3.5
Mineral substances 0.7 0.57-0.83
Organic acids 0.17 0.12-0.21
Miscellaneous 0.15 -

1.1.1. Lactose
Lactose is the major carbohydrate of milk and it is unique to milk; other
carbohydrates are present in milk only in traces. Lactose is a disaccharide
composed of glucose and galactose. It is the principal carbon source for most of
the microorganisms that grow in milk. Lactose is metabolized to compounds of
lower molecular weight by various microorganisms. The most important
fermentations from the technological point of view are those that produce lactic
acid, which is the basis for production of fermented dairy products (Walstra &
Jenness, 1984).
The concentration of lactose in milk is the most constant compared with fat
and protein content (Grimley et al., 2009; Heck et al., 2009; Lindmark-Månsson,
2003).
1.1.2. Milk fat
More than 98% of milk fat is made up of triglycerides. Other lipids include
phospholipids (0.8%), cholesterol (0.3%), diglycerides (0.3%), free fatty acids
(0.1%), and cerebrosides (0.1%). The fatty acids of milk lipids vary widely in
chain length (4-20 carbon atoms) and in saturation (0-4 double bonds). The
content of saturated fatty acid residues is about 63%. Milk fat contains a

12
relatively high proportion of short-chain fatty acid residues with 4-10 carbon
atoms. Oleic acid is the most abundant of the unsaturated fatty acid residues
(about 70%). The other unsaturated fatty acid residues are present in a wide
variety of chain length, unsaturation and isomers (Walstra & Jenness, 1984).
Majority of milk fat is included into fat globules (see below).
The concentration of fat in milk (Grimley et al., 2009; Heck et al., 2009;
Lindmark-Månsson et al., 2003) and fatty acid composition (Heck et al., 2009)
are substantially variable, and most important cause of variation is feeding
pattern of cows, which varies with season, region, farming practice and so on.
1.1.3. Milk proteins
The main milk proteins are categorized as caseins and whey proteins, which
comprise roughly 80% and 20% of total protein in milk, respectively (Walstra &
Jenness, 1984). The concentrations and properties of milk principal proteins are
summarised in Table 2.
Table 2. Concentrations and properties of major milk proteins (adapted from Farrel
et al., 2004, Walstra & Jenness, 1984 and Walstra et al., 2006).
Properties Caseins Whey proteins
αs1-CN αs2-CN β-CN κ-CN α-LA β-LG
Conc. in milk, g·kg-1 10.7 2.8 8.6 3.1 1.2 3.2
Molecular weight, kD 23.6 25.2 24.0 19.0 14.2 18.3
Amino acid res./mol. 199 207 209 169 123 162
Pro, res./mol. 17 10 35 20 2 8
P-Ser, res./mol. 8-9 10-13 5 0-3 0 0
Cys, res./mol. 0 2 0 2 8 5
Carbohydrates 0 0 0 + 0 0
Isoelectric pH 4.5 5.0 4.8 5.6 4.3 5.2

Caseins are those phosphoproteins that precipitate from raw skim milk by
acidification to pH 4.6 at 20°C. Caseins are identified according to the homology
of their primary structures (amino acid sequences) into the following families:
αs1-, αs2-, β- and κ-casein (-CN), which in turn have several genetic variants
(Farrel et al., 2004). Caseins have many prolines in their primary structure,
which prevents the formation of secondary and tertiary structures. Lack of
highly organized structure makes casein stable against denaturation but
susceptible to proteolysis. The hydrophobic, polar and charged residues are not
uniformly distributed throughout the molecular sequences but occur as
hydrophobic or hydrophilic patches. αs1-, αs2- and β-CN are highly
phosphorylated. The phosphate groups are esterified to serine residues of caseins
and bind strongly divalent ions. αs1-, αs2- and β-CN tend to aggregate in the
presence of Ca2+. Unlike other caseins, κ-CN contains normally only one
phosphoseryl residue and is insensitive to Ca2+. Most of the κ-CN molecules are
glycosylated to various extents, and they contain up to four tri- or tetrasaccharide

13 
 
moieties attached to its C-terminal region increasing the hydrophilicity of that
region (Fox et al., 2000).
The term whey proteins has been used to describe the group of milk proteins
that remain soluble in milk serum or whey after precipitation of casein at pH 4.6
at 20°C. Traditionally, β-lactoglobulin (β-LG), α-lactalbumin (α-LA), bovine
serum albumin, immunoglobulins, and proteose-peptone fractions have been
considered the major characterized components of this fraction (Farrel et al.,
2004). In contrast to the caseins, the whey proteins possess high levels of
secondary, tertiary, and quaternary structures. They are typical globular proteins,
which are denatured upon heating. Whey proteins are not phosphorylated and
are insensitive to Ca2+ (Fox et al., 2000). Denatured whey proteins may form
aggregates with each other or with κ-CN (Vasbinder & de Kruif, 2003), which
can markedly impair the rennet coagulation properties of milk during
cheesemaking and alter the rheological properties of acid gel based fermented
dairy products.
Milk protein composition is variable due to intrinsic genetic polymorphism of
milk proteins, differences in their degree of phosphorylation and glycosylation
(Farrel at al., 2004). In addition, protein content and relative concentrations of
different fractions may vary with breed, stage of lactation and season (Jõudu et
al., 2008; Wedholm et al., 2006).
1.1.4. Milk salts
Almost all of the salts are in the serum (diffusible fraction) or in the casein
micelles, and very little is bound to the fat globules (Walstra & Jenness, 1984).
Sodium, potassium and chloride ions are essentially diffusible, although
calcium, inorganic phosphate and magnesium are partly bound to the casein
micelles. About one-third of calcium, half the inorganic phosphate, two-thirds of
magnesium and over 90% of citrate are in the aqueous phase of milk.
Ions in the diffusible fraction are not totally “free” in solution and different
associations of ions occur. Calcium exists as ionic calcium (free ion) and a stable
complex mainly with citrate (as the trivalent citrate), and to a lesser degree with
inorganic phosphate (as a mixture of H2PO4– and HPO42–) and with chloride.
Sodium and potassium exist mainly as free ions, but a small part of these ions
are associated with citrate, inorganic phosphate and chloride (Gaucheron, 2005).
A part of the salts in milk is present in or on the casein micelles. Part of those
is considered as counterions. The casein molecules are negatively charged at the
milk pH and are associated with positive counterions (Walstra et al., 2006),
which comprise almost all of the K+, Na+, Mg2+, and about 20% of the Ca2+ in
the micelles (Walstra & Jenness, 1984). The rest of the ions, which is mainly
calcium and phosphate together with a little citrate, is present in a state of
colloidal calcium phosphate (CCP). The CCP consists of small, non-crystalline
regions and is, moreover, bound to the proteins (Walstra et al., 2006). The
colloidal calcium in milk can be defined as a mixture of calcium caseinate

14
(involving organic phosphate of phosphoserine residues) and calcium phosphate
(which is an inorganic phosphate) (Gaucheron, 2005).
The salts of milk are in dynamic equilibrium: among themselves in solution,
between solution and colloidal phosphate, and between solution and proteins
(Walstra et al., 2006). Minerals play an important role in the structure and
stability of the casein micelles. Small changes of the physico-chemical
conditions can induce some repartition and consequently can alter the stability of
casein micelles.
The mineral composition of milk is considered as relatively constant, but
slight variations can still be observed. Calcium and phosphate contents are
higher in milk rich in proteins. Also variations during different stages of
lactation and in case of mastitis may occur (Gaucheron, 2005).

1.2. Milk structure

Milk is an aqueous solution of lactose, serum proteins, salts and other minor
components. On the other hand, milk is a dispersion of fat globules and casein
micelles. All particles exhibit Brownian motion and are negatively charged at
natural milk pH (Walstra et al., 2006). Main structural elements and some of
their properties are presented in Table 3.
Table 3. Main structural elements of milk and their properties (adapted from Walstra
et al., 2006).
Milk
Plasma
Serum
Structural element Fat globules Casein micelles Globular proteins
Main components Fat Casein, water, salts Serum proteins
Volume fraction 0.05 0.1 0.006
Particle diameter 0.1-10 μm 20-400 nm 3-6 nm
Number per mL 1010 1014 1017
Surface area, cm2 mL-1 700 40,000 50,000
Isoelectric pH 3.8 4.6 4-5

1.2.1. Milk fat globules

The particles of the largest size in milk are fat globules. Nearly all of the fat in
milk exists in fat globules, only about 0.4% of the fat is found outside the
globules (Walstra & Jenness, 1984). The milk fat globules vary in diameter from
about 0.1 to 10 μm. Milk contains very many small globules, which comprise
only a small fraction of the total fat: globules smaller than 1 μm make up about
75% of the total number of globules, but only about 2% of the total globular fat,
and about 7% of the fat globule surface area (Walstra et al., 2006). Average fat
globule size varies among breeds, among individual cows and with stage of

15
lactation (Walstra & Jenness, 1984). The positive relation was found between
milk fat globule sizes and fat content in raw milk (Wiking et al., 2004).
Each fat globule is surrounded by a thin protective layer or membrane, which
consists mainly of polar lipids and proteins, including enzymes. The mass of the
membrane is about 2% of that of the total fat globules. Membrane prevents the
fat globules from flocculation and coalescence, and protects the fat against
lipolysis and oxidation (Walstra & Jenness, 1984). Milk handling during and
after milk harvesting as air inclusion, agitation (pumping and stirring), cooling,
heating, homogenisation and aging may alter fat globule membrane composition
and properties (Evers, 2004).
1.2.2. Casein micelles
Despite their name, casein micelles are not the real “micelles” in terms of colloid
science. A typical casein micelle is made up of nearly 20,000 individual protein
molecules associated into complex colloidal particles, whose interior is formed
by the highly phosphorylated caseins (αs- and β-CN) interacting and aggregating
with calcium phosphate, and whose sizes are controlled by the layer of κ-CN
bound to their surfaces (Dalgleish, 2011). The average size of casein micelles in
pooled milk was measured to be 120 nm (de Kruif et al., 2012), and is
determined by the proportion of κ-CN in the mixture of caseins (Dalgleish,
2011). Average casein micelle size was found to be constant for a given cow
during milking, lactation and over the years (de Kruif et al., 2012).
The structure of the casein micelle has been discussed for many years and has
not been fully agreed until now. Among most widespread and debated models
are the “submicelle model”, the “nanocluster model” and the “dual-bonding
model”. The classical “submicelle model” (Walstra, 1990) suggests that a casein
micelle is built of smaller units, which are of two main types, one primarily
consisting of αs- and β-CN, the other of αs- and κ-CN, and which are linked
together by CCP. The sub-micelles rich in κ-CN are located on the surface of the
micelles and κ-CN protrudes out from the micelles core into the solvent as
flexible “hairs”. The “nanocluster model” was first introduced by Holt (1992)
and considered the calcium phophate nanoclusters to be responsible for cross-
linking individual proteins and holding the gel-like casein network together. This
model was in some sense refined by “dual-bonding model” of Horne (1998),
which takes into consideration two types of interactions keeping micelles
structure together: a) binding of CCP to negatively charged phosphoserine
clusters of caseins, reducing their charge, and b) hydrophobic interactions
between the hydrophobic regions of the caseins.
The submicellar model has received much criticism and was shown to be
nonconsistent with electron micrographs (Dalgleish et al., 2004; McMahon &
McManus, 1998) and the experimental scattering based data (de Kruif et al.,
2012). An advanced model based on calcium phosphate nanoclusters was
considered to be the most relevant in the recent publications of Dalgleish (2011)
and de Kruif et al. (2012).

16
 De Kruif et al. (2012) considered the casein micelle as a homogeneous matrix
of caseins in which the CCP nanoclusters are dispersed as very small “cherry
stones” with the radius of 1.72 to 2.27 nm at an average distance of 18.6 nm
(Figure 1). Based on the calculations, there are about 372 CCP nanoclusters per
casein micelle in pooled milk. Attached to the surfaces of the nanoclusters are
the centres of phosphorylation (3-5 nearby phosphorylated amino acid residues)
of the caseins. The hydrophobic tails of the caseins, much larger than the CCP
clusters, stick out. These tails are entangled with other hydrophobic tails forming
small hydrophobic and denser protein regions of about 2 nm in radius. The
association of the tails is driven by a collection of weak interactions, which
engage hydrophobic interactions, hydrogen bonding, ion bonding, weak
electrostatic Van der Waals attraction and other factors (but not the strong
calcium phosphate interaction) leading to self-association. The κ-CN limits the
process of self-association leading to stabilization of the native casein micelle.

Figure 1. Schematic representation of the internal structure of casein micelle (by

kind permission of C. G. de Kruif and Elsevier B.V.1). Black spheres are calcium
phosphate nanoclusters, gray spheres are protein clusters.
While the internal structure of the casein micelles remains the matter of the
debates for more than 50 years by now, the existence of the hairy layer of the
micelles is universally accepted. However, the distribution of κ-CN on the

1
Reprinted from Advances in Colloid and Interface Science, Vol. 171-172. De Kruif,
C. G., Huppertz, T., Urban, V. S., & Petukhov, A. V., Casein micelles and their internal
structure, Pages No. 36-52, Copyright (2012), with permission from Elsevier.

17
surface of the casein micelles is not fully understood. It was proposed that the
surface of the micelles is only partially covered by κ-CN, which is
heterogeneously distributed (Dalgleish, 1998). The evidence for the organization
of the surface of the casein micelles into tubular structures (bunches of κ-CN)
was provided on the basis of the scanning electron micrographs, and it was
proposed that the surface contains gaps between the substructures (Dalgleish et
al., 2004). Eventually, it was proposed that the interior of the casein micelles is
inaccessible for very large molecules and other casein micelles and only the
outer layer of κ-CN will be detected as the particles approach each other.
However, for smaller molecules, including individual proteins such as enzymes,
the surface of the micelle will include also a part of the interior due to clefts or
pores on the surface of the micelles (Dalgleish, 2011).
Milk can be moderately heated or cooled without significant aggregation of
casein micelles or disruption of their basic structure. On the other hand, the
micelles can be easily destabilized by treatment with proteolytic enzymes or by
acidification resulting in the formation of coagulum, which is the basis of the
production cheese and yoghurt-type products. In that sense, most of the
important functional properties of the casein micelles are determined by the
properties of the surface, rather than those of the interior. However, the interior
of the casein micelles becomes important in postcoagulation rearrangements
taking place in cheese curd or acid-induced gels (Dalgleish & Corredig, 2012).

1.3. Milk coagulation

Milk coagulation can be achieved with rennet or another proteolytic enzyme

preparation, with acid, by heating, or by combinations of these factors. The
manufacture of traditional ripened semi-hard and hard cheeses includes
preculturing of the milk before rennet addition – rennet coagulation is facilitated
by acid produced if a high percentage of starter bacteria is added. A combined
acid and rennet coagulation is used in the manufacture of fresh and ripened soft
cheeses; however, gel formation is mainly induced by acid. Queso Blanco
cheese is made by heating milk to between 80 and 85°C and further acid
addition (acetic, citric, phosphoric, etc., or simply lime juice) while gently
stirring the milk, which leads to the formation of a coagulum (Walstra et al.,
2006).
1.3.1. Rennet-induced coagulation
Casein micelles are stabilized by steric and electrostatic repulsion generated by
the polyelectrolyte layer of κ-CN, which prevents micelles from aggregation
(Dalgleish & Corredig, 2012; Dejmek & Walstra, 2004). Any process that
removes the C-terminal part of κ-CN causes a decrease in the colloidal stability
of the micelles (Dalgleish & Corredig, 2012).
Two stages, overlapping to some extend, can be raughly distinguished during
renneting (Fox, et al., 2000):

18
1. κ-CN ⎯enzyme
⎯⎯→ para-κ-CN + glycomacropeptide
Ca 2 +
2. para-κ-CN covered micelles ⎯⎯⎯→ gel

The primary phase of renneting involves the specific enzymatic modification of

the casein micelles, and the second phase involves the aggregation of the rennet-
altered micelles.
Several proteinases can coagulate milk, but most are too proteolytic (high
level of non-specific proteolysis (Fox et al., 2000)). The main clotting enzyme
used in cheesemaking is chymosin (E.C. 3.4.23.4., MW 30,700, pI 4.6-4.7). At
milk pH 6.7, chymosin splits only κ-CN Phe105-Met106 bond, resulting in para-κ-
CN, which remains attached to casein micelles, and glycomacropeptide (GMP),
which is soluble in the whey fraction. The positive charge of the region of the κ-
CN chain accounts presumably for a rapid binding of the negatively charged
active site of the enzyme molecule (Walstra & Jenness, 1984). The sequence κ-
CN f98-111 around the Phe105-Met106 bond, rather than the residues in the bond
itself, determines the binding affinity of the enzyme (Fox et al., 2000).
During renneting, there is about one enzyme molecule per 100 casein
micelles and a micelle contains in the order of 1000 κ-CN molecules (Dejmek &
Walstra, 2004). After the addition to milk (Figure 2a), the enzyme approaches
the micelles “gradually” by means of diffusion, part of the diffusion path being
in the hairy layer of the micelle, as the Phe-Met bond to be split is quite close to
the micelle surface. This increases the diffusion time, and hence, the enzyme
molecules bind gradually onto the surface of casein micelles. The reaction
velocity thus is considered diffusion-limited. At milk physiological pH the
“hairs” are removed at random (Walstra et al., 2006) (Figure 2b). Removal of
the “hairs” results in a decrease of the hydrodynamic diameter of the casein
micelles and causes a slight minimum in the viscosity during the initial phase of
renneting (Lucey, 2009).
The release of the GMP from the surface of the casein micelles leading to a
decrease of the zeta potential by ~ 50%, from -10/-20 to -5/-7 mV (Fox et al.,
2000), reduces electrostatic as well as steric repulsion between rennet-altered
casein micelles (Lucey, 2009). Then, the micelles can approach closer to one
another and flocculate. The flocculation does not proceed until nearly 70% of κ-
CN “hairs” are hydrolysed; after this point, para-casein micelles interact
increasingly strongly with an increase in the extent of proteolysis (Dejmek &
Walstra, 2004; Lucey, 2009). The term flocculation refers to weak reversible
aggregation and coagulation to irreversible aggregation (Walstra et al., 2006).
Only after more extensive removal of the protective κ-CN layer does true
aggregation occur (Lucey, 2009). At first irregular (Figure 2c), but then
somewhat thread-like (Figure 2d) aggregates are formed, which grow to form
large tenuous flocks and eventually a continuous network (Dejmek & Walstra,
2004). A three-dimensional particle gel formed as a result of aggregation is
dynamic in its nature. Further rearrangements of the bonds (fusion of the

19
micelles), particles and clusters of the particles result in shrinkage and
coarsening of the gel network and induce syneresis (Mellema et al., 2002).

