47 
 

Journal homepage: www.fia.usv.ro/fiajournal 
Journal of Faculty of Food Engineering,  

Ştefan cel Mare University of Suceava, Romania  
Volume XVI, Issue 1- 2017, pag. 47  - 53 

 

 

THE RATIONALIZATION OF THE PARAMETERS OF MILK PROTEINS’ 

THERMO ACID COAGULATION BY BERRY COAGULANTS 

 
Olena GREK1, Olena ONOPRIICHUK1, *Tetiana PSHENYCHNA1 

1Educational and Research Institute of Food Technology, National University of Food Technologies, 
01601, Volodymyrska str.68, Kyiv, Ukraine  

Tanya5031@ukr.net  
*Corresponding author 

Received February 24th 2017, accepted March 28th 2017 

 
Abstract: This paper presents the results related to the influence of berry coagulant amount, its 
proactive acidity and duration of thermo acid coagulation on the process of milk proteins’ 
sedimentation. In the present work, the regression equations and response surface analysis were used 
to design and optimize an industrial bioprocess. Increase in the berry coagulant amount to 11 % and 
reduction of active acidity to 2.4 units were determined. pH up to 3 minutes is characterized by the 
highest processes of destabilization. Moreover, it improves the organoleptic properties and has the 
biggest impact on the yield of protein-berry clot (to 25 %) and active acidity. 
 
Keywords: thermo acid coagulation, milk proteins, berry coagulant, protein-berry clots, 
rationalization of parameters 
 
 
1. Introduction 
 
Insufficient amount of dairy raw materials 
and seasonality of milk manufacturing 
affect the volumes of dairy-protein 
products manufacturing (cottage cheese 
and dairy products on its basis, hard and 
soft cheeses, etc.) during the year [1]. The 
acidic, rennet, acid-rennet, thermo calcium, 
thermo acid coagulation and 
ultrafiltrational concentration of milk are 
used in the manufacturing technology of 
these products. The chemical nature of 
milk proteins’ coagulation is complicated 
and not studied in detail up to now. 
According to some researchers, 
fundamental changes of protein 
macrostructure are occurring at 
denaturational transformations. These 
changes are related to the weakening of the 
interaction forces between the protein 
chains of amino acid residues [2-3]. The 
first stage of denaturation consists in 

change of the original type of polypeptide 
chains. This change is caused by different 
methods, but it has a common pattern – the 
gap of the minimal amount of 
intramolecular ties for globule deploying. 
All typical manifestations of denaturation 
are explained by the release of single 
radicals, which in the native state of the 
protein are closely grouped among them. 
Secondary effects after denaturation - 
association of deployed globules and their 
chemical change lead to the allotment of 
proteins. Denatured whey proteins, in the 
form of aggregates with irregular shape, 
surround changed casein particles and 
probably partially connect with it. 
Together with the temperature increase, the 
degree of whey proteins’ denaturation 
increases and a complex of denatured 
whey proteins with casein amplify. The 
influence of high temperature cannot lead 
to coagulation at the pH of whole milk. At 

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Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XVI, Issue 1  – 2017 

Olena Grek, Olena Onopriichuk, Tetiana Pshenychna, The rationalization of the parameters of milk proteins’ thermo acid 
coagulation by berry coagulants, Food and Environment Safety, Volume XVI, Issue 1 – 2017, pag. 47 – 53 
 
 

48

the acidification whey proteins coagulate 
as casein [4-5]. 
From all existing ways of proteins 
allotment only the thermo calcium, thermo 
acid coagulation and ultrafiltrational 
concentration of milk provide 
comprehensive selection of proteins that 
allows receiving dairy-protein product with 
increased biological value. The expansion 
of protein products manufacturing made by 
intensive technologies is actual. In 
particular thermo acid method of milk 
processing, which is based on the 
simultaneous coagulation of milk casein 
and whey proteins under the influence of 
acid and high temperature deserves 
attention. The degree of proteins 
application in this method is up to 
95...97 %, while about 90 % at acid 
coagulation, and about 85 % at rennet [6-
8]. Food acids (hydrochloric, acetic, lactic, 
rarer citric) or acidic whey are used as 
coagulants [9]. The innovative 
developments where berry raw materials 
are used as a coagulant of milk protein are 
known. Berries are a source of vitamins, 
minerals and food fibers [10-11]. 
Berry raw materials coagulants have been 
used more in dairy technology, especially 
in cheese-making. Modern principles of 
high quality products manufacturing are 
based on selection and justification of 
appropriate raw materials kinds in 
proportions which would ensure expected 
quality, consumer and functional 
properties and maximal food components 
balance in chemical composition with 
increased nutritional and biological value. 
The aim of research is to study the 
rationalization of the parameters of milk 
proteins’ thermo acid coagulation with the 
help of mathematical modeling methods. 
Beside this, to study the effect of these 
parameters on the yield and quality 
indicators of protein-berry clot (PBC). 
The object of research is thermo acid 
coagulation of milk proteins by berry 
coagulant. 