Figure 2. Schematic description of the various stages of enzymatic coagulation of

milk (by kind permission of Elsevier B.V.2): a) initial mixture of casein micelles and
enzyme, b) cleavage of κ-CN, c) initial aggregation into small clusters, d) reaching a gel
point at percolation.
During cheesemaking, there are a number of compositional and
environmental factors that affect the kinetics of rennet coagulation as well as the
strength of the resultant gel (curd firmness): milk protein and fat content (Guinee
et al., 1997), pasteurization temperature (Leaver et al., 1995; Singh &
Waungana, 2001; Vasbinder et al., 2003), cooling and cold storage of milk
(Malacarne et al., 2013; Raynal & Remeuf, 2000), renneting temperature (Ong et
al., 2011) and pH (Leaver et al., 1995; Ong et al., 2012), addition of CaCl2
(Bringe & Kinsella, 1986; Famelart et al., 1999; Sandra et al., 2012) etc.
1.3.2. Acid-induced coagulation
Acidification of milk can be induced by lactic acid bacteria (LAB), which
produce lactic acid from lactose, by the addition of acids such as HCl, or by the
use of glucono-δ-lactone (GDL), where the hydrolysis of GDL to gluconic acid
results in a reduction of pH (Lucey & Singh, 1997). The final pH that is attained
in GDL-induced gels depends on the amount initially added to milk, whereas
starter bacteria can produce acid until they become inhibited by the low pH.

2
Reprinted from Cheese: chemistry, physics and microbiology, Vol. 1 (3rd ed.), P. F.
Fox, P. L. H. McSweeney, T. Cogan, & T. P. Guinee (Eds.). Horne, D. S., & Banks, J.
M., Rennet-induced coagulation of milk, Pages No. 47-70, Copyright (2004), with
permission from Elsevier.

20
 Acidification causes a number of changes in casein micelles, which can be
divided into three arbitrary pH regions during acidifying milk from pH 6.7 to
4.6. The decrease of pH from 6.7 to ~6.0 causes a decrease in the net negative
charge on the casein micelles and reduces electrostatic repulsion. Further
decrease of pH from ~6.0 to ~5.0 causes further decrease in the charge of κ-CN
“hairs” on the micelles surface, so these “hairs” may shrink and steric
stabilisation fails. The decrease of pH to ~5.0 is also accompanied by dissolution
of CCP and consequent loosening of the molecular interactions between caseins.
Aggregation of casein particles in unheated milk occurs at around pH 4.9. If
acidification is performed at very high temperatures, a higher gelation pH is
observed (Lucey, 2009). In heated milk, gelation occurs at higher pH (5.2-5.4),
which can be attributed to the higher isoelectric pH (~5.2) of denatured whey
proteins, associated with casein micelles upon heating, compared to that of
caseins (pH 4.6) (Lucey, 2004b). In Publication IV, a novel approach – front-
face fluorescence spectroscopy – was used for non-destructive dynamic
monitoring of changes in the milk proteins during acidification by starter
bacteria.
In dairy industry, cultured milk products are formed mainly as a result of the
production of lactic acid during fermentation of lactose by the starter culture
(Lucey, 2004a). Proteolytic enzymes of the bacteria can possibly be also
engaged in this process because some of those can split κ-casein; however, the
main mechanism is casein becoming insoluble near its isoelectric pH (Walstra et
al., 2006). Acidification activity of milk is a very important characteristic of the
quality of dairy starters. The methods used for the characterization of the
acidification activity of starter bacteria are usually based on measuring pH
changes or on determination of the accumulation of lactic acid per time unit
(Zanatta & Basso, 1992). In addition to acid production, the LAB also play a key
role in the formation of aroma, texture and flavour of the end-product. The
metabolic activity of the bacteria can be characterized also by the heat produced
during their growth, and this can be measured by microcalorimetry. In
Publication II, an isothermal batch microcalorimeter was used to obtain the
precise and detailed information on the growth of thermophilic LAB in
continuous mode in situ (in small ampoules) during the formation of yoghurt.

1.4. Variation in composition and technological properties of milk

Cow’s milk is rather constant in a qualitative sense, but there are considerable
quantitative variations in chemical composition, size and stability of the
structural elements, and properties of different lots of milk (Walstra & Jenness,
1984). These differences originate from a natural variation in milk composition
(due to breed, individual cow etc.), contaminants entering milk via the cow from
the feed or during and after milking (e.g. antibiotics, detergents and
disinfectants) and various other changes occurring during and after milking and
during further processing.

21
 1.4.1. Natural variation
Three kinds of factors can lead to natural variation of milk composition: genetic
factors (differences among breeds and individuals within breed), physiological
conditions (stage of lactation, health state, age of the animal) and environmental
factors (feed, weather conditions, treatment) (Walstra & Jenness, 1984).
Throughout the years, cows of Estonian Holstein-Friesian (EHF) breed were
shown to have higher yield but lower protein and fat content than cows of
Estonian Red (ER) or Estonian Native breed (Estonian Animal Recording
Centre, 2012). In addition it has been shown that total CN, αs2- and κ-CN, β-LG
and the relative contents of αs1- and κ-CN in total casein were lower in milk
from cows of the EHF breed than those of the ER breed (Jõudu et al., 2008).
Likewise, in spite of the higher milk productivity of Holstein-Friesian breed,
milk from Italian Friesian cows was shown to have lower protein content, also
lower curd firming rate, weaker coagulum and lower cheese yield than milk
from Italian Brown breed (Malacarne et al., 2006). On the other hand, milk from
Holstein-Friesian (HF) cows from Finnish herds was shown to have lower
protein and fat content but better coagulation ability in comparison with Finnish
Ayrshire (FA) cows (Tyrisevä et al., 2004).
Milk from about 30% of FA and 12% of HF cows was found categorized as
poorly coagulating, while milk from 8.6% of FA and 1.3% of HF cows was non-
coagulating (Tyrisevä et al., 2004). Similarly, more than 30% of the Swedish
and Danish cows sampled produced poorly coagulating or noncoagulating milk
(Wedholm et al., 2006) that was associated with a low concentration of κ-CN
and a low proportion of κ-CN in relation to total casein.
Total protein content and ratios of different protein fractions have been
reported to change during lactation (Caffin et al., 1985; Jõudu et al., 2008;
Ostersen et al., 1997). The proportion of total casein was higher in mid-lactation;
proportions of αs- and κ-CN decreased and β-CN increased during lactation
(Ostersen et al., 1997). A shift to higher pH of milk was also observed
throughout the lactation (Tsioulpas et al., 2007a; Tyrisevä et al., 2004). The
longest renneting time and lowest curd firmness were observed during the mid-
lactation period (Ostersen et al., 1997; Tyrisevä et al., 2004).
The variability of milk composition of individual cows is greater than the
variability of milk lots received at dairy factory – pooling of milk reduces the
variations. The main differences of lots of pooled milk are assigned to seasonal
differences and those between regions, which may differ in predominant breed
or feeding practice (Walstra & Jenness, 1984).
Only few studies have been published which characterize the composition of
cows’ milk in a whole country with respect to seasonal, geographical or annual
variations (Lindmark-Månsson et al., 2003; Heck et al., 2009). In milk collected
from nine Swedish dairies, minimum protein content was observed in spring and
maximum in autumn, fat content not showing any significant seasonal variation
(Lindmark-Månsson et al., 2003). Heck et al. (2009) reported seasonal variations
in the composition of average dairy milk in the Netherlands based on a single

22
pooled milk sample collected and analysed weekly within one year. Summer
milk was characterized by low protein and low fat content, and the highest
values were observed in winter in the Netherlands (Heck et al., 2009). Despite
the clear variation in the total protein content during the year, the protein
composition of Swedish and Dutch pooled milk was rather constant (Lindmark-
Månsson et al., 2003; Heck et al., 2009). Unfortunately, these studies were
performed without turning a special attention on the rennetability of milk.
The coagulation characteristics of Estonian raw bulk milk were evaluated in
Publication I by direct measurement of rennet flocculation time (RFT) and curd
firmness – correlations of these properties and milk composition were observed.
Results of our study provide information on rennetability of bulk milk and its
variability from a large number of farms representing the whole country.
1.4.2. Disinfectants
Effective cleaning and sanitizing of dairy equipment is a prerequisite for
producing high quality dairy products with low bacterial counts. Disinfectant
formulations including quaternary ammonium or halogen-containing
compounds, and H2O2 are still used in dairy industry (Tamime & Robinson,
1999). Besides being not acceptable in European countries, the retardation of
growth of unwanted bacteria in raw milk by adding substantial amounts of H2O2
is permitted in some developing countries with warm climate. The recommended
quantities of H2O2 (if allowed to be added to milk) range from 100 to 800 mg L-1
(Björck, 1987). Yet, lower concentrations of disinfectant residues may get into
the milk due to faulty cleaning systems, improper dosage, insufficient rinsing,
unsuitable pipeline design, or in some cases can be deliberately added by
producers into milk to ’improve’ its bacterial quality.
Early studies of adding high concentrations of H2O2 (100 mg L-1 to 15 g L-1)
to milk prior to cheesemaking showed that the treatment resulted in softer cheese
mass (Roundy, 1958; Kosikowski & Fox, 1968), higher solubility of casein, and
higher susceptibility of casein to proteolysis by rennet (Fox & Kosikowski,
1967) and proteolytic enzymes of Pseudomonas fluorescens (Fish et al., 1969).
Schmidt et al. (1969) reported reduced rate and lower completeness of casein
clotting by rennet in the presence of high concentrations of H2O2.
Effects of low concentrations of H2O2 (< 10 mg L-1) in milk have been
commonly considered in scientific literature in conjunction with the
lactoperoxidase (LPO) system, which prevents the growth of bacteria by
catalyzing the oxidation of thiocyanate (SCN-) to hypothiocyanite (OSCN-)
believed to possess antimicrobial properties, using H2O2 as the electron acceptor
(Özer, 1999). The use of LPO system is recommended by FAO/WHO as a
standard method for retarding bacterial growth in raw milk during collection and
transportation to dairy plants in situations where refrigeration is not feasible
(Codex Alimentarius Commission, 1991). Recommended concentrations of
H2O2 added for activation of LPO system are up to 10 mg L-1 as noted. On the
other hand, the activation of LPO system has been associated with inhibiting

23
effects on cheese and yoghurt starter cultures (reviewed by Seifu et al., 2005).
Moreover, an impairment of rheological properties of yoghurt was reported,
alterations being assigned to the oxidation of protein thiol groups (-SH) by
OSCN- leading to decreased number and/or strength of cross-linking (Hirano et
al., 1998a, b). Antimicrobial and oxidizing effects were not related in those
studies to the action of H2O2 itself but to other components of LPO system.
Application of higher than recommended concentrations of H2O2 may cause
irreversible inactivation of LPO, naturally occurring in raw milk (Björck, 1987;
Kussendrager & van Hooijdonk, 2000; Fonteh et al., 2005). Some slight
decrease in tyrosine content in milk, showing susceptibility of amino acids to
oxidation, and significant increase in peroxide values, indicating lipid oxidation,
have been reported in yoghurt produced from milk treated with considerably
high levels of H2O2 (100-140 mg L-1), which demonstrates the evidence of H2O2-
promoted and apparently LPO-independent oxidation of milk proteins and lipids
(Özer & Atamer, 1999). Nevertheless, the oxidative and inhibiting influences
against starter bacteria of intermediate concentrations of H2O2 added to milk at
higher levels (10-100 mg L-1) than normally required for activation of LPO
system, and independently of LPO has not been evaluated as yet and were
addressed in Publication II.
1.4.3. Changes occurring during and after milking
Processes occurring in milk after it is drawn include creaming of fat globules
and crystallization of part of the fat on cooling; inclusion of air bubbles, which
may cause a disruption of fat globules; inclusion of oxygen, which may induce
lipid oxidation; lipolysis and proteolysis initiated by milk or bacterial enzymes
(Walstra & Jenness, 1984).
Deterioration of milk microbiological quality and decrease in curd yield
during storage of milk in bulk milk tanks at the farm and in dairy silos have been
reported (Leitner et al., 2008). A comprehensive information on the influence of
different storage temperature/time combinations on the physico-chemical and
microbiological characteristics as well as processing properties of milk was
provided in the recent publication (Malacarne et al., 2013) – the storage of milk
at the lowest observed temperature 4-6°C impaired the rennet coagulation
properties of milk the most.
The differences between protein composition and the coagulation properties
of milk sampled at the farms and the same milk received at the dairy (milk
originated from the same milkings), which has undergone mechanical
perturbations such as pumping and refrigerated transportation in trucks, were
studied in Publication I.

1.5. Methods for monitoring milk coagulation

One of the earliest and simplest methods for the estimation of milk rennet
coagulation properties is the visual determination of the onset of flocculation in a

24
glass tube (Berridge, 1952). In contrast to cheese manufacture, where milk is
renneted under quiescent conditions to ensure gel formation, this method
determines the time of coagulation under agitation. Yet, the effect of constant
stirring on the enzyme action and aggregation kinetics is not clear, as well as this
method is destructive towards the formation of milk coagulum. The registration
of the onset of flocculation by human eye occurs later than optically determined
onset of the aggregation of casein micelles (Tabayehnejad et al., 2012).
The most direct way to measure gel formation during milk coagulation is to
monitor the evolution of rheological properties. Dynamic low-amplitude
rheometry applies a low-amplitude oscillatory shear stress (τ0) or strains (γ0) and
records the response of the developing gel (Horne & Banks, 2004). Under these
conditions the gel strands of the gelling milk are strained to a fixed displacement
(within their elastic limit) and recover instantaneously when the stress is
removed (Fox et al., 2000). The measurements yield the elastic or storage
modulus (G'), which is a measure of the energy stored per oscillation cycle and
reflects how the sample behaves as an elastic solid, and the viscous or loss
modulus (G"), which is a measure of the energy dissipated per cycle and
indicates how much the sample behaves as a viscous liquid. The ratio of G" to G'
is tan δ, the tangent of the phase angle of the response to the applied stress or
strain. The shear moduli are defined as follows:

cos

sin

In uncoagulated milk δ > 45° and the viscous component dominates; when
coagulation proceeds δ < 45° and the sample appears more like an elastic.
Initially the G' lags behind growth of viscosity or viscous modulus, G", but
quickly crosses and dominates as gel elasticity rapidly develops (Horne &
Banks, 2004). The stress required to achieve a fixed strain (displacement of a gel
strand at a given position) increases as the gel strands become more elastic and
firm; hence, measurement of stress energy provides a measure of the gel
strength. The G' is a direct measure of curd firmness and is thus of high
significance in cheese manufacture (Fox et al., 2000). The increase in the shear
moduli after gelation reflects ongoing fusion of micelles, which results in an
increase in the contact area between aggregated particles, and possibly the
incorporation of additional particles into the gel network (Lucey, 2009). Low-
amplitude dynamic rheometry gives a precise estimation of the rheological
changes that occur during renneting without altering the process of gel
formation; hence, it accurately reflects the changes in curd firmness that occur
upon renneting milk in the cheese vat under quiescent conditions. However,
limitations of such measurements include the high cost of instrumentation, the

25 
 
high level of operator skill required for accurate measurement, the fact that only
one sample can be analysed at a time, and instruments currently available cannot
be used for online measurement of gel formation in the cheese vat (Fox et al.,
2000).
Other instrumental methods with different principles have been used to
monitor milk coagulation in laboratory scale research for better understanding of
this process, and many attempts have been made to automate the cheese
production by developing a variety of different on-line sensors for monitoring
milk coagulation and prediction of cutting time. The last reviews of the off-line
and on-line methods for assessment of milk coagulation – their operating
principles, advantages and drawbacks were published a decade ago (Lucey,
2002; O’Callaghan et al., 2002). The techniques for monitoring of milk
coagulation have been based mainly on a) measuring physical parameters related
to the changes of rheological properties of milk using mechanical (e.g.
“Formagraph”; McMahon & Brown, 1982), vibrational (e.g. “Sofraser”,
“Viscolite”; O’Callaghan et al., 2000), ultrasonic (Dwyer et al., 2005; Wang et
al., 2007), thermal conductivity probes (“Hot-Wire”; O’Callaghan et al., 2000)
or b) optical techniques detecting the changes in the sizes of micelles using near
infrared (NIR) transmission or reflectance (e.g. “TxPro” and “CoAguLite”;
Castillo et al., 2000; Klandar et al., 2007, O’Callaghan et al., 2000). Among the
promising techniques to study coagulation of milk a diffusing wave
spectroscopy was highlighted, which provides additional information on particle
interactions and their dynamics during flocculation of casein micelles
(Alexander & Corredig, 2007; Hemar et al., 2004; Huppertz & de Kruif, 2007).
Concerning the use of sensors in practice, the installation of mechanical and
vibrational probes through the wall of a cheese vat is rather problematic; also
proper cleaning of such systems is an issue. Furthermore, mechanically moving
or vibrational action of the probe may be intrusive and thus only a limited degree
of coagulation could be measured (O’Callaghan et al., 2002). Conversely, the
optical methods have definite advantages as they are non-destructive, do not
perturb coagulation, are integratable in-line and easily applicable on plant scale
from the hygienic viewpoint. At the same time, the ability of optical methods to
provide the information on the rheological properties of forming coagulum and
prediction of resistance to cutting may be limited (Lucey, 2002). Variations in
protein composition can also confound the results of optical measurements of
curd firming (O’Callaghan et al., 2000).
Most recent research has been focussed specifically on the development of
combined sensors for continuous non-invasive monitoring of milk coagulation
and syneresis, based on ultrasonic measurements (Taifi et al., 2006) or a sensor
utilizing a large field of view relative to curd particle size based on detecting
NIR light backscatter (Fagan et al., 2007b, c; 2008). Recent publication of Fagan
et al. (2011) has shown the potential applicability of front-face fluorescence
spectroscopy to monitor both coagulation and curd syneresis phases in
laboratory cheese vat.

26
 Although many instrumental techniques have been developed for monitoring
milk coagulation, the implementation of such systems in the dairy industry has
been still modest overall (O’Callaghan et al., 2002). The objective of the
Publication III was to study the potential of the front-face fluorescence
spectroscopy to monitor the changes in milk proteins occurring during rennet-
induced coagulation and to derive the coagulation time parameter, which could
be used to monitor the coagulation ability of milk.

1.6. Front-face fluorescence spectroscopy in dairy research

After the absorption of light by a fluorescent molecule (fluorophore), electrons

from the singlet ground electronic level S0 move to an excited state Sn (n > 1).
After that the molecule in Sn moves to the lowest excited state S1 by dissipating a
part of its energy in the surrounding environment, which is called internal
conversion. The molecule returns from the lowest excited state S1 to the ground
state S0 and emission of a photon may occur – this phenomenon is fluorescence
(Valeur, 2002). The energy absorbed by a fluorophore is higher than the energy
of the emitted photon. Thus, the emission spectrum has its maximum shifted to
longer wavelengths compared to the maximum of absorption spectrum (Albani,
2007).
Each electronic state has several associated vibrational levels, which means
that excitation does not occur at only one single wavelength, but rather over a
distribution of wavelengths corresponding to different vibrational transitions.
The deactivation of the excited state occurs only from the vibrational ground
level, but emission also occurs at several wavelengths as it may reach different
vibrational levels in the electronic ground state. Therefore, all fluorophores have
specific spectral excitation and emission profiles characterizing their unique
fluorescent properties (Christensen et al., 2006). These profiles can be measured
as excitation and emission spectra separately or as a complete excitation-
emission matrix, also known as fluorescence landscapes or spectral fluorescence
signatures (SFS).
If absorbance is less than 0.1 optical density units, the intensity of the emitted
light is proportional to fluorophore concentration and excitation and emission
spectra are accurately recorded by classical right-angle fluorescence device. If
the absorbance is higher than 0.1 optical density units, a dilution of samples is
needed. But in this case the organisation of the food matrix is lost. To avoid
these problems, the method of front-face fluorescence spectroscopy can be used
when measuring turbid, powdered or solid samples (Karoui et al., 2003). In
front-face illumination mode, the incident beam is set to the sample at an angle
other than right angle, and the measurement of fluorescence is performed from
the surface of the sample (Christensen et al., 2006).
Fluorophores are mainly aromatic or linear conjugated molecules, in which
the transition of de-localized π-electrons to the excited state is observed (Valeur,
2002). Milk and milk products contain a wide variety of naturally occurring

27
intrinsic fluorophores as aromatic amino acids – tryptophan, tyrosine and
phenylalanine in proteins, vitamin A and B2, cofactors etc. (Karoui et al., 2003).
In proteins, tryptophan fluorescence dominates; zero or weak tyrosine and
phenylalanine fluorescence results from energy transfer to tryptophan and/or
neighbouring amino acids (Albani, 2007). The major proteins in milk (αs1-, αs2-,
β-, κ-CN, α-LA and β-LG) contain at least one tryptophan residue each (Fox et
al., 2000).
Intrinsic tryptophan residues in proteins are sensitive fluorophores, those
fluorescence depends on the polarity of the environment and existence of
quenching molecules in the vicinity of tryptophan (Ladokhin, 2000). Polar
amino acid side chains (Asp, Glu, Lys, Arg and protonated His), amide groups
of Gln and Asn, phenol group of Tyr, as well as disulfide (Cys-Cys) bridges or
single Cys can effectively quench tryptophan fluorescence (Chen & Barkley,
1998; Engelborghs, 2003; Ladokhin, 2000). Changes in microenvironment,
induced for example by ligand binding or conformational changes in proteins
may result either in enhancement or quenching of fluorescence and in shifts in
the spectrum to shorter or longer wavelengths (Ladokhin, 2000). Several
inherent advantages of fluorescence spectroscopy for characterisation of
molecular interactions and reactions have been drawn out: 1) it is 100-1000
times more sensitive than other spectrophotometric techniques; 2) fluorescent
compounds are highly sensitive to their environment; 3) fluorescence method is
relatively rapid (Karoui et al., 2003).
At present there are only a few publications concerning the application of
fluorescence spectroscopy to monitor coagulation of milk (Fagan et al., 2011;
Herbert et al., 1999; Laligant et al., 2003; Lopez & Dufour, 2001). The specific
feature of front-face fluorescence spectroscopy is that the technique is
potentially able to reflect changes taking place with proteins at the molecular
level during renneting, apart from the other optical methods (e.g. measurement
of NIR absorbance (Klandar et al., 2007) or NIR light backscatter profile (Fagan
et al., 2007b)) that rely on the changes in particle size. Herbert et al. (1999) have
shown that front-face fluorescence spectroscopy was able to distinguish acid-
induced coagulation of milk from rennet-induced process and allowed the
detection of structural changes in casein micelles during coagulation. Laligant et
al. (2003) used a dynamic approach for characterization of acid gelation of milk
combining front-face fluorescence and other measurements. Lopez and Dufour
(2001) successfully applied front-face fluorescence spectroscopy for
investigation of the influence of the composition of fat globule surface on the
rennet-induced coagulation of reconstituted milk, however, interpretation of the
changes of tryptophan fluorescence intensity profiles recorded during the
renneting was not provided by the authors. It is known that in the course of the
manufacture of skimmed milk powder, the equilibrium of calcium between
serum and colloidal phases as well as the size of casein micelles could be
irreversibly altered (Martin et al., 2007), which was suggested to explain the
differences in rennet-induced coagulation properties of raw and reconstituted

28
milk samples (Martin et al., 2008). Hence, analytical techniques applied for
monitoring of reconstituted milk should be separately validated also in the case
of natural bulk milk. Recently, Fagan et al. (2011) have shown that spectral
changes of naturally occurring milk fluorophores in bulk milk allowed
monitoring of both coagulation and curd syneresis phases by mounting a
fluorescence probe into a laboratory cheese vat. The evolution of tryptophan
fluorescence has been shown to have the largest response during coagulation
compared with other intrinsic fluorophores of milk – riboflavin and vitamin A. A
need for further study under a wider range of processing conditions was
indicated by the authors (Fagan et al, 2011). In Publication III we aimed to
provide a deeper insight into different phases of renneting during coagulation of
different milk samples with various coagulation properties (reconstituted
skimmed milk, whole and skimmed raw bulk milk) using front-face fluorescence
spectroscopy together with reference methods.

29
2. AIMS OF THIS DISSERTATION
The aims of this dissertation were to investigate the indigenous and exogenous
factors affecting technological properties of milk and to apply a novel front-face
fluorescence spectroscopy method for monitoring of milk coagulation. The
specific aims were as follows:
I. To determine the quality of raw bulk milk collected from individual
farms in Estonia in relation to cheesemaking properties (Publication I)
with the special emphasis on:
• seasonal variations;
• comparison between bulk milk from individual farms and
pooled milk from dairy silos;
• verification of correlations of bulk milk composition and
renneting properties.
II. To study the influence of a common disinfectant used in dairy industry,
H2O2, on the growth of thermophilic starter bacteria and acid gelation of
milk (Publication II).
III. To apply front-face fluorescence spectroscopy to study rennet-
(Publication III) and acid- (Publication IV) induced coagulation of milk.

30
3. MATERIALS AND METHODS
The detailed descriptions of the materials and methods applied are available in
the publications. The following sections are provided to make this dissertation
more accessible.

3.1. Milk supply and treatment

3.1.1. Milk samples from farms, tank trucks and dairy silos
(Publication I)
More than thousand raw bulk milk samples from over 170 individual dairy farms
of geographically different locations in Estonia delivering milk to two large
dairies were collected during the period February 2004 – June 2007. Samples
were collected in 34 batches distributed randomly throughout all seasons, and
each time approximately 30 milk samples were analysed. On most sampling
days, samples were also taken from tank trucks and dairy silos where the milk
from examined individual farms was pooled on that day. The details of the
sampling procedure are provided in Publication I.
3.1.2. UHT milk samples (Publication II)
Utilization of ultra-high temperature treated (UHT) milk (Kalev Paide Tootmine
AS, Paide, Estonia) with fat contents of 3.5% and 0.05% (referred as UHT 3.5%
and UHT 0.05%, respectively) enabled the investigation of H2O2 oxidative or
antimicrobial influence independently of LPO system, since high temperature is
known to inactivate the natural antimicrobial LPO of milk (Kussendrager & van
Hooijdonk, 2000).
To samples of UHT 3.5% and UHT 0.05% various dilutions of H2O2 (Riedel-
de Haën, Seelze, Germany) were added to yield final concentrations of 0, 10, 25,
50, 75, 100, 150, 200 and 250 mg L-1 in milk. The treated milk samples were left
at 40°C for one hour to promote chemical reaction between H2O2 and milk
constituents before starter bacteria were added. To assure complete utilization of
H2O2 before inoculation, the aliquots of UHT 3.5% samples incubated with H2O2
were treated with excess bovine liver catalase (Sigma-Aldrich, St. Louis, MO,
USA) prior to the addition of starter culture.
3.1.3. Milk samples for monitoring coagulation by front-face
fluorescence spectroscopy and reference methods (Publication
III)
Coagulation experiments were performed with reconstituted skimmed milk
(RSM) and both, whole and skimmed raw bulk milk samples.
RSM was prepared from low-heat skimmed milk powder (LHSMP; Valio
Ltd., Helsinki, Finland) by dissolution in distilled water at 10% (w/v) milk
solids, with addition of CaCl2 to yield 5 or 10 mM final concentration. RSM
without CaCl2 addition was also used in some trials.

31
 Nine samples of whole bulk milk with varying coagulation properties were
collected from four dairy farms supplying milk for cheese production at
Saaremaa Dairy (Kuressaare, Estonia) on different sampling days. Skimmed
milk was prepared by centrifuging aliquots of the whole milk at 3,000 g for 10
min at 4°C.
3.1.4. RSM from untreated and irradiated LHSMP (Publication IV)
A part of LHSMP (Valio Ltd.) was irradiated by gamma rays at 10 kGy using a
dosimetric system GEX WinDose (Centennial, CO, USA). Untreated LHSMP
and irradiated LHSMP (irrLHSMP) were reconstituted in distilled water to yield
a final concentration of 10% (w/v) milk solids, mixed thoroughly for one hour,
heated at 90°C for 30 min and cooled to 40°C immediately before experiments,
resulting in regular RSM or RSM prepared from irrLHSMP (irrRSM) ready for
inoculation.

3.2. Starter cultures and inoculation rates (Publication II and IV)

Liquid bulk starter containing Streptococcus (St.) thermophilus and

Lactobacillus (Lb.) delbrueckii subsp. bulgaricus (CH XY-11, Chr. Hansen,
Hørsholm, Denmark) was used at inoculation rate of 1% in Publication II.
St. thermophilus ST12 (ST12; Chr. Hansen) was used in Publication IV. Milk
samples were inoculated at 1% (v/v). Inoculation rate of 105 cfu mL-1 was used
in dynamic rheological and front-face fluorescence measurements during
acidification. Preparation of the inoculum is provided in detail in Publication IV.

3.3. Analysis of milk composition (Publication I)

3.3.1. General characteristics of milk

Analysis of fat, protein, lactose, freezing point and urea of milk of individual
dairy farms was performed with infrared spectrometry, according to IDF
standard 141C:2000 (International Dairy Federation, 2000), using a MilcoScan
(Foss Electric, Hillerød, Denmark), somatic cell count (SCC) was determined
using a Fossomatic (Foss Electric), and total bacterial count (TBC) was
determined using a BactoScan FC (Foss Electric) in Milk Analysis Laboratory of
Estonian Animal Recording Centre (Tartu, Estonia). The pH of all milk samples
was also measured.
3.3.2. Analysis of soluble cations and anions in rennet whey
Rennet whey was separated from the coagulated milk samples, prepared as
described in section 3.6.4, after the measurements of curd firmness. Rennet
whey was filtered and mixed 1:1 with isopropanol, centrifuged at 14,000 g for 5
minutes and diluted with the proper eluent before analysis. A high-performance
liquid chromatographic system (Waters Corporation, Milford, MA, USA)
equipped with Waters 1515 isocratic pump and Waters 432 conductivity detector

32
was used for determination of ions. Cations were separated on Waters IC-Pac C
column (3.9 x 150 mm) at 25°C and eluent 3.0 mM nitric acid, flow rate 1.0 mL
min-1. Anions were separated using Waters IC-Pac A column (4.6 x 50 mm) at
25°C; eluent lithium borate/gluconate containing n-butanol and acetonitrile, flow
rate 1.2 mL min-1.
3.3.3. Capillary electrophoresis of milk proteins
Protein fractions of milk were analysed by capillary electrophoresis (CE)
according to the method described for milk by Ardö and Polychroniadou (1999).
CE analyses were carried out with a Beckman P/ACETM MDQ instrument
controlled by 32 KaratTM version 8.0 software (Beckman Coulter, Inc., Brea,
CA, USA). The separations were performed using a neutral PVA coated
capillary column (Agilent Technologies Finland OY, Espoo, Finland) of 40 cm
effective length and 50 μm i.d., with a slit opening of 100 x 800 μm, at a
temperature of 45°C with a linear voltage gradient from 0 to 20 kV in 3 min,
followed by constant voltage of 20 kV. The samples were injected by pressure
injection at 3.4 kPa for 20 s. Protein fractions were detected by UV absorbance
at 214 nm.
Identification of proteins was done by injection of milk protein standards (α-
LA, β-LG, κ-, β- and αs-CN; Sigma, St. Louis, MO, USA) and by comparison of
the electropherograms obtained with those presented in the literature (Otte et al,
1997; Miralles et al., 2003; Heck et al., 2008). The six major protein fractions
(α-LA, β-LG and αs1-, αs2-, β-, κ-CN) were detected and quantified.
Concentrations of the protein fractions were expressed via relative concentration
(Heck et al., 2008):

100% ,
∑

where Cx is the relative concentration of fraction x (%), Ax – the area in the

electropherogram of fraction x, tx – the migration time of fraction x and n – the
total number of identified peaks that comprise 100% of the area. The κ-CN
exists in milk in one major carbohydrate-free form (Farrel et al., 2004), which is
separated well by CE and comprises ~50% of the total κ-CN peak area (Otte et
al., 1997), and at least six minor fractions, which migrate at the same time as
other caseins and are not separated by CE in milk. The αs2-CN has multiple
phosphorylation states (Farrel et al., 2004), and the major fraction with 11
phosphates (αs2-CN-11P) comprises also near 50% of the total αs2-CN area
(Heck et al., 2008). Only the major fractions of κ- and αs2-CN were quantified in
Publication I, therefore, to obtain an adequate relative protein composition of
raw milk, the integrated areas of identified major peaks of κ- and αs2-CN were
multiplied by two.

33
 3.4. Rennet coagulation of milk (Publication I and III)

Samples of milk were equilibrated in glass tubes at 35°C (Publication I) or at

30°C (Publication III) for at least 30 min using a circulating water bath, followed
by addition of 1% (v/v) 10 g L-1 (Publication I) or 2.5, 5 or 10 g L-1 (Publication
III) aqueous solutions of chymosin (Sigma, St. Louis, MO, USA).
After the addition of rennet, samples were mixed by rapid inversion of test
tubes three-five times. The rennet flocculation time was determined by visual
observation (Publication I and Publication III) or coagulated milk samples were
transferred to the rheometer’s cup or fluorimetric optical cell for immediate
rheological or fluorescence measurements (Publication III). In Publication III,
each milk sample was divided into aliquots and renneted separately for visual
determination of rennet flocculation time, dynamic rheological or fluorescence
measurements.

3.5. Dynamic characterisation of bacterial growth and milk

coagulation

3.5.1. Microcalorimetry (Publication II)

Isothermal batch microcalorimeter TAM III (Thermometric, Järfälla, Sweden)
was used to determine the starter growth-related heat evolution during
fermentation at 40°C. After addition of the starter culture, samples were stirred
and transferred into the autoclaved ampoules. In the exponential growth phase
the relationship between biomass concentration (X) and specific growth rate (µ)
may be described by the first order kinetics dX/dt = μX. Assuming that the rate
of biomass formation (dX/dt) is proportional to the rate of heat production
(dQ/dt), maximum specific growth rate (µmax) of the starter was calculated from
power-time curves as a slope of ln dQ/dt over time (t) (Maskow & Babel, 2003):

ln ln

3.5.2. Dynamic rheological measurements (Publication II, III and

IV)
Low-amplitude oscillatory measurements were conducted using a Physica MCR
301 rheometer (Anton Paar GmbH, Graz, Austria) with the direct strain
oscillation option, the Peltier temperature control unit C-PTD200 and coaxial
cylinder measuring system CC27 (outer and inner diameters 28.92 and 26.66
mm, respectively).
After the addition of starter (Publication II and Publication IV) or rennet
(Publication III), milk samples were stirred and an appropriate volume was
transferred into the measuring system. In Publication II and Publication IV the
gelation assays were performed at 40°C and followed for up to 24 h. In

34
Publication III milk was coagulated at 30°C and rheological parameters were
determined in 10 s intervals until coagulation occurred. Rheological parameters
were determined in oscillation mode at a frequency of 0.1 Hz (Publication II and
Publication IV) or 1 Hz (Publication III) and the strain of 0.01, which was within
the linear viscoelastic region and did not perturb the coagulation process. The
elastic modulus (G'), viscous modulus (G'') and loss tangent (tan δ) were
recorded during the measurements.
3.5.3. Front-face fluorescence spectroscopy (Publication III and IV)
The Instant Screener® Compact spectrofluorimeter (LDI Ltd., Tallinn, Estonia)
equipped with pulsed xenon lamp was used for the front-face fluorescence
measurements of renneted or acidified milk samples. Measurements were
performed in a 10-mL optical cell with quartz bottom window. The spectral
fluorescence signatures (SFS) were recorded as a matrix of fluorescent
intensities depending on excitation and emission wavelengths (λex and λem,
respectively) resulting in 3D records.
In Publication III the SFS were recorded at 30°C every 10-11 s after rennet
addition throughout coagulation. The fluorescence signal was measured near
maximum emission of tryptophan at λex ranging from 280 to 283 nm and λem
ranging from 325 to 328 nm with 1 nm slits.
In Publication IV the SFS were recorded at 40°C from milk samples
inoculated with ST12 every 15 min throughout the fermentation. The SFS were
recorded changing λex in the range 230-400 nm and λem in the range 250-615 nm,
with excitation and emission slits set at 5 nm.