Subject of research - skimmed milk, 
blackcurrant puree produced by LiQberry 
(Ukraine Technical Conditions 15.3-
24110704-003:2011), the parameters of 
thermo acid coagulation process, quality 
indicators of protein-berry clots.  
Moreover, selecting an optimum amount of 
berry coagulant with different values of 
active acidity for thermo acid coagulation 
processing and to achieve maximum 
efficiency protein-berry clot has been 
evaluated. 
 
2. Matherials and methods 
 
To achieve the goal the full factorial 
experiment (FFE) was used. Skimmed 
milk with solid mass fraction – 11.2 %, 
protein mass fraction – 3.7 % active 
acidity – 6.7 units was used. pH was 
chosen as a basis for the thermo acid 
coagulation. Blackcurrant puree with 
active acidity 2.8...2.4 units was used. pH 
was used as coagulant. The amount of 
puree is 3...11 % of milk weight. 
Homogenized blackcurrant puree was 
produced in industrial conditions by 
advanced technology, using hydrodynamic 
(cavitation) raw materials, processed by 
devices of TEK-SM. The berries were 
treated in the said above setup according to 
the designed regime until a necessary level 
of homogenization and industrial sterility 
was achieved [12-13]. 
The determination of physico-chemical 
indicators of skimmed milk, protein-berry 
clot and whey was made. Moisture mass 
fraction was determined by drying the 
sample to the constant weight. The active 
acidity of all samples was measured by 
potentiometric pH meter PB-20 Sartorius; 
the protein mass fraction of skimmed milk 
by formalin titration method, which is 
based on previous amino groups of the 
protein neutralization by formalin. Formed 
in this conditions metilenamino acid is 
titrated by 0.1 N of sodium hydroxide 
solution. Water-retaining capacity PBC 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XVI, Issue 1  – 2017 

Olena Grek, Olena Onopriichuk, Tetiana Pshenychna, The rationalization of the parameters of milk proteins’ thermo acid 
coagulation by berry coagulants, Food and Environment Safety, Volume XVI, Issue 1 – 2017, pag. 47 – 53 
 
 

49

was determined by the Grau-Hamm 
method in modification of A. А. Alekseev, 
which is based on the determination of the 
water amount (mass that is allocated from 
the product at its mild pressing) and is 
absorbed by filter paper. 
At the department of milk and dairy 
products technology of the National 
University of Food Technologies the 
technology of protein-berry clots (PBC) 
obtaining, with a pH of clots 5.15...5.25 
was developed. This technology provides 
berry coagulant introduction (pH 2.6±0.2) 

to the milk with the temperature (75±1) °C 
for the process of thermo acid coagulation 
implementation. The qualitative indicators 
of the ready product depend on the 
realization of conditions of the above 
mentioned process. A parameter scheme of 
the process of milk proteins thermo acid 
coagulation by berry coagulants (Fig. 1) 
was prepared; input (controlling) and 
outbound (operated) factors were defined; 
the rationalization criteria were chosen and 
substantiate.

.  
 

 
Fig. 1. Parameter scheme of the process of milk proteins thermo acid coagulation by berry coagulants 

 
For parameters of rationalization, zero 
level of factors was defined. It is based on 
prior information and the interval of 
factors variation in such a way that it was 
outside of measurement error but not too 
wide. 
The Box-Uilson on cube method of the 
mathematical modeling was used in the 
rationalization of the thermo acid 
coagulation process. Determination of the 
functional dependencies was made by the 
least square method. This method is based 
on the determination of the regression 
equation coefficients which provides a 
minimum sum of squared deviations of 
experimental data (Ye) from the values 
calculated by regression equation [14-16]. 
For the role of controlling factors which 
have a significant effect on PBC quality 

indicators were chosen: C1 – amount of 
berry coagulant, %, C2 – active acidity of 
berry coagulant, units. pH, C3 – duration of 
thermo acid coagulation of milk proteins, 
min. Outbound (operated) parameters that 
mostly significantly characterize the 
thermo acid coagulation and have  
influence on the formation of protein berry 
clot quality indicators are: Y1 – mass 
(yield), %, Y2 – active acidity units. pH, Y3 
– moisture mass fraction, %, Y4 – water-
retaining capacity, %. 
The amount of berry coagulant varied in 
the range from 3 to 11 % with discrete 
4 %; active acidity of berry coagulant – 
from 2.4 to 2.8 units. pH; step of varying - 
0.2 units; duration of thermo acid 
coagulation – from 1 min to 3 min with the 
1 min interval. The coefficients of 