3.6. Determination of coagulation time parameters and

characterisation of rennet-induced gels

3.6.1. Rennet flocculation time (Publication I and III)

The rennet flocculation time (RFT) of milk was measured visually using a
modified Berridge method (Berridge, 1952). After the addition of rennet,
samples were gently stirred in a water bath at 35°C (Publication I) or at 30°C
(Publication III) until RFT was determined as the time from rennet addition until
the formation of the first visible flocks of coagulated protein on the walls of the
glass tube.
3.6.2. Coagulation time parameters determined from dynamic
rheological measurements (Publication III)
Based on dynamic rheological measurements, the time indicating the start of
aggregation of casein micelles was derived from the time when G" started to
deviate from the baseline (tG") in coagulating milk; the gelation time (tg) was
defined as time needed to reach the crossover point of G' and G".

35
 3.6.3. Coagulation time derived from fluorescence measurements
(Publication III)
For smoothing the raw tryptophan fluorescence intensity data and estimation of
possible coagulation time parameters from the fluorescence patterns an
algorithm was developed in MatLab version R2009a (The MathWorks, Inc.,
Natick, MA, USA), in which a Savitzky-Golay smoothing filter was used to
reduce the amount of high frequency noise component in the signal (Savitzky &
Golay, 1964). The Savitzky-Golay filter was chosen, as it tends to preserve
features of the original distribution such as relative maxima, minima and width
that are required for precise estimation of coagulation times. For details see
Publication III.
The filtering was followed by calculating the derivatives of the filtered
signals and finding local extreme points. Exact point corresponding to
coagulation time was chosen by comparison of tryptophan fluorescence intensity
data to coagulation time parameters determined by reference methods as
described above. The time to the minimum of the second derivative of
tryptophan fluorescence profile (t2min) was proposed to be used as a coagulation
time parameter.
3.6.4. Curd firmness (Publication I)
Rennet gels were prepared by addition of 100 L of a 10 g L-1 chymosin solution
to 10 mL of skimmed milk (3,000 g at 10C for 10 min) previously equilibrated
at 35°C for 30 min. Skimmed milk was used to eliminate the effect of fat content
on coagulum strength. Gels were set in plastic tubes with inner diameter of 22.5
mm at 30°C for 50 min and equilibrated at 22°C for 10 min before measurement.
Penetration test with 10-mm displacement at a crosshead speed of 1 mm s-1 was
performed using a TA.XT2i Texture Analyzer (Stable Micro Systems Ltd,
Godalming, UK), equipped with a 5 kg load cell and a P0.5 cylinder ebonite
probe and operated using Texture Expert Exceed software. The firmness of pre-
set gels was determined as the force (g) at structure breaking, defined as the first
significant discontinuity produced in a force-time curve.

3.7. Determination of gelation time and characterisation of acid-

induced milk gelation (Publication II and IV)

Based on dynamic rheological measurements, the gelation time (tg) was defined
as the time necessary for the rheometer response (torque) to reach the value of
0.01 Nm, considered to be the first reliable increase in viscosity that exceeded
the signal noise indicating that the milk started to gel. A gelation rate was
obtained from the maximum rate of increase in G' over time, (dG'/dt)max. The
structure of acid gels formed was described by the values of G', G'' and tan δ
measured 6 h after the start of gelation.

36 
 
4. RESULTS AND DISCUSSION
The results of this dissertation are presented and discussed in four sections,
which correspond to four publications this work is based on.

4.1. Technological quality of raw bulk milk in Estonia

(Publication I)

A survey on raw milk microbiological quality (Stulova et al., 2010) and

chemical composition together with renneting characteristics (Publication I) was
conducted in 2004-2007 using the bulk milk samples collected from farms
located in 7 of the 15 counties of Estonia, covering more than a half (53%) of the
total land area, and thus, was regarded as representing milk quality in Estonia.
The study on the microbiological quality (Stulova et al., 2010) showed that
although overall bacterial numbers in raw milk had decreased significantly in the
past decades, the numbers of technologically harmful bacteria (psychrotrophic
and sporeforming species) increased over lactic acid bacteria (LAB), which
might potentially cause problems in case of usage of milk for cheese production.
The primary objective of Publication I was to evaluate the rennetability of
Estonian raw bulk milk by the direct measurement of coagulation characteristics
(rennet flocculation time – RFT and curd firmness), to establish seasonal and
non-seasonal variations and to find correlations between these properties and
milk composition. The comparison of the properties of milk sampled at the
farms and the same milk received at the dairy, which had undergone mechanical
perturbations as pumping and refrigerated transportation in tank trucks, was also
carried out.
4.1.1. Milk renneting properties
Milk pH is known to affect the first stage of milk renneting – an enzyme-
catalyzed reaction as well as nonenzymatic phase of rennet coagulation (Fox et
al., 2000). The mean pH of Estonian bulk milk obtained from individual farms
has become 0.1 units higher in the past two decades: in 2004-2007 the mean pH
was 6.76 ± 0.04 (Table 4) and in 1984-1991 – 6.67 ± 0.03. The mean pH of raw
bulk milk in Estonia in 2004-2007 was also 0.05-0.1 units higher than reported
for milk from individual cows in UK (Tsioulpas et al, 2007a) or bulk milk
analysed in France, Sweden and Italy (Martin et al, 1997; Lindmark-Månsson et
al, 2003; Malacarne et al., 2006). Taking into account rather strong negative
correlation between milk pH and curd firmness, r = -0.49 (see section 4.1.5),
higher losses of curd fines in whey during vat process and lower cheese yield
could be expected with the increase in the pH of raw bulk milk.
Higher pH of milk has been associated in the literature with the increased
somatic cell count (SCC) of over 800,000 cells mL-1 (Moslehishad et al., 2010).
Only one sample exceeded that boundary and the mean SCC (277,000 cells mL-1)
was three times lower than the critical level in bulk milk collected from

37 
 
Estonian farms in 2004-2007. Indeed, the microbiological quality of milk has
definitely improved in the last two decades in Estonia (Stulova et al., 2010).
However, even two decades ago the raw milk quality was not so poor that this
could have been assumed to be the reason for observing lower pH than in 2004-
2007. Dairy farms in Estonia have been traditionally rather big and raw milk has
been usually stored at the farm not more than 24 h after milking. Hence, the
LAB had not have sufficient time to grow to considerable numbers in cooled
milk to produce enough lactic acid to decrease milk pH by 0.1 units in the past in
comparison with the situation in 2004-2007. Furthermore, we did not find any
correlation between pH and total bacterial count (TBC) in bulk milk. The LAB
counts were also determined in all received samples during the survey (data not
included in Publication I) and no correlation was found between pH and LAB
counts. Thus, it was assumed that the observed increase of average milk pH
could be associated with the changes in feeding strategies of cows (increased
share of concentrates) as a result of industrialisation of dairy farming.
Table 4. The renneting properties of raw bulk milk samples collected from
individual farms and pooled milk samples collected from trucks and dairy silos in 2004-
2007 in Estonia. The p-values characterize the difference between the mean values of
bulk milk from individual farms and pooled milk from trucks and silos.
Parameter Raw bulk milk collected from Pooled milk from p-value
individual farms trucks and dairy silos
Mean ± SD Range Mean ± SD
Min Max
value value
pH 6.76 ± 0.04 6.56 6.86 6.76 ± 0.05 NS
RFT (min) 5.45 ± 1.06 2.07 10.35 6.48 ± 1.11 <0.001
Curd firmness (g) 21.5 ± 4.5 9.7 36.4 23.5 ± 5.1 <0.01

The direct milk renneting characteristics of the individual milk samples

analysed in 2004-2007 varied in a wide range: values of RFT varied five-fold
and curd firmness – more than three-fold (Table 4). Although curd firmness of
the milk samples from different farms varied to a great extent, pooling of milk
decreased the variability a lot, i.e. firmness of the curd obtained from the milk
from dairy silos varied from 19 to 27 g depending on the sampling occasion.
Only one bulk milk sample from one sampling did not result in any measurable
coagulum during the survey; milk from that farm produced coagulum with
satisfactory time and of good firmness in all other sampling days.
The mean values of renneting properties characterizing individual farms were
calculated for the dairy farms sampled at least three times during the observed
period. The greatest variation was observed in case of curd firmness: the mean
values for bulk milk of individual farms differed two-fold from 14 to 29 g. The
mean RFT for milk of different farms varied from 4.5 to 6.8 min, and mean pH
varied from 6.68 to 6.81.

38 
 
 4.1.2. Seasonal variations in milk general composition and renneting
properties
Remarkable seasonal variations in milk composition and renneting properties
were found in Estonian farm-tank milk (Table 5). The mean protein and fat
contents in Estonian milk were the lowest in summer and the highest in autumn.
Although the climate, calving patterns and feeding strategies are relatively
similar in Estonia compared to the Netherlands and Sweden (Heck et al., 2009;
Lindmark-Månsson et al., 2003), Estonian milk showed somewhat different
seasonal changes in the two main components, protein and fat. Regarding the
entire data set presented in Table 5, milk with the best cheese-making properties
(low pH, low TBC, high protein and fat, firmer coagulum) was produced in
autumn. On the other hand, the shortest mean RFT in Estonian milk was
recorded in summer; the lowest mean urea and the highest mean SCC were also
observed in summer (Table 5). The highest mean urea was observed in spring,
which is in line with the results of Grimley et al. (2009) showing elevated urea
concentrations in milk during turn-out from dry feed to spring pasture.
Table 5. The mean ± SD values of the general characteristics and renneting
properties of raw bulk milk samples collected from individual farms through seasons.
The p-values characterize the difference between the mean values obtained in case of
milk samples collected during different seasons.
Parameter Season when raw bulk milk samples were collected p-value
Winter Spring Summer Autumn
General characteristics
Protein (g 100 g-1) 3.31 ± 0.13 3.27 ± 0.16 3.25 ± 0.13 3.41 ± 0.17 <0.001
Fat (g 100 g-1) 4.17 ± 0.25 4.14 ± 0.27 3.98 ± 0.20 4.30 ± 0.27 <0.001
Lactose (g 100 g-1) 4.73 ± 0.10 4.78 ± 0.08 4.77 ± 0.07 4.67 ± 0.10 <0.001
Urea (mg L-1) 254 ± 58 268 ± 65 220 ± 53 231 ± 68 <0.001
Freezing point (°C) -0.528 ± -0.528 ± -0.526 ± -0.527 ± NS
0.005 0.005 0.004 0.005
TBC (103 cfu mL-1) 33 ± 96 33 ± 79 34 ± 81 19 ± 15 NS
SCC (103 cells mL-1) 266 ± 89 273 ± 105 300 ± 93 287 ± 134 <0.05
Renneting properties
pH 6.77 ± 0.04 6.77 ± 0.04 6.74 ± 0.03 6.72 ± 0.05 <0.001
RFT (min) 5.65 ± 0.99 5.37 ±1.08 5.35 ± 0.93 5.54 ± 1.23 <0.01
Curd firmness (g) 20.0 ± 4.1 20.6 ± 3.2 22.0 ± 4.3 24.0 ± 4.9 <0.001

Principal component analysis (PCA) was applied to visualise the

differentiation of farm milk quality by seasons. Principal component one (PC1;
21.4% of the total variation) distinguished autumn milk (higher scores) from
summer milk (lower scores). High PC1 values were related mainly to high
firmness of the rennet curd and higher protein and fat content of the milk. The
best discrimination of the samples between the seasons was observed in the plot
of principal component two (PC2, 13.4% of the total variation) versus principal
component three (PC3, 12.7% of the total variation) (Figure 3). Autumn samples

39
scored positively on PC2 and PC3 axes; spring and winter milk samples were
shifted to the negative PC2 values; and summer milk samples were shifted to the
positive PC2 and negative PC3 values. Hence, autumn milk was characterized
by higher fat, protein, longer RFT, and lower pH; conversely, spring samples
were discriminated due to higher pH. Summer milk showed lower pH, lower fat,
protein, and RFT.
5 0.6
0,6
a 2
1
b
0 fat freezing
-1 0,4
0.4
3 -2 RFT point

Loadings PC3 (12.7%)

-2 -1 0 1 2 protein log SCC
Scores PC3 (12.7%)

0.2
0,2 curd
1 pH firmness
0,0
0.0
-1
-0.2
-0,2 log TBC

-3 lactose urea
-0,4
-0.4

-5 -0.6
-0,6
-5 -3 -1 1 3 5 -0.6
-0,6 -0.1
-0,1 0.4
0,4
Scores PC2 (13.4%) Loadings PC2 (13.4%)

Figure 3. Scores (a) and loadings (b) of the second (PC2) and third (PC3) principal
components obtained by PCA of general composition and renneting properties of raw
bulk milk samples collected from individual farms in spring (■), summer (∆), autumn
(○) and winter (x). Averages (“centres of gravities”) of the PCA scores of milk collected
in different seasons are presented in the insert.
What are the possible reasons of seasonal differences in milk composition
and the renneting properties? The calving of cows is distributed in Estonia
evenly throughout the year (Estonian Animal Recording Centre, 2010). Hence,
changes in milk composition could be explained by certain changes in cows’ diet
in warm and in cold months – more concentrates in winter and more fresh fodder
in summer. The majority of milk (55.2%) is produced by the herds of a size
ranging from 100 to 600 cows (Estonian Animal Recording Centre, 2010),
which are predominantly held in the barn throughout the year. Still, the majority
of cows receive fresh grass as a part of their diet in summer independently of
whether the herd is freely grazing or held indoors. The transition from forage
feeding to free pasturing (if any occurs) takes place in late spring, and the
grazing period lasts normally until the end of September.
It was clearly shown in Publication I for the first time that the renneting
properties of milk also displayed a seasonal pattern – the seasonal variability has
been reported earlier only for the chemical composition of milk (Heck et al.,
2009; Lindmark-Månsson et al., 2003).
4.1.3. Stability of the quality of milk produced in Estonia
Cheese producers can take into account seasonal changes in milk quality, but
random fluctuations in milk composition, not related to season, cannot be
forecasted. Farms engaged in the survey and sampled at least three times in the
period 2004-2007 were evaluated for the stability of the composition and

40 
 
renneting characteristics of the milk they produced. The quality of milk was
considered variable if the values of standard deviation (SD) calculated for a farm
were higher than the SD calculated for all analysed milk samples throughout the
whole period.
More than half of the farms produced milk with the uniform quality
concerning cheesemaking, showing stable protein, fat, pH and rennet curd
firmness during the observation period. The quality of milk was variable in
42.5% of the farms concerning RFT, and in 20-25% of the farms concerning fat,
urea content and curd firmness; 13% of the farms were variable in protein and
7% in lactose content of milk. Large variations in the composition of milk
produced by the same farm indicated most probably the lack of the proper
management of cows feeding.
4.1.4. Comparison of raw bulk milk samples from individual farms
with pooled milk from tank trucks and dairy silos
The mean relative concentrations of whey proteins and caseins, as well as their
ratios were significantly different in milk from tank trucks and dairy silos in
comparison with the samples from individual farms (p < 0.05) (Table 6).
Table 6. The relative protein composition of raw bulk milk samples collected from
individual farms and pooled milk samples from trucks and dairy silos in one batch. The
p-values characterize the difference between the mean values of bulk milk from
individual farms and pooled milk of the same milking collected from trucks and silos.
Component Raw bulk milk collected from individual Pooled milk p-value
farms from trucks
and dairy silos
Mean ± SD Range Mean ± SD
Min. value Max. value
α-LA (%) 2.8 ± 0.4 1.7 3.7 2.9 ± 0.6 NS
β-LG (%) 8.8 ± 1.2 6.9 12.0 10.1 ± 1.3 <0.05
αS1-CN (%) 26.5 ± 1.4 23.7 29.9 26.7 ± 1.4 NS
αS2-CNa (%) 5.3 ± 0.7 4.0 7.1 6.2 ± 0.4 <0.001
κ-CNb (%) 10.0 ± 1.0 7.9 12.1 9.5 ± 0.6 <0.05
β-CN (%) 37.1 ± 1.4 30.9 38.9 35.3 ± 2.1 <0.05

WP (%) 11.7 ± 1.4 9.3 15.5 13.0 ± 1.8 <0.05

CN (%) 78.9 ± 1.4 75.1 81.3 77.6 ± 1.8 <0.05
WP:CN 0.148 ± 0.021 0.115 0.206 0.168 ± 0.028 <0.05
a
recalculated assuming that the major fraction comprises 50% of the total αS2-CN area
b
recalculated assuming that the major fraction comprises 50% of the total κ-CN area

Notably higher mean TBC in pooled milk (45,000 cfu mL-1) than in milk
obtained from separate farms (32,000 cfu mL-1) was also observed, however, the
difference was not statistically significant (p > 0.05). Psychrotrophic bacteria
have been reported as predominant in Estonian raw milk (Stulova et al., 2010).
These bacteria also possess considerable proteolytic activity on milk proteins

41
(Ercolini et al., 2009), and they can activate plasmin activators under cheese-
making conditions (Frohbieter et al., 2005) enhancing further degradation of β-
casein by plasmin. A protease isolated from Pseudomonas fluorescence, the
most common species of psychrotrophic bacteria found in milk, was shown to
hydrolyse preferentially κ- and β-CN if incubated with artificial micelles (Koka
& Weimer, 2000). Lower relative total casein content in pooled milk from tank
trucks and dairy silos and essentially lower content of κ- and β-CN fractions (see
Table 6) refer to a certain degree of proteolysis that could have taken place in
pooled milk during transportation to the dairy. The appearance of para-κ-CN,
which has a migration time close to that of β-LG, has been discovered in raw
bulk milk after few days of refrigerated storage, causing an overestimation of the
β-LG fraction in milk if proteolysis had taken place (Miralles et al., 2003).
Similarly, our results showed significant increase in estimated β-LG fraction in
pooled milk from tank trucks and dairy silos compared to bulk milk collected at
the farms (p < 0.05).
Pooled milk from tank trucks and dairy silos had one minute longer mean
RFT (p < 0.001), which is almost 20% longer than the mean RFT value
measured for the bulk milk from individual farms (see Table 4). We propose that
the action of proteases originating from psychrotrophic bacteria on κ-CN located
on the surface of the casein micelles could have affected the affinity of the
chymosin binding sites for rennet action. In contrast, the mean curd firmness
turned to be even slightly higher (p < 0.01) if prepared from pooled milk.
However, considering high overall variability of curd firmness among individual
farm milk samples and among samples from tank trucks (differences between
boundary values of 27 and 23 g, respectively), and among milk from dairy silos
(difference of 8 g), the difference of nearly 2 g between the mean curd firmness
of farm milk and the pooled milk from tank trucks and silos seemed to be rather
inessential. It should be noted, however, that as the amounts of milk collected
from every farm, truck or silo were not taken into account in the calculation of
the means in the present study (the mean values were not weighted), the
conclusions drawn could not be considered completely impeccable.
Differences in the relative protein composition and properties of bulk milk
obtained from individual farms and pooled milk from tank trucks and dairy silos
provides us with awareness that, in addition to the original milk composition and
microbiological quality, the factors encountered after milking at the farm and
before processing at dairy plant, such as refrigerated transportation of milk to
dairy silos, milk vigorous pumping, occurrence of disinfectant residues etc., may
become also important in determining milk coagulability.
4.1.5. Correlation between chemical composition and renneting
properties of bulk milk
The Pearson’s coefficients between the renneting properties, milk composition
and ions in rennet whey are shown in Table 7. The pH of milk correlated
negatively with curd firmness and protein content and positively with RFT (p <

42
0.001). The pH of milk also correlated significantly with ions determined in
rennet whey: negative correlations with Na+, K+ and Cl- were observed. The
RFT did not correlate significantly with any of the determined parameters except
pH, and only a small negative correlation with urea was also observed. A high
positive correlation was observed between curd firmness and protein content (p
< 0.001). Correlations between curd firmness, and fat and lactose content were
also noticed. However, this was probably due to the close relationship of fat,
lactose and protein contents. The positive correlation between curd firmness and
K+ content in rennet whey was also established (p < 0.05). The relative κ-CN
composition correlated with curd firmness even more than the total protein
content, r = 0.45 (p < 0.001). These results obtained in case of bulk milk
correspond to those reported by Wedholm et al. (2006) based on milk from
individual cows, which showed the association of low concentration of κ-CN
with poor clotting properties of milk, resulting in weak curd and lower cheese
yield. The results of Publication I have shown that correlations between milk
composition and technological properties exist not only in milk from individual
cows but also in pooled milk utilised for processing.
Table 7. Correlation matrix of Pearson’s coefficients of milk renneting properties,
general and relative protein composition, and ions in rennet whey.
pH RFT Curd firmness
Renneting properties
pH 1.00
RFT 0.17*** 1.00
Curd firmness -0.49*** -0.07 1.00
Milk general characteristics
Protein -0.13*** 0.01 0.41***
Fat 0.07* -0.01 0.24***
Lactose 0.08* 0.00 0.19***
Urea 0.02 -0.09** 0.03
Freezing point 0.02 0.06 -0.15**
TBC -0.06 -0.03 0.02
SCC 0.01 0.00 0.01
Ions in rennet whey
Na+ -0.32*** 0.10 0.11
K+ -0.52*** -0.06 0.19*
Cl- -0.20* 0.12 -0.03
Relative protein composition
α-LA 0.06 -0.19 0.31
β-LG 0.02 0.05 -0.17
αS1-CN 0.24 -0.07 -0.11
αS2-CN -0.23 -0.07 0.04
κ-CN -0.10 -0.23 0.45**
β-CN -0.09 0.29 -0.18
WP:CN 0.04 -0.02 -0.05
* correlation is significant at the 0.05 level; ** correlation is significant at 0.01 level;
*** correlation is significant at 0.001 level

43
 4.2. Effect of H2O2 on the growth of starter bacteria and structure
of acid gels (Publication II)

The retardation of the growth of starter bacteria by H2O2 may take place via the
following paths: 1) direct action on microbial cells; 2) activation of
lactoperoxidase (LPO) system; 3) oxidation of substrates – oxidation of vitamins
or amino acids, cross-linking of proteins making them unavailable for bacteria,
or accumulation of toxic compounds, e.g. lipid oxidation products. The direct
action of H2O2 on the cells of LAB can be precluded or at least largely alleviated
by incubating milk treated with H2O2 at elevated temperatures for adequate time,
or using catalase before inoculation – the approaches applied in Publication II to
degrade H2O2. The natural LPO system of milk is inactivated during high
temperature treatment (Kussendrager & van Hooijdonk, 2000); hence, any LPO-
related oxidative or antimicrobial action is not likely to occur in ultra-high
temperature treated (UHT) milk used in Publication II. Therefore, oxidation of
milk proteins, fats etc., and not the direct action of H2O2 on the bacterial cells
was assumed to be the main cause of inhibition of the growth of starters and
formation of gel in UHT milk. A proof for this hypothesis was sought by
exploring the growth of thermophilic starter bacteria in UHT milk by using
isothermal batch microcalorimetry and comparing the results with the
rheological profiles of milk gelation during acidification.
4.2.1. Inhibition of growth of starter bacteria by H2O2
A typical power-time curve of UHT milk samples inoculated with thermophilic
starter containing St. thermophilus and Lb. bulgaricus showed two peaks
corresponding to two growth phases of the starter bacteria (Figure 4). St.
thermophilus may exhibit diauxic growth in milk, displaying two exponential
growth phases separated by a nonexponential phase, during which proteinase is
synthesized; during the first exponential growth phase free amino acids and
small peptides and during second phase caseins are utilized as the source of
nitrogen (Letort et al., 2002). Lb. bulgaricus grows in milk in the presence of St.
thermophilus exhibiting no diauxia (Courtin et al., 2002). Hence, the occurrence
of two peaks on power-time curves could be explained by diauxic growth of St.
thermophilus.
All power-time curves of UHT 3.5% and UHT 0.05% milk samples, treated
with H2O2 at concentrations from 0 to 100 mg L-1 and one hour afterwards
inoculated with thermophilic starter, retained the two-stage growth pattern
(Figure 4a and b). However, increasing the concentrations of H2O2 led to the
prolongation of the lag phase. The maximum specific growth rate (µmax) of the
thermophilic starter decreased significantly in both the first and the second
exponential growth phases (p < 0.05) with the increase of H2O2 concentration at
which milk was treated (see Table 1 in Publication II). In case of 150 mg L-1
H2O2 the heat production associated with the growth of starter bacteria was
initiated only on the second day of experiment in UHT 3.5% and UHT 0.05%.

44
No heat production was recorded during 5 days in case of 200 and 250 mg L-1
H2O2, indicating total inhibition of the growth of the starter bacteria.
0.4
0,4 0.4
0,4
a b 0 mg L-1

0.3
0,3 10 mg L-1
0.3
0,3
0 mg L-1

25 mg L-1 100 mg L-
dQ/dt (mW mL-1)

dQ/dt (mW mL-1)

1
10 mg L-1 100 mg L-1 50 mg L-1
0,2
0.2 0,2
0.2 75 mg L-1
25 mg L-1
50 mg L-1

75 mg L-1
0.1
0,1 0.1
0,1

0,0
0.0 0,0
0.0
0 5 10 15 20 0 5 10 15 20
Time (h) Time (h)
0,4
0.4
c

0,3
0.3 0 mg L-1

10 mg L-1
25 mg L-1
dQ/dt (mW mL-1)

50 mg L-1
0,2
0.2
100 mg L-1
75 mg L-1
200 mg L-1
150 mg L-1
250 mg L-1
0.1
0,1

0,0
0.0
0 5 10 15 20
Time (h)

Figure 4. Mean power-time curves of UHT 3.5% (a) or UHT 0.05% (b) inoculated
milk samples pre-treated with H2O2 at different concentrations one hour before starter
addition. Some aliquotes of UHT 3.5% milk pre-treated with H2O2 were also treated
with catalase before starter addition (c).
Nevertheless, looking carefully at power-time curves of milk samples treated
with higher amounts of H2O2 (over 50 mg L-1), one can see some heat production
in the beginning of the curves, before the first and second peaks associated with
the growth of the bacteria (Figure 4a, b). This comparatively small descending
heat production was also recorded from milk samples containing H2O2 but with
no starter added and correlated with the amount of H2O2. Hence,
abovementioned heat evolution should be an indication of H2O2 decomposition
proceeding further in milk even after inoculation with starter. In contrast to our
initial assumption, incubation of milk for one hour at 40°C was not enough for
added H2O2 to undergo complete decomposition. Moreover, in UHT 3.5% milk

45 
 
samples, treated with catalase one hour after H2O2 addition and prior to
inoculation, the starter had the same lag-phase (Figure 4c) and no change in µmax
in the first exponential growth phase (p > 0.05) compared to the control samples
without treatment with H2O2, in contrast to milk with added H2O2 but lacking
catalase treatment. It was obvious from calorimetrical data that bacteria started
to grow in milk samples not treated with catalase only after H2O2 had been fully
decomposed (Figure 4a, b). Therefore, inhibition of growth of starter bacteria
and prolongation of their lag phase in samples with H2O2 added to milk and not
subsequently treated with catalase was assigned to the direct inhibitory effect of
residual H2O2.
Still, power-time curves of inoculated milk samples, pre-treated for one hour
with H2O2 at different concentrations and followed by catalase treatment before
inoculation, showed rather modified patterns compared to control ones (Figure
4c). A significant decrease of the µmax (p < 0.05) in the second exponential
growth phase from 0.8 h-1 in control sample to 0.5-0.6 h-1 after H2O2 pre-
treatment at 150-250 mg L-1 in milk was observed. In other words, the
elimination of residual H2O2 from milk with catalase removed the inhibitory
effect on the first exponential growth phase, but retained the inhibition in the
second exponential growth phase of starter bacteria. Relying on calorimetrical
data, we assumed that the oxidative action of H2O2 in UHT milk was directed
presumably on the components essential for microbial growth particularly during
the second exponential growth phase. Milk contains a wide variety of
components, including proteins that can act like antioxidants and can serve as
the primary target for H2O2 action (Østdal et al., 2000). Protein oxidation may
occur through the oxidation of amino acid side chains or protein backbone
resulting in protein-protein cross-linkage (e.g. through the formation of
dityrosine) or cleavage of the peptide bond (Berlett & Stadtman, 1997; Giulivi et
al., 2003; Takasaki et al., 2005). Recently, it was demonstrated that the treatment
of milk with transglutaminase had a growth-slowing effect on yoghurt starter
bacteria, and it was proposed that low molecular weight peptides and/or amino
acids were cross-linked by the enzyme and became partially unavailable for
bacteria (Özer et al., 2007). Similarly, we assumed that H2O2 caused oxidative
changes in proteins, used as a source of nitrogen during the second exponential
growth phase, diminishing their availability for starter bacteria. Therefore, also
in samples with H2O2 added to milk and not treated with catalase before starter
addition, the retardation of bacterial growth might occur also via the oxidation of
substrate in addition to primary inhibitory effect – direct bacteriostatic influence
of H2O2.
Comparison of the power-time curves recorded in case of UHT 3.5% and
UHT 0.05% milk fermented with thermophilic starter (Figure 4a and b) and also
the data from Table 1 in Publication II showed that the duration of the lag phase
and growth in the first exponential phase were more severely affected in skim
milk than in 3.5% milk. Conversely, second exponential growth phase was more
prolonged on increasing concentrations of H2O2 in 3.5% milk compared to skim

46 
 
milk. The rate of decomposition of H2O2 was reported to be higher in whole
milk than in skimmed milk (Amin & Olson, 1967). Significant increase in
peroxide values in yoghurt produced from H2O2-treated raw milk (Özer &
Atamer, 1999) and in H2O2-treated raw cream (Özer et al., 2000) refer to
possible stimulation of lipid oxidation in high-fat samples. Produced lipid
hydroperoxides may be subsequently decomposed to yield the reactive aldehyde
species that can amplify damage to proteins by alternation of amino acids side
chains (Uchida, 2003). Therefore, the retardation of bacterial growth due to the
oxidation of substrate initiated by H2O2 is more evident to be the case in milk
containing fat than in low-fat milk.
4.2.2. Milk gelation and small deformation rheological properties of
yoghurts prepared from milk treated with H2O2
Gelation curves of UHT 3.5% and UHT 0.05% milk samples, treated with H2O2
at concentrations from 0 to 100 mg L-1 and one hour later inoculated with
thermophilic starter are shown in Figure 5. No change in viscoelastic moduli (no
gelation) was observed in milk samples with H2O2 added in concentrations over
150 mg L-1 during 24 hours.
250 250
a 0 mg L-1 b

200 200
10 mg L-1
25 mg L-1
50 mg L-1
75 mg L-1
150 150
G' and G'' (Pa)
G' and G'' (Pa)

0 mg L-1
25 mg L-1 75 mg L-1
100 mg L-1 50 mg L-1
10 mg L-1 100 mg L-1
100 100

50 50

0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time (h) Time (h)

Figure 5. Mean curves of elastic (G') (■) and viscous (G'') moduli (▲) as a function
of time in inoculated milk samples pre-treated with H2O2 (0 to 100 mg L-1) one hour
before starter addition: a) UHT 3.5% and b) UHT 0.05% milk samples.
A significant prolongation of the time of initiation of gel formation (tg) was
caused by increasing H2O2 concentration in the case of both, UHT 3.5% and
UHT 0.05% milk samples (Table 8). Already the lowest concentration of H2O2
utilized (10 mg L-1) prolonged the lag phase of gel formation for at least one
hour. The tg of milk treated at 100 mg L-1 of H2O2 reached approximately 17
hours, extending the onset of gel formation fourfold compared to the control
samples. Treatment with H2O2 led to the decrease of gelation kinetics (decrease
of (dG'/dt)max) and the decrease of the solid-like character of the gels formed

47 
 
(decrease of elastic modulus – G' and increase of loss tangent – tan δ). We
assumed that the remarkably affected growth of starter bacteria due to residual
H2O2 in milk (Figure 4a and b) was the primary reason for delayed and slowed
down acid production, which consequently postponed the onset of milk gelation
and also might have influenced the texture of yoghurts.
Table 8. The means ± SD of gelation time (tg), storage modulus (G'), loss tangent
(tan δ) and gelation rate ((dG'/dt)max) for control and H2O2 pre-treated UHT 3.5% or
UHT 0.05% samples treated or not with catalase before starter addition.
Milk H2O2 (mg L-1) Catalase tg (h) G' (Pa) tan δ (dG'/dt)max (Pa h-1)
UHT 0 - 4.6a 209a 0.191a 64.9a
3.5% 10 - 6.4b 173ab 0.194a 51.2b
25 - 7.4c 156bc 0.200a 43.7bc
50 - 9.4d 128c 0.221b 33.3cd
75 - 12.7e 115c 0.224b 29.7d
100 - 16.9f 145bc 0.203a 45.2bc
UHT 0 - 4.3a 135a 0.250a 37.9a
0.05% 10 - 5.7b 95a 0.265a 22.7a
25 - 6.4c 115a 0.261a 29.3a
50 - 7.9d 98a 0.273a 23.7a
75 - 11.2e 68a 0.301b 17.4a
100 - 17.2f 100a 0.256a 28.0a
UHT 0 + 3.2a 237a 0.182a 89.6a
3.5% 75 + 3.3a 239a 0.186a 79.2a
100 + 3.6a 226a 0.187a 72.6a
150 + 3.8a 242a 0.187a 82.4a
200 + 3.8a 233a 0.186a 79.3a
250 + 4.0a 232a 0.188a 74.7a

The (dG'/dt)max and G' decreased in UHT 3.5% samples to the minimum
value on increasing the H2O2 concentration in milk up to 75 mg L-1, and resulted
in almost twofold weaker gels compared to control sample (Table 8). Milk
samples pre-treated with H2O2 at the level of 100 mg L-1 one hour before starter
addition exhibited slightly higher G' values again, but still did not reach the
values of the control sample. Both, high and low fat milk samples exhibited an
increase in tan δ at 75 mg L-1 of H2O2 added into milk one hour before
inoculation, and then diminished at concentration of 100 mg L-1 up to the level
in control gels.
The dynamic rheological properties of gels produced from low fat samples
were less influenced by H2O2 addition compared to milk with higher fat content
(Figure 5, Table 8). The (dG'/dt)max and G' 6 h after onset of gelation showed in
UHT 0.05% milk similar dependence on H2O2 concentration as in 3.5% UHT
samples, but the differences were not statistically significant according to
ANOVA (p = 0.084 and p = 0.068, respectively).

48
 It could be assumed that the stronger negative influence of H2O2 pre-
treatment of milk exerted on the rheological characteristics of gel observed in
case of UHT 3.5% could be explained by more intensive protein oxidation taken
place in high-fat milk which might hinder proper gel structure formation in
addition to ‘simple’ deterioration of gel formation because of reduced rate of
acid production by starter bacteria. Nevertheless, UHT 3.5% milk samples
incubated with H2O2 (10 to 250 mg L-1) for one hour and subsequently treated
with catalase prior to inoculation, i.e. milk samples free of residual H2O2,
showed a negligible increase in tg compared to the control ones (p = 0.146) and
did not exhibit any significant effect on (dG'/dt)max or rheological characteristics
of gels (G', G'', tan δ) 6 hours after the onset of gelation (p > 0.05) (Table 8).
Thus, our assumption, considering enhanced weakening of gel structure in H2O2-
treated high-fat milk due to the changes in milk proteins participating in gel
matrix formation, was not supported.
It was shown in Publication II that even low concentrations of H2O2 led to the
prolongation of initiation time of gelation and deterioration of gel quality, acted
presumably mainly through the direct inhibition of starter bacteria by residual
H2O2. Milk samples pre-treated with H2O2 and then with catalase, ensuring
complete removal of the oxidant, showed nearly the same gelation times and
overall rheological characteristics as in the control indicating lack of influence of
pre-treatment on the formation of protein gel matrix. The data obtained indicated
that H2O2 did not alter the structure of milk proteins determining coagulation
processes but rather changed the bioavailability of amino acids included in the
peptides and proteins for starter bacteria. Nevertheless, in milk treated with H2O2
followed by catalase, modified growth profile of yoghurt starter bacteria
recorded during second exponential growth phase may potentially result in
yoghurt with e.g. altered flavour or other defects, even though gel properties
would not be affected.

4.3. Front-face fluorescence spectroscopy monitoring rennet-

induced coagulation (Publication III)

A novel front-face fluorescence spectroscopy method was introduced and

applied to investigate the rennet coagulation of reconstituted skimmed milk
(RSM) and raw bulk milk. Tryptophan fluorescence intensity patterns allowed us
to distinguish different phases during the enzymatic coagulation of milk, and
determine the specific time corresponding to the onset of aggregation of casein
micelles.
4.3.1. Characteristic tryptophan fluorescence intensity profiles
during rennet coagulation
Typical tryptophan fluorescence intensity profiles recorded during rennet
coagulation of different types of milk (RSM, skimmed and whole raw bulk milk)
are presented in Figure 6. The general pattern of the evolution of tryptophan

49
fluorescence during rennet coagulation of RSM (Figure 6a) was rather similar to
that reported by Lopez and Dufour (2001). However, the experiments performed
with RSM with addition of different amounts of CaCl2 and with different
concentrations of enzyme used to induce coagulation indicated that the
fluorescence profiles can look quite different from those reported earlier.
Moreover, the behaviour of tryptophan fluorescence observed in bulk milk
(Figure 6b and c) was clearly different from that measured in RSM – instead of
an overall descending character of the signal in RSM, ascending profile was
observed in bulk milk during coagulation within the time span of the
measurements. However, if the fluorescence response during renneting of bulk
milk was recorded for longer period, a decline in tryptophan emission could also
be observed similarly to that obtained in RSM. Instead of initially descending
signal observed in skimmed bulk milk (as well as in RSM), tryptophan
fluorescence rose quickly in the whole milk during the first minutes of renneting,
but the further profile was very similar to that of skimmed bulk milk.
In Publication III we divided the tryptophan fluorescence profiles recorded
during rennet-induced coagulation into four stages and tried to relate the changes
in measured signal with combined result of release of tryptophan fluorescence
and factors that quench the effect, as briefly described below.
1. After the addition of rennet, a decrease of tryptophan fluorescence is seen
(but only in the case of skimmed milk – Figure 6a and b).
The quenching substances – Cys-Cys bridges of chymosin and Asp
residues located in the active site of the enzyme (Gilliland et al., 1990) – get
closer to the tryptophans of κ-CN located on the surface of the micelles and
therefore can reduce their fluorescence. The process is gradual and
sequential because, firstly, the number of enzyme molecules is far less than
potential cutting sites on the surface of the casein micelles, and secondly,
the enzyme approaches the micelles by diffusion, whose path being partly
located in the hairy layer, which increases the diffusion time (Walstra et al.,
2006).
In non-homogenized whole milk, the creaming effect causes a decrease
in the concentration of fat globules at the bottom of the optical cell, from
there the fluorescence emission is measured in the front-face mode. Photons
emitted by tryptophan can be absorbed by vitamin A contained in fat
globules in the course of resonance energy transfer (Lopez & Dufour, 2001;
Peterson & Rask, 1971) explaining the depression of tryptophan
fluorescence in milk containing fat. During the first 2-3 minutes of
measurement in the whole milk, the increase in tryptophan fluorescence
was observed presumably due to the gradual depletion of vitamin A content
at the bottom of the optical cell with time, not depending whether milk was
being renneted or not. Hence, a decrease in tryptophan fluorescence caused
by successive binding of coagulating enzyme could not be seen in the
whole milk because of counteracting and overbalancing effect of creaming
(Figure 6c).

50
 Figure 6. Tryptophan fluorescence data (●) recorded during rennet coagulation of
milk and their corresponding second order derivatives: a) RSM enriched with 0.01 M
CaCl2, b) skimmed or c) whole raw bulk milk. Renneting was induced by addition of 5
(a) or 10 (b, c) mg mL-1 solution of chymosin at 1% (v/v). Smoothed curves and their
second derivatives are indicated with black solid or dotted lines, respectively. The times
to the minimum of the second order derivative (t2min) are indicated with vertical dashed
lines. Evolution of elastic (G') (♦) and viscous (G") (▲) moduli during rennet
coagulation RSM and skimmed milk are also presented. Time corresponding to initial
increase in G" is defined as tG", time to crossover point of G' and G" is defined as
gelation time (tg). Approximate time of visually determined rennet flocculation time
(RFT) is also indicated.

51
2. Some time later the initial decrease in tryptophan fluorescence stops (if any
occurred) and signal starts to increase (see Figure 6).
The C-terminal part of κ-CN (GMP), removed from the surface of the
micelles by the enzyme action, does not contain any aromatic amino acids
but includes twelve polar (Asp, Glu and Lys) and five amide side chain
groups (Asn and Gln) – potential quenchers of tryptophan fluorescence. In
contrast, the N-terminal part of κ-CN (para-κ-CN), containing one Trp, nine
Tyr and four Phe residues, remains attached to the micelle. Thus, the release
of GMP can cause an increase of fluorescence of aromatic amino acid
residues retained in the structure of para-κ-CN. In addition, the dissociation
of the enzyme-substrate complex after the reaction should also
counterbalance an initial decrease in tryptophan fluorescence.
Eventually, during enzymatic phase of renneting, there are two
counteracting effects influencing fluorescence signal that occur
simultaneously: absorption of chymosin onto the surface of the micelles
causing a decrease, and removal of GMP and release of enzyme resulting in
an increase of tryptophan emission. In skimmed milk, the increase in
tryptophan fluorescence becomes noticeable when the enhancing effect
caused by cleavage overcomes the depression induced by enzyme binding.
3. At some specific point of time, the fluorescence signal starts to decline as in
RSM (Figure 6a), or the slope of the ascending fluorescence curve
decreases markedly as in bulk milk (Figure 6b and c).
If the majority of κ-CN is cleaved, para-κ-CN micelles can approach
each other and start to form small chain-like aggregates (Fox et al., 2000).
The fluorescence of tryptophan residues located near the surface of micelles
can be suppressed by the appearance of additional protein-protein
interactions between aggregating particles. The fluorescence signal showed
clear decrease in case of RSM enriched with CaCl2 and only a reduction of
the slope of the ascending signal in case of bulk milk, which suggests more
intensive aggregation of micelles in RSM which contained more calcium
than raw bulk milk usually did.
4. After reaching the maximum, the fluorescence signal starts to decrease
continuously (in case of bulk milk), or the decline of fluorescence initiated
at the previous stage continues with a different slope (in RSM).
Three-dimensional gel, which is dynamic in its nature, is formed in the
result of aggregation. After the onset of gel formation, further changes in
protein matrix – incorporation of additional caseins, fusion and
rearrangements of particles result in the thickening and hardening of the gel
(Mellema et al., 2002) accompanied by lasting decrease in fluorescence,
which supports a dynamic character of the gel.

52
 4.3.2. Determination of coagulation time parameter by fluorescence
measurements
For better understanding of the changes in the tryptophan fluorescence signal
taking place during renneting, the dynamic rheological measurements were
conducted in parallel with recordings of fluorescence profiles.
The time indicating the start of aggregation of casein micelles was derived
from the time when G" started to deviate upwards from the baseline (tG"). In case
of RSM samples the moment when the fluorescence started to descend
practically coincided with the tG" (Figure 6a). In case of skimmed bulk milk the
tG" roughly coincided with the turning point on the fluorescence curve after
which an ascending signal continued to increase with a slower rate (Figure 6b).
As for the whole bulk milk, the dynamic rheological measurements failed to
show the profile typical to skimmed milk – G' was initially higher than G" and
their respective curves did not have a crossover, as well as viscoelastic moduli
increased from the onset of the measurements and the point at which the
aggregation of the micelles might proceed was not detectable on rheological
profiles. Nevertheless, taking into account the evident similarity of the overall
shapes of the fluorescence profiles of skimmed and whole bulk milk (except the
very beginning of the curves), the moment of change in the slope of ascending
fluorescence signal was suggested to correspond to the onset of aggregation
likewise in whole milk (Figure 6c).
In case of RSM samples the moment when the change in the slope of the
descending fluorescence signal occurred coincided with the time of the
crossover of G' and G" curves (tg in Figure 6a), meaning that the three-
dimensional gel was established at this point. The gelation of bulk milk occurred
approximately when tryptophan fluorescence intensity reached its maximum
value, but a specific point analogous to rheological gel point was not clearly
discernible on fluorescence profiles, as the curves did not have well-marked
transition from ascending to descending behaviour.
The data obtained in Publication III showed that the changes in tryptophan
fluorescence intensity were different during coagulation of RSM, skimmed and
whole bulk milk. Nevertheless, the universal approach for the determination of
the coagulation time parameters utilising the minimum of the second order
derivative (t2min) of fluorescence signal was introduced, as also illustrated in
Figure 6. In RSM samples, the t2min designated the time point when fluorescence
curve started to decrease (Figure 6a). In the bulk milk samples the t2min perfectly
fitted the time point when tryptophan fluorescence intensity curve changed the
slope.
In contrast to our findings, Fagan et al. (2011) proposed an approach utilizing
the time to the maximum of the first derivative of tryptophan fluorescence
profile (t1max) for monitoring milk coagulation. The comparison of the
fluorescence and rheological patterns recorded in the present study indicated that
a measurable aggregation of casein micelles proceeded later than t1max typically

53
occurred. On the contrary, the t2min parameter may potentially refer to the start of
aggregation of casein micelles, irrespectively of the nature of the milk sample.
4.3.3. Coagulation time parameters determined by different
methods
The enrichment of RSM with different amounts of CaCl2 and the addition of
rennet at different concentrations resulted in a wide range of coagulation kinetics
– estimated t2min displayed nearly a tenfold difference between the fast and
slowly coagulating samples. In skimmed and whole bulk milk the t2min parameter
ranged from 8.7 ± 0.5 to 12.8 ± 1.0 min and from 7.4 ± 0.8 to 10.6 ± 0.6 min,
respectively. As the fluorescence profiles obtained during coagulation of RSM
and bulk milk samples were substantially different, the Pearson’s correlation
coefficients (r) between coagulation time parameters derived from fluorescence
or reference measurements were calculated separately for different types of milk
(Table 8).
Table 8. Pearson’s correlation matrix of milk coagulability estimating parameters,
obtained by different methods, measured in RSM, skimmed or whole raw bulk milk.a
Milk Variable t2min RFT tG" tg
RSM t2min 1
(n = 7) RFT 0.98*** 1
tG" 0.98*** 0.95** 1
tg 0.94** 0.92** 0.99*** 1
Skimmed raw bulk t2min 1
milk RFT 0.77* 1
(n = 9) tG" 0.98*** 0.78* 1
tg 0.96*** 0.73* 0.94*** 1

Whole raw bulk milk t2min 1

(n = 9) RFT 0.12 1
tG" - - -
tg - - - -
a
Parameters in the table are defined as follows:
t2min – time to the minimum of the second derivative of tryptophan fluorescence intensity
signal,
RFT – visually determined rennet flocculation time,
tG" – time to the initial increase of G" derived from dynamic rheological measurements,
tg – time to the crossover of G' and G" derived from dynamic rheological measurements.
*
significant at the 0.05 level; ** significant at 0.01 level; *** significant at 0.001 level.

In RSM the t2min had the strongest relationship with rennet flocculation time
(RFT) and tG" (r = 0.98). The parameters determined by reference methods
(visual and rheological) appeared to correlate with each other less well than with
t2min determined by front-face fluorescence spectroscopy. The strongest
relationship in the case of skimmed bulk milk was observed between t2min and
rheologically determined parameters, particularly tG" (r = 0.98). Noteworthy, the

54
correlations between t2min and RFT and between RFT and rheological
coagulation parameters were notably poorer, but still significant. Unfortunately,
no significant correlation between t2min and RFT was observed in the case of
whole bulk milk, and rheological parameters could not be derived due to the
altered patterns of rheological profiles (see above). Possible explanation of the
different relationships obtained between t2min and RFT when measured in
different types of milk is given below.
The visual determination of RFT is very subjective, and the specific moment
of coagulation process, which RFT value corresponds to, is not well defined.
The estimation of RFT in different types of milk may reflect a different degree
of flocculation due to differences in viscosity and turbidity of the samples. Our
results indicated that the strongest correlations between RFT and coagulation
parameters determined by other methods were obtained if measured in the case
of RSM samples. Considerably poorer correlation of RFT with t2min, tG" and tg in
skimmed bulk milk samples may be associated with its thicker nature compared
to RSM, which might influence the reliability of RFT determination. At the
same time coagulation parameters other than RFT still demonstrated strong
correlations with each other in skimmed bulk milk. The determination of RFT
value in case of the whole bulk milk was quite difficult due to adherence of
flakes of milk fat to the glass walls during stirring of the milk samples in thin
test tubes, which could be confused with the first signs of protein flocculation.
Nevertheless, the shape of tryptophan fluorescence pattern was very
characteristic and determination of t2min in the whole milk samples was as simple
as in skimmed bulk milk samples. Hence, only the determination of RFT, rather
than fluorescence measurements in whole milk was considered unsuccessful in
Publication III. Further research is still needed for proving the capability of the
fluorescence method to assess coagulation time characteristics in whole bulk
milk.

4.4. Front-face fluorescence spectroscopy monitoring milk

acidification (Publication IV)

The application of front-face fluorescence spectroscopy in the study of the

differences of the growth of St. thermophilus ST12 in milk reconstituted from
the untreated or irradiated at 10 kGy low-heat skim milk powder (RSM and
irrRSM, respectively) is reported in Publication IV.
Firstly, the comparison of spectral fluorescence signatures (SFS) of the
control and irradiated milk powders and the reconstituted milk samples provided
the first insight into the changes which had taken place with milk proteins during
irradiation. The fluorescence intensity corresponding to tryptophan emission
maximum was threefold lower in irradiated LHSMP (irrLHSMP) than in
LHSMP and the coordinates of the maxima λex/λem were 285/330 and 285/325
nm, respectively. After reconstitution, almost a twofold difference in the
fluorescence intensity between the spectra of irrRSM and RSM were observed.

55 
 
We suggested that the decreased tryptophan fluorescence in irrLHSMP and
irrRSM was associated with the oxidation of aromatic amino acids or production
of other oxidation products (Stadtman & Levine, 2003), which could quench the
fluorescence, or with conformational changes of proteins caused by irradiation
(Cieśla at al., 2004). A distinctive additional fluorescence signal with a
maximum at λex/λem of 350/410-420 nm, which corresponded most probably to
the oxidation or Maillard reaction products (Becker et al., 2003; Liu & Metzger,
2007), was observed on the SFS spectra of irrLHSMP (see Fig. 1 in Publication
IV) and irrRSM samples.
The changes in maximum fluorescence intensity of tryptophan recorded
during fermentation of RSM and irrRSM by ST12 were similar, except for the
fact that these occurred much faster in irrRSM, which was in line with the faster
drop of pH in irrRSM sample (Figure 7a). Changes in fluorescence intensity
corresponded presumably to the modifications of the environment of the
tryptophan residues caused by pH decrease during acidification, whereas
changes in the protein network associated with gelation phenomenon were not
clearly distinguishable. The gelation points of RSM and irrRSM determined by
the rheological measurements (marked with an arrow in Figure 7a) did not
coincide with the same specific points on the tryptophan fluorescence intensity
profiles, but were different for the two milk samples. The gelation point in
acidified RSM sample occurred between the maximum and the local minimum
and in irrRSM – coincided with the local minimum on the fluorescence pattern.
The tryptophan fluorescence intensity patterns obtained in this study during
fermentation with ST12 were similar to those shown in the case of milk acidified
by glucono-δ-lactone (Lopez & Dufour, 2001). This suggested that the profile
presented may be more or less universal and independent of whether
acidification was induced by addition of glucono-δ-lactone or growth of starter
culture.
The SFS spectral data recorded during fermentation of RSM and irrRSM
with ST12 were pooled in one matrix and analysed by PCA (Figure 7b). Two
fermentations were fully discriminated along PC1 axis. Changes of SFS in time
were observed along PC1 and PC2 axes. The discrimination of the SFS spectra
during fermentation was associated with PC1 and PC2 due to specific λex of
tryptophan fluorescence peak at maximum λem of near 330 nm. Considering
PC1, the fluorescence intensity increased after excitation at 295-300 nm during
the first phase of fermentation, and after the turning point a change in the
opposite direction was observed. Considering PC2, the fluorescence intensity
mainly decreased after excitation at 275 nm during the time course of
fermentation. Earlier the tryptophan fluorescence signal measured in processed
cheese samples was reported to be composed of two tryptophan components
with λex/λem of 300/347 and 280/339 nm, which were assigned to the
fluorophores in different protein structures (Christensen et al., 2003). Comparing
our results with those cited above, two types of tryptophan residues included in

56
different protein structures could also be differentiated in milk during
fermentation.

a b

Figure 7. a) Development of the maximum fluorescence intensity of tryptophan

during fermentation of RSM (black) and irrRSM (grey) with Streptococcus thermophilus
ST12 at inoculation rate of 105 cfu mL-1. b) The PCA similarity map defined by two first
principal components for the SFS data recorded during fermentation of RSM (■) and
irrRSM (♦). Arrows mark the mean gelation times determined rheologically.
The layout of the scores on both axes during the time span of fermentations
of RSM and irrRSM samples followed rather different profiles. Two phases with
turning point at pH 5.6-5.7 and pH 6.1 in the case of RSM and irrRSM,
respectively, were observed on the similarity map. These data showed that the
turning point separating two phases on the similarity map did not necessarily
correspond to the gelation point. From the data obtained we can conclude that
depending on milk used as a substrate for ST12 growth the comparable
reduction of pH could lead to different changes in protein structures depending
on the history of the samples.
Based on the results presented in Publication IV, we suggest that front-face
fluorescence spectroscopy could be potentially applicable and useful to elucidate
the peculiarities of acidification processes in milk. Front-face fluorescence
spectroscopy can provide additional structural information in dynamic mode,
which is not readily obtainable by other commonly used experimental
techniques.

57
5. CONCLUSIONS
The two main aims of the dissertation were: a) to gain better insight into the
factors affecting technological properties of milk, including original composition
of bulk milk and external factors as transportation, pooling of milk, ingress of
contaminants i.e. H2O2, and b) to apply front-face fluorescence spectroscopy for
monitoring enzymatic and acid-induced coagulation of milk.
The main results of this dissertation could be summarized as follows:
I. The renneting properties together with the composition of bulk milk
from a representative number of Estonian dairy farms were
determined and compared with those of pooled milk from tank trucks
and dairy silos for the first time. It was shown that
• pH of the Estonian bulk milk has increased 0.1 units during the
past decades, and it is higher than that of milk produced in other
countries in Europe;
• the renneting characteristics of bulk milk of different farms varied
in a wide range; moreover, clear seasonal changes in milk
rennetability and firmness of coagulum were observed;
• although pooling of milk reduces the variations, the important
correlations between the composition and renneting
characteristics of bulk milk were found to correspond to those
reported earlier for milk from individual cows. Milk pH correlated
statistically significantly with curd firmness and RFT; curd
firmness correlated also with protein content and with κ-casein
fraction in particular (r = 0.45);
• changes taking place in milk after milking at the farm and before
processing at the dairy plant influence the technological properties
of milk. The ratio of whey protein to casein was higher, due to
partial degradation of κ- and β-casein fractions, and RFT was
longer in pooled milk from tunk trucks and dairy silos. These
aspects deserve further investigation.
II. Application of microcalorimetry enabled comprehensive evaluation
of the action of a common disinfectant H2O2 on the growth of
thermophilic starter bacteria in milk. It was shown that
• the residual H2O2 caused the direct inhibition of bacterial growth
that resulted in prolonged gelation time and deteriorated gel
quality at concentrations starting from 10 mg L-1 even if added
into milk one hour before starter inoculation;
• the pre-treatment of raw milk with H2O2 for one hour followed by
its complete decomposition by catalase did not interfere with
acid-induced coagulation, but changed presumably the
bioavailability of nutrients for starter bacteria affecting their
growth.

58
III. A novel front-face fluorescence spectroscopy method was applied for
detailed investigation of rennet coagulation of reconstituted skimmed
milk (RSM) and raw bulk milk. The method could be applied in a
laboratory-scale research of milk coagulation, and a non-destructive
sensor for in-line monitoring of milk coagulation during
cheesemaking or for off-line evaluation of milk suitability for cheese
production in dairies could be developed. It was shown that
• the changes in tryptophan fluorescence intensity patterns recorded
during renneting depended on the nature of milk, presence of milk
fat and enrichment with CaCl2;
• tryptophan fluorescence intensity patterns allowed to distinguish
different phases of enzymatic coagulation, and to determine a
specific point (t2min), which corresponded to the moment when
aggregation of casein micelles started;
• t2min was found to correlate with coagulation time parameters
obtained by reference methods in RSM and skimmed bulk milk;
the characteristic shape of fluorescence pattern also allowed the
determination of t2min in the case of whole bulk milk, but
applicability of the method for the determination of the
coagulation characteristics of whole milk needs additional
verification.
IV. Front-face fluorescence spectroscopy allowed to differentiate milk
samples undergone different treatments (e.g. irradiation) and to trace
acid-induced changes occurring with milk during fermentation with
starter. It was shown that
• tryptophan fluorescence intensity patterns recorded during
acidification corresponded to changes in milk proteins caused
primarily by pH decrease and not by gelation phenomenon as
such;
• depending on the nature of milk samples the comparable
reduction of milk pH led to different changes in the structures of
proteins during acid gel formation.

59
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68
ACKNOWLEDGMENTS
First of all I wish to thank my first supervisor Dr. Tiiu-Maie Laht for introducing
me to the world of dairy science nine years ago and for providing me an
opportunity to deepen my knowledge in this field. I appreciate her confidence in
me and support during my bachelor, master and doctoral studies.
I am very grateful to Prof. Raivo Vilu for supervising my dissertation, his
valuable comments, our interesting discussions and his ability to help me
looking at the issues from somewhat different point of view.
I specially thank Anastassia, Irina and Natalja for excellent experimental
work done together, their support and friendship. I rely on our further
cooperation.
I wish to thank Olga – yours inspirational capacity for work has always been
an example for me. I also thank my brother and cousin who were great examples
of being keen on something – I probably owe my love of science to them.
I also thank my former students: Jaanika, Kristi, Tanja, Viktoria – they aided
me the deeper understanding of what I have been doing.
I am grateful to all my colleagues in the Competence Centre of Food and
Fermentation Technologies from whom I always learned something new and
who opened my mind.
I wish to thank Signe for reading the manuscript and valuable advice.
Financial support for this work was provided by grant SF0140090s08 of
Estonian Ministry of Education and Research, and by the European Regional
Development Fund projects EU28912 and EU29994.

69
ABSTRACT
The ability of milk to coagulate is essential for manufacturing of cheese and
fermented dairy products, which provides the added value to milk.
First of all, this dissertation aimed to evaluate the rennetability of Estonian
raw bulk milk by direct measurement of rennet flocculation time (RFT) and curd
firmness and to find correlations between these properties and milk composition.
The direct information on seasonal and non-seasonal variations in rennetability
of bulk milk collected from a large number of farms was provided. The results
showed that the pooling of milk at the farms, i.e. averaging of milk composition,
did not change basic correlations between milk composition and cheesemaking
properties reported earlier in case of milk of individual dairy cows. Pooled milk
from tank trucks and dairy silos originating from the same milkings was also
analysed. Changes in whey proteins to casein ratio due to partial degradation of
κ- and β-casein fractions in pooled milk from tank trucks and dairy silos in
comparison with the farm milk were observed. Considering changes in the
technological properties of milk subjected to mechanical perturbations as
pumping and refrigerated transportation in trucks, renneting times of farm milk
samples were in average one minute shorter than those of samples from dairy
silos. Further, the influence of disinfecting agent, hydrogen peroxide (H2O2), on
the diauxic growth of starter bacteria and formation of acid-induced gel was
investigated by applying microcalorimetry and dynamic low-amplitude
rheology, respectively. Even the lowest concentrations of H2O2 (starting with 10
mg L-1) added into milk one hour before inoculation caused the direct inhibition
of thermophilic bacteria, retarded the onset of milk gelation, and resulted in
formation of weaker gels. In milk samples pre-treated with H2O2 and
subsequently with catalase, ensuring complete removal of the oxidant, the first
exponential growth phase of starter bacteria was not affected, and hardly any
difference in gelation and no changes in rheological characteristics of mature
gels were observed. Nevertheless, clear bacteriostatic influence of H2O2 pre-
treatment on the second exponential growth phase was observed in milk treated
with H2O2 followed by catalase, which may potentially result in the end-product
with altered flavour or other defects, even though gel properties would not be
affected. These studies highlighted that in addition to the original milk
composition the external factors encountered between milking and processing at
dairy plant (refrigerated transportation of milk from farms to dairy silos, milk
vigorous pumping or any other mechanical treatment, occurrence of disinfectant
residues etc.) may also affect enzymatic and/or acid-induced coagulation and
properties of the end-product.
Another aim of this dissertation was to apply a novel front-face fluorescence
spectroscopy method for detailed investigation of rennet coagulation as well as
acid coagulation induced by starter bacteria. Tryptophan fluorescence intensity
patterns allowed distinguishing different phases of enzymatic coagulation,
starting with first interactions of enzyme with casein micelles and ending with

70
gel aging. Furthermore, this study showed the ability of fluorescence method to
estimate coagulation properties of different milk substrates: reconstituted
skimmed milk with wide range of coagulation kinetics as well as skimmed bulk
milk samples. A characteristic shape of fluorescence pattern was also obtained
during coagulation of whole milk, but the applicability of this method to
determine coagulation characteristics in whole milk is still to be verified.
Considering the application of front-face fluorescence spectroscopy to trace
acid-induced changes occurring with milk proteins during fermentation with
starter, tryptophan fluorescence was shown to be highly sensitive to the
reduction of milk pH and clearly showed differences in acidification profiles
between different reconstituted skimmed milks.

71
KOKKUVÕTE
Piima kalgendumisvõime on esmatähtis omadus, millele põhineb juustu ja
fermenteeritud piimatoodete valmistamine ja mis annab piimale lisandväärtuse.
Käesolevas töös hinnati Eesti farmide segupiima kalgendatavust
kalgendumisaja (RFT) ja kalgendi tugevuse määramise kaudu ja leiti seosed
segupiima koostise ja kalgendatavuse vahel. Töös analüüsiti paljudest farmidest
võetud segupiima proove läbi nelja aasta, mis võimaldas kirjeldada ka
sesoonseid kõikumisi Eesti farmipiima kalgendumisomadustes. Saadud
tulemuste põhjal näidati, et üksikute lehmade piima kokkusegamine farmis
(piima koostise keskmistamine) ei muuda olulisi seoseid piima koostise ja
kalgendatavuse vahel (piima pH ja kalgendi tugevuse ja RFT vahel, kalgendi
tugevuse ja valgusisalduse, eriti κ-kaseiini sisalduse vahel), mida oli näidatud
üksikute lehmade piima puhul. Samuti võrreldi üksikute farmide segupiima ja
samade farmide kokku segatud piimade (proovid võetud piimakogumisautodest
ja tööstuste silotankidest) koostist ja kalgendatavust. Tööstusesse jõudnud ja
silotangi piimas oli keskmiselt kõrgem vadakuvalkude ja kaseiinide suhe, mis
võib viidata β-kaseiini osalisele hüdrolüüsile. Piim, mida oli pumbatud
farmimahutist piimakogumisautosse, veetud tööstusesse ja pumbatud tööstuse
silotanki, kalgendus keskmiselt minuti võrra kauem võrreldes farmist võetud
segupiima proovidega. Käesolevas töös uuriti ka desinfektandina kasutatava
vesinikperoksiidi (H2O2) mõju termofiilsete juuretisebakterite kasvule ja
happekalgendi moodustumisele, kasutades mikrokalorimeetriat ja dünaamilist
reomeetriat. Juba väga madalate H2O2 kontsentratsioonide (alates 10 mg L-1)
lisamine piima tund enne inokuleerimist põhjustas juuretisebakterite otsese
inhibeerimise, piima geelistumise aeglustamise ja nõrgema geeli struktuuri
moodustumise. H2O2 lisamine, millele järgnes katalaasiga töötlemine  vahetult
enne inokuleerimist kindlustamaks oksüdeeriva aine täieliku eemaldamise, ei
mõjutanud juuretisebakterite kasvu esimese eksponentsiaalse faasi jooksul ja ei
mõjutanud valmis geeli reoloogilisi omadusi. Samas oli bakterite
kasvudünaamika teises eksponentsiaalses faasis selgelt mõjutatud, mis võib
tähendada muutusi lõpp-produkti aroomi- või maitseprofiilis või muid defekte,
vaatamata sellele, et geeli struktuur ei muutunud. Käesoleva töö tulemused
näitavad, et lisaks piima algsele koostisele võivad piima ensümaatilist ja/või
happelist kalgendamist ning valmistoote kvaliteeti mõjutada ka sellised
esmapilgul minoorsed välised mõjud nagu lüpsijärgne jahutamine, pumpamine,
transportimine, desoainete jääkide sattumine piima jms.
Doktoritöö teiseks eesmärgiks oli välja selgitada pinna-
fluorestsentsspektroskoopia meetodi kasutatavust piima ensümaatilise
kalgendamise ja hapendamise jälgimiseks. Trüptofaani fluorestsentsi
intensiivsuse muutuste järgi eristati erinevaid kalgendumise faase, alustades
ensüümi seostamisega kaseiini mitsellidega ja lõpetades kalgendi vananemisega.
Lisaks sellele oli fluorestsentsmeetodi abil võimalik määrata piima
kalgendumisomadusi, mida kinnitati nii taastatud piima kui ka kooritud

72 
 
toorpiima katsetes. Rasvase toorpiima kalgendamise puhul registreeriti samuti
iseloomulik fluorestsentsi profiil, siiski vajab fluorestsentsmeetodi kasutatavus
täispiima kalgendumisaja määramiseks edasist valideerimist. Samuti näidati, et
trüptofaani fluorestsents on väga tundlik juuretisebakterite poolt esile kutsutud
piima pH languse suhtes. Hapendamise jooksul registreeritud fluorestsentsi
muutuste profiilide järgi oli võimalik selgelt eristada erinevalt töödeldud
taastatud piimas toimuvaid protsesse.

73 
 
  
 
PUBLICATION I

Kriščiunaite, T., Stulova, I., Taivosalo, A., Laht, T.-M., and Vilu, R.
Composition and renneting properties of raw bulk milk in Estonia
International Dairy Journal, 23(1), 45-52 (2012)3

3
Copyright 2011, reprinted with permission from Elsevier.

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PUBLICATION II

Kriščiunaite, T., Stulova, I., Kabanova, N., Laht, T.-M., and Vilu, R.
The effect of hydrogen peroxide on the growth of thermophilic lactic
starter and acid gelation of UHT milk
International Dairy Journal, 21(4), 239-246 (2011)4

4
Copyright 2010, reprinted with permission from Elsevier.

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PUBLICATION III

Kriščiunaite, T., Seiman, A., Laht, T.-M., and Vilu, R.

Front-face fluorescence spectroscopy studying rennet coagulation of
milk
Manuscript

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PUBLICATION IV

Stulova, I., Kabanova, N., Kriščiunaite, T., Taivosalo, A., Laht, T.-M., and
Vilu, R.
Fermentation of reconstituted milk by Streptococcus thermophilus:
effect of irradiation on skim milk powder
International Dairy Journal (2013)
http://dx.doi.org/10.1016/j.idairyj.2013.02.0045

5
Copyright 2013, reprinted with permission from Elsevier.

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CURRICULUM VITAE
1. Personal data
Name Tiina Kriščiunaite
Date of birth 13.08.1982
Place of birth Tallinn
Citizenship Estonian

2. Contact information
Address AS TFTAK, Akadeemia tee 15a, 12618 Tallinn
Phone +37255987805
E-mail tiina@tftak.eu

3. Education
2006-... Tallinn University of Technology, chemistry and gene technology,
doctoral studies
2004-2006 Tallinn University of Technology, bio- and food technology, MSc
2000-2004 Tallinn University of Technology, bio- and food technology, BSc
1988-2000 Tallinn High School of Humanities

4. Language skills
Russian mother tongue
Estonian fluent
English fluent
German basic skills
Lithuanian basic skills

5. Special Courses
19-24.08.2007 PhD course „Ripening of Northern European Cheeses”,
University of Copenhagen, Denmark
21-25.08.2006 PhD course „From Milk to Cheese”, Norwegian University
of Life Sciences, Norway
28.03-8.04.2004 Socrates/Erasmus Intensive Course "Food Safety",
Larenstein University of Professional Education, the Netherlands

6. Professional Employment
2005-… Competence Centre of Food and Fermentation Technologies, researcher

7. Defended theses
Master thesis: Development of Front-Face Fluorescence Spectroscopy for
Studying Milk Renneting – Monitoring Influence of Strong Oxidants
Bachelor thesis: Influence of Protein Composition and pH on Coagulation
Properties of Milk

137
ELULOOKIRJELDUS
1. Isikuandmed
Ees- ja perekonnanimi Tiina Kriščiunaite
Sünniaeg 13.08.1982
Sünnikoht Tallinn
Kodakondsus Eesti

2. Kontaktandmed
Aadress AS TFTAK, Akadeemia tee 15a, 12618 Tallinn
Telefon +37255987805
E-posti aadress tiina@tftak.eu

3. Hariduskäik
2006-… Tallinna Tehnikaülikool, keemia ja geenitehnoloogia doktoriõpe
2004-2006 Tallinna Tehnikaülikool, bio- ja toiduainetehnoloogia magistriõpe
2000-2004 Tallinna Tehnikaülikool, bio- ja toiduainetehnoloogia bakalaureuse-
õpe
1988-2000 Tallinna Humanitaargümnaasium

4. Keelteoskus
Vene keel emakeel
Eesti keel kõrgtase
Inglise keel kõrgtase
Saksa keel algtase
Leedu keel algtase

5. Täiendusõpe
19-24.08.2007 PhD kursus „Põhja-Euroopa juustude valmimine”,
Kopenhaageni Ülikool, Taani
21-25.08.2006 PhD kursus „Piimast juustuni”, Norra Maaülikool, Norra
28.03-8.04.2004 Socrates/Erasmus intensiivkursus "Toiduohutus", Larenstein
Ülikool, Holland

6. Teenistuskäik
2005-… Toidu- ja Fermentatsioonitehnoloogia Arenduskeskus, teadur

7. Kaitstud lõputööd
Magistritöö: Uue front-face fluorestsentsmeetodi väljatöötamine piima
kalgendatavuse hindamiseks – tugevate oksüdeerijate mõju uurimine
Bakalaureusetöö: Piima kalgendumis- ja hapnemisomaduste sõltuvus
valgulisest koostisest ja pH-st

138
 DISSERTATIONS DEFENDED AT
TALLINN UNIVERSITY OF TECHNOLOGY ON
NATURAL AND EXACT SCIENCES
1. Olav Kongas. Nonlinear Dynamics in Modeling Cardiac Arrhytmias. 1998.
2. Kalju Vanatalu. Optimization of Processes of Microbial Biosynthesis of
Isotopically Labeled Biomolecules and Their Complexes. 1999.
3. Ahto Buldas. An Algebraic Approach to the Structure of Graphs. 1999.
4. Monika Drews. A Metabolic Study of Insect Cells in Batch and Continuous
Culture: Application of Chemostat and Turbidostat to the Production of
Recombinant Proteins. 1999.
5. Eola Valdre. Endothelial-Specific Regulation of Vessel Formation: Role of
Receptor Tyrosine Kinases. 2000.
6. Kalju Lott. Doping and Defect Thermodynamic Equilibrium in ZnS. 2000.
7. Reet Koljak. Novel Fatty Acid Dioxygenases from the Corals Plexaura
homomalla and Gersemia fruticosa. 2001.
8. Anne Paju. Asymmetric oxidation of Prochiral and Racemic Ketones by
Using Sharpless Catalyst. 2001.
9. Marko Vendelin. Cardiac Mechanoenergetics in silico. 2001.
10. Pearu Peterson. Multi-Soliton Interactions and the Inverse Problem of
Wave Crest. 2001.
11. Anne Menert. Microcalorimetry of Anaerobic Digestion. 2001.
12. Toomas Tiivel. The Role of the Mitochondrial Outer Membrane in in vivo
Regulation of Respiration in Normal Heart and Skeletal Muscle Cell. 2002.
13. Olle Hints. Ordovician Scolecodonts of Estonia and Neighbouring Areas:
Taxonomy, Distribution, Palaeoecology, and Application. 2002.
14. Jaak Nõlvak. Chitinozoan Biostratigrapy in the Ordovician of Baltoscandia.
2002.
15. Liivi Kluge. On Algebraic Structure of Pre-Operad. 2002.
16. Jaanus Lass. Biosignal Interpretation: Study of Cardiac Arrhytmias and
Electromagnetic Field Effects on Human Nervous System. 2002.
17. Janek Peterson. Synthesis, Structural Characterization and Modification of
PAMAM Dendrimers. 2002.
18. Merike Vaher. Room Temperature Ionic Liquids as Background Electrolyte
Additives in Capillary Electrophoresis. 2002.
19. Valdek Mikli. Electron Microscopy and Image Analysis Study of Powdered
Hardmetal Materials and Optoelectronic Thin Films. 2003.
20. Mart Viljus. The Microstructure and Properties of Fine-Grained Cermets.
2003.
21. Signe Kask. Identification and Characterization of Dairy-Related
Lactobacillus. 2003
22. Tiiu-Mai Laht. Influence of Microstructure of the Curd on Enzymatic and
Microbiological Processes in Swiss-Type Cheese. 2003.

139
23. Anne Kuusksalu. 2–5A Synthetase in the Marine Sponge Geodia cydonium.
2003.
24. Sergei Bereznev. Solar Cells Based on Polycristalline Copper-Indium
Chalcogenides and Conductive Polymers. 2003.
25. Kadri Kriis. Asymmetric Synthesis of C2-Symmetric Bimorpholines and
Their Application as Chiral Ligands in the Transfer Hydrogenation of Aromatic
Ketones. 2004.
26. Jekaterina Reut. Polypyrrole Coatings on Conducting and Insulating
Substracts. 2004.
27. Sven Nõmm. Realization and Identification of Discrete-Time Nonlinear
Systems. 2004.
28. Olga Kijatkina. Deposition of Copper Indium Disulphide Films by
Chemical Spray Pyrolysis. 2004.
29. Gert Tamberg. On Sampling Operators Defined by Rogosinski, Hann and
Blackman Windows. 2004.
30. Monika Übner. Interaction of Humic Substances with Metal Cations. 2004.
31. Kaarel Adamberg. Growth Characteristics of Non-Starter Lactic Acid
Bacteria from Cheese. 2004.
32. Imre Vallikivi. Lipase-Catalysed Reactions of Prostaglandins. 2004.
33. Merike Peld. Substituted Apatites as Sorbents for Heavy Metals. 2005.
34. Vitali Syritski. Study of Synthesis and Redox Switching of Polypyrrole and
Poly(3,4-ethylenedioxythiophene) by Using in-situ Techniques. 2004.
35. Lee Põllumaa. Evaluation of Ecotoxicological Effects Related to Oil Shale
Industry. 2004.
36. Riina Aav. Synthesis of 9,11-Secosterols Intermediates. 2005.
37. Andres Braunbrück. Wave Interaction in Weakly Inhomogeneous
Materials. 2005.
38. Robert Kitt. Generalised Scale-Invariance in Financial Time Series. 2005.
39. Juss Pavelson. Mesoscale Physical Processes and the Related Impact on the
Summer Nutrient Fields and Phytoplankton Blooms in the Western Gulf of
Finland. 2005.
40. Olari Ilison. Solitons and Solitary Waves in Media with Higher Order
Dispersive and Nonlinear Effects. 2005.
41. Maksim Säkki. Intermittency and Long-Range Structurization of Heart
Rate. 2005.
42. Enli Kiipli. Modelling Seawater Chemistry of the East Baltic Basin in the
Late Ordovician–Early Silurian. 2005.
43. Igor Golovtsov. Modification of Conductive Properties and Processability of
Polyparaphenylene, Polypyrrole and polyaniline. 2005.
44. Katrin Laos. Interaction Between Furcellaran and the Globular Proteins
(Bovine Serum Albumin β-Lactoglobulin). 2005.
45. Arvo Mere. Structural and Electrical Properties of Spray Deposited Copper
Indium Disulphide Films for Solar Cells. 2006.

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46. Sille Ehala. Development and Application of Various On- and Off-Line
Analytical Methods for the Analysis of Bioactive Compounds. 2006.
47. Maria Kulp. Capillary Electrophoretic Monitoring of Biochemical Reaction
Kinetics. 2006.
48. Anu Aaspõllu. Proteinases from Vipera lebetina Snake Venom Affecting
Hemostasis. 2006.
49. Lyudmila Chekulayeva. Photosensitized Inactivation of Tumor Cells by
Porphyrins and Chlorins. 2006.
50. Merle Uudsemaa. Quantum-Chemical Modeling of Solvated First Row
Transition Metal Ions. 2006.
51. Tagli Pitsi. Nutrition Situation of Pre-School Children in Estonia from 1995
to 2004. 2006.
52. Angela Ivask. Luminescent Recombinant Sensor Bacteria for the Analysis
of Bioavailable Heavy Metals. 2006.
53. Tiina Lõugas. Study on Physico-Chemical Properties and Some Bioactive
Compounds of Sea Buckthorn (Hippophae rhamnoides L.). 2006.
54. Kaja Kasemets. Effect of Changing Environmental Conditions on the
Fermentative Growth of Saccharomyces cerevisae S288C: Auxo-accelerostat
Study. 2006.
55. Ildar Nisamedtinov. Application of 13C and Fluorescence Labeling in
Metabolic Studies of Saccharomyces spp. 2006.
56. Alar Leibak. On Additive Generalisation of Voronoï’s Theory of Perfect
Forms over Algebraic Number Fields. 2006.
57. Andri Jagomägi. Photoluminescence of Chalcopyrite Tellurides. 2006.
58. Tõnu Martma. Application of Carbon Isotopes to the Study of the
Ordovician and Silurian of the Baltic. 2006.
59. Marit Kauk. Chemical Composition of CuInSe2 Monograin Powders for
Solar Cell Application. 2006.
60. Julia Kois. Electrochemical Deposition of CuInSe2 Thin Films for
Photovoltaic Applications. 2006.
61. Ilona Oja Açik. Sol-Gel Deposition of Titanium Dioxide Films. 2007.
62. Tiia Anmann. Integrated and Organized Cellular Bioenergetic Systems in
Heart and Brain. 2007.
63. Katrin Trummal. Purification, Characterization and Specificity Studies of
Metalloproteinases from Vipera lebetina Snake Venom. 2007.
64. Gennadi Lessin. Biochemical Definition of Coastal Zone Using Numerical
Modeling and Measurement Data. 2007.
65. Enno Pais. Inverse problems to determine non-homogeneous degenerate
memory kernels in heat flow. 2007.
66. Maria Borissova. Capillary Electrophoresis on Alkylimidazolium Salts.
2007.
67. Karin Valmsen. Prostaglandin Synthesis in the Coral Plexaura homomalla:
Control of Prostaglandin Stereochemistry at Carbon 15 by Cyclooxygenases.
2007.

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68. Kristjan Piirimäe. Long-Term Changes of Nutrient Fluxes in the Drainage
Basin of the Gulf of Finland – Application of the PolFlow Model. 2007.
69. Tatjana Dedova. Chemical Spray Pyrolysis Deposition of Zinc Sulfide Thin
Films and Zinc Oxide Nanostructured Layers. 2007.
70. Katrin Tomson. Production of Labelled Recombinant Proteins in Fed-Batch
Systems in Escherichia coli. 2007.
71. Cecilia Sarmiento. Suppressors of RNA Silencing in Plants. 2008.
72. Vilja Mardla. Inhibition of Platelet Aggregation with Combination of
Antiplatelet Agents. 2008.
73. Maie Bachmann. Effect of Modulated Microwave Radiation on Human
Resting Electroencephalographic Signal. 2008.
74. Dan Hüvonen. Terahertz Spectroscopy of Low-Dimensional Spin Systems.
2008.
75. Ly Villo. Stereoselective Chemoenzymatic Synthesis of Deoxy Sugar Esters
Involving Candida antarctica Lipase B. 2008.
76. Johan Anton. Technology of Integrated Photoelasticity for Residual Stress
Measurement in Glass Articles of Axisymmetric Shape. 2008.
77. Olga Volobujeva. SEM Study of Selenization of Different Thin Metallic
Films. 2008.
78. Artur Jõgi. Synthesis of 4’-Substituted 2,3’-dideoxynucleoside Analogues.
2008.
79. Mario Kadastik. Doubly Charged Higgs Boson Decays and Implications on
Neutrino Physics. 2008.
80. Fernando Pérez-Caballero. Carbon Aerogels from 5-Methylresorcinol-
Formaldehyde Gels. 2008.
81. Sirje Vaask. The Comparability, Reproducibility and Validity of Estonian
Food Consumption Surveys. 2008.
82. Anna Menaker. Electrosynthesized Conducting Polymers, Polypyrrole and
Poly(3,4-ethylenedioxythiophene), for Molecular Imprinting. 2009.
83. Lauri Ilison. Solitons and Solitary Waves in Hierarchical Korteweg-de
Vries Type Systems. 2009.
84. Kaia Ernits. Study of In2S3 and ZnS Thin Films Deposited by Ultrasonic
Spray Pyrolysis and Chemical Deposition. 2009.
85. Veljo Sinivee. Portable Spectrometer for Ionizing Radiation
“Gammamapper”. 2009.
86. Jüri Virkepu. On Lagrange Formalism for Lie Theory and Operadic
Harmonic Oscillator in Low Dimensions. 2009.
87. Marko Piirsoo. Deciphering Molecular Basis of Schwann Cell
Development. 2009.
88. Kati Helmja. Determination of Phenolic Compounds and Their
Antioxidative Capability in Plant Extracts. 2010.
89. Merike Sõmera. Sobemoviruses: Genomic Organization, Potential for
Recombination and Necessity of P1 in Systemic Infection. 2010.

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90. Kristjan Laes. Preparation and Impedance Spectroscopy of Hybrid
Structures Based on CuIn3Se5 Photoabsorber. 2010.
91. Kristin Lippur. Asymmetric Synthesis of 2,2’-Bimorpholine and its 5,5’-
Substituted Derivatives. 2010.
92. Merike Luman. Dialysis Dose and Nutrition Assessment by an Optical
Method. 2010.
93. Mihhail Berezovski. Numerical Simulation of Wave Propagation in
Heterogeneous and Microstructured Materials. 2010.
94. Tamara Aid-Pavlidis. Structure and Regulation of BDNF Gene. 2010.
95. Olga Bragina. The Role of Sonic Hedgehog Pathway in Neuro- and
Tumorigenesis. 2010.
96. Merle Randrüüt. Wave Propagation in Microstructured Solids: Solitary and
Periodic Waves. 2010.
97. Marju Laars. Asymmetric Organocatalytic Michael and Aldol Reactions
Mediated by Cyclic Amines. 2010.
98. Maarja Grossberg. Optical Properties of Multinary Semiconductor
Compounds for Photovoltaic Applications. 2010.
99. Alla Maloverjan. Vertebrate Homologues of Drosophila Fused Kinase and
Their Role in Sonic Hedgehog Signalling Pathway. 2010.
100. Priit Pruunsild. Neuronal Activity-Dependent Transcription Factors and
Regulation of Human BDNF Gene. 2010.
101. Tatjana Knjazeva. New Approaches in Capillary Electrophoresis for
Separation and Study of Proteins. 2011.
102. Atanas Katerski. Chemical Composition of Sprayed Copper Indium
Disulfide Films for Nanostructured Solar Cells. 2011.
103. Kristi Timmo. Formation of Properties of CuInSe2 and Cu2ZnSn(S,Se)4
Monograin Powders Synthesized in Molten KI. 2011.
104. Kert Tamm. Wave Propagation and Interaction in Mindlin-Type
Microstructured Solids: Numerical Simulation. 2011.
105. Adrian Popp. Ordovician Proetid Trilobites in Baltoscandia and Germany.
2011.
106. Ove Pärn. Sea Ice Deformation Events in the Gulf of Finland and This
Impact on Shipping. 2011.
107. Germo Väli. Numerical Experiments on Matter Transport in the Baltic Sea.
2011.
108. Andrus Seiman. Point-of-Care Analyser Based on Capillary
Electrophoresis. 2011.
109. Olga Katargina. Tick-Borne Pathogens Circulating in Estonia (Tick-Borne
Encephalitis Virus, Anaplasma phagocytophilum, Babesia Species): Their
Prevalence and Genetic Characterization. 2011.
110. Ingrid Sumeri. The Study of Probiotic Bacteria in Human Gastrointestinal
Tract Simulator. 2011.
111. Kairit Zovo. Functional Characterization of Cellular Copper Proteome.
2011.

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112. Natalja Makarytsheva. Analysis of Organic Species in Sediments and Soil
by High Performance Separation Methods. 2011.
113. Monika Mortimer. Evaluation of the Biological Effects of Engineered
Nanoparticles on Unicellular Pro- and Eukaryotic Organisms. 2011.
114. Kersti Tepp. Molecular System Bioenergetics of Cardiac Cells:
Quantitative Analysis of Structure-Function Relationship. 2011.
115. Anna-Liisa Peikolainen. Organic Aerogels Based on 5-Methylresorcinol.
2011.
116. Leeli Amon. Palaeoecological Reconstruction of Late-Glacial Vegetation
Dynamics in Eastern Baltic Area: A View Based on Plant Macrofossil Analysis.
2011.
117. Tanel Peets. Dispersion Analysis of Wave Motion in Microstructured
Solids. 2011.
118. Liina Kaupmees. Selenization of Molybdenum as Contact Material in
Solar Cells. 2011.
119. Allan Olspert. Properties of VPg and Coat Protein of Sobemoviruses.
2011.
120. Kadri Koppel. Food Category Appraisal Using Sensory Methods. 2011.
121. Jelena Gorbatšova. Development of Methods for CE Analysis of Plant
Phenolics and Vitamins. 2011.
122. Karin Viipsi. Impact of EDTA and Humic Substances on the Removal of
Cd and Zn from Aqueous Solutions by Apatite. 2012.
123. David Schryer. Metabolic Flux Analysis of Compartmentalized Systems
Using Dynamic Isotopologue Modeling. 2012.
124. Ardo Illaste. Analysis of Molecular Movements in Cardiac Myocytes.
2012.
125. Indrek Reile. 3-Alkylcyclopentane-1,2-Diones in Asymmetric Oxidation
and Alkylation Reactions. 2012.
126. Tatjana Tamberg. Some Classes of Finite 2-Groups and Their
Endomorphism Semigroups. 2012.
127. Taavi Liblik. Variability of Thermohaline Structure in the Gulf of Finland
in Summer. 2012.
128. Priidik Lagemaa. Operational Forecasting in Estonian Marine Waters.
2012.
129. Andrei Errapart. Photoelastic Tomography in Linear and Non-linear
Approximation. 2012.
130. Külliki Krabbi. Biochemical Diagnosis of Classical Galactosemia and
Mucopolysaccharidoses in Estonia. 2012.
131. Kristel Kaseleht. Identification of Aroma Compounds in Food using
SPME-GC/MS and GC-Olfactometry. 2012.
132. Kristel Kodar. Immunoglobulin G Glycosylation Profiling in Patients with
Gastric Cancer. 2012.
133. Kai Rosin. Solar Radiation and Wind as Agents of the Formation of the
Radiation Regime in Water Bodies. 2012.

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134. Ann Tiiman. Interactions of Alzheimer`s Amyloid-Beta Peptides with
Zn(II) and Cu(II) Ions. 2012.
135. Olga Gavrilova. Application and Elaboration of Accounting Approaches
for Sustainable Development. 2012.
136. Olesja Bondarenko. Development of Bacterial Biosensors and Human
Stem Cell-Based In Vitro Assays for the Toxicological Profiling of Synthetic
Nanoparticles. 2012.
137. Katri Muska. Study of Composition and Thermal Treatments of
Quaternary Compounds for Monograin Layer Solar Cells. 2012.
138. Ranno Nahku. Validation of Critical Factors for the Quantitative
Characterization of Bacterial Physiology in Accelerostat Cultures. 2012.
139. Petri-Jaan Lahtvee. Quantitative Omics-level Analysis of Growth Rate
Dependent Energy Metabolism in Lactococcus lactis. 2012.
140. Kerti Orumets. Molecular Mechanisms Controlling Intracellular
Glutathione Levels in Baker’s Yeast Saccharomyces cerevisiae and its Random
Mutagenized Gluthatione Over-Accumulating Isolate. 2012.
141. Loreida Timberg. Spice-Cured Sprats Ripening, Sensory Parameters
Development, and Quality Indicators. 2012.
142. Anna Mihhalevski. Rye Sourdough Fermentation and Bread Stability.
2012.
143. Liisa Arike. Quantitative Proteomics of Escherichia coli: From Relative to
Absolute Scale. 2012.
144. Kairi Otto. Deposition of In2S3 Thin Films by Chemical Spray Pyrolysis.
2012.
145. Mari Sepp. Functions of the Basic Helix-Loop-Helix Transcription Factor
TCF4 in Health and Disease. 2012.
146. Anna Suhhova. Detection of the Effect of Weak Stressors on Human
Resting Electroencephalographic Signal. 2012.
147. Aram Kazarjan. Development and Production of Extruded Food and Feed
Products Containing Probiotic Microorganisms. 2012.
148. Rivo Uiboupin. Application of Remote Sensing Methods for the
Investigation of Spatio-Temporal Variability of Sea Surface Temperature and
Chlorophyll Fields in the Gulf of Finland. 2013.

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