• ВС amount , % 
(С1)

• BC active 
acidity, units рН 
(С2)

• Duration of 
thermo acid 
coagulation of
milk proteins, min 
(С3)

Protein-berry 
clot, obtained by 

thermo acid 
coagulation of milk 

proteins 

• PBC mass 
(yield), % (Y1)

• PBC active 
acidity, units рН 
(Y2)

• PBC moisture 
mass fraction, % 
(Y3)

• PBC water-
retaining 
capacity, % (Y4)



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XVI, Issue 1  – 2017 

Olena Grek, Olena Onopriichuk, Tetiana Pshenychna, The rationalization of the parameters of milk proteins’ thermo acid 
coagulation by berry coagulants, Food and Environment Safety, Volume XVI, Issue 1 – 2017, pag. 47 – 53 
 
 

50

regression equation were calculated, whose 
significance analysis was defined by the 
Fisher’s criterion (Fr). The essence of 
criterion consists in decomposition of total 
variance of statistical complex on 
constituent elements. Their next evaluation 
gives the opportunity to determine the 
proportion of effective sign change from 
the effect of factor signs, in other words to 
narrow the area of research. That proves 
the adequacy of resulting equations. 
Experimental data were processed by the 
method of mathematical statistics - 
STATISTIKA (StatSoft) [17]. For 
determination of functional dependence 
which recreates the most accurately the 
change of indicators, the coefficient of 

approximation reliability (R2) of each 
function was found. The accuracy of the 
results obtained is provided by three or 
five time’s repeatability of experiments. 
 
3. Results and discussion 
 
Processing of results allows getting the 
analytical dependencies in the form of 
regression equations to calculate the effect 
of the active acidity, amount of berry 
coagulant and duration of milk proteins’ 
thermo acid coagulation on yield (1) and 
quality indicators of protein-berry clots 
(PBC) – active acidity (2), moisture mass 
fraction (3), and water–retaining capacity 
(4):

 
Y1 (m) = 193,500 + 6,250·Х1 + 3,500·Х1

2 + 27,000·Х2+ 3,500·Х2
2 – 13,600·Х3 

– 3,500·Х3
2 + 7,750·Х1·Х2  + 13,500·Х1·Х3  + 12,250·Х2·Х3 

Вкр і  = 0,530; Вкр іi  = 1,068; Fr = 0,187 

(1) 

Y2 (рН) = 5,229 – 0,309·Х1 – 0,0492·Х2  – 0,066·Х1·Х2 

Вкр і  = 0,149; Вкр іj  = 0,054; Вкр іi  = 0,098; Fr = 1,105 

(2) 

Y3 (W) = 70,254 – 0,647·Х1 – 0,379·Х1
2  – 0,863·Х2 – 0,379·Х2

2  – 0,401·Х3 + 

0,378·Х3
2  + 0,825·Х1·Х2 + 0,557·Х1·Х3 + 0,096·Х2·Х3 

Вкр і  = 0,1; Вкр іi  = 0,1; Fr = 0,031 

(3) 

Y4 (ВУЗ) = 43,383 + 2,552·Х1 +3,182·Х1
2 – 7,349·Х2 +3,187·Х2

2  + 5,278·Х3 – 

3,187·Х3
2  + 1,964·Х1·Х2  – 8,414·Х1·Х3  – 2,504·Х2·Х3 

Вкр і  = 0,095; Вкр іi  = 0,191; Fr = 0,001 

(4) 

The graphical interpretation (of the 
response surface) of the obtained data is 
shown in the form of ternary charts 
(Fig. 2), which are commonly used in 
experimental research of the response 
dependencies from the relative content of 
three options (in this case, amount of BC, 

% (C1) active acidity BC, units. pH (C2), 
duration of milk proteins’ thermo acid 
coagulation, min (C3)). The correlation of 
these parameters is changed in order to 
determine rational values of initial 
parameters (yield (Y1) and quality 
indicators of protein-berry clots (Y2-4)). 

 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XVI, Issue 1  – 2017 

Olena Grek, Olena Onopriichuk, Tetiana Pshenychna, The rationalization of the parameters of milk proteins’ thermo acid 
coagulation by berry coagulants, Food and Environment Safety, Volume XVI, Issue 1 – 2017, pag. 47 – 53 
 
 

51

  
а) b) 

 
 

c) d) 
 

Fig. 2. The response surfaces of protein–berry clot mass change (a), of moisture mass fraction (b), of active 
acidity (c), of water-retaining capacity (d) of protein–berry clot depending on the parameters of milk proteins’ 

thermo acid coagulation 
 
The conducted research has shown that at 
the thermo acid coagulation the increasing 
of berry coagulant (BC) amount decreases 
its pH level and at the increasing of 
process duration, the PBC yield growth 
(Fig. 2a) and the moisture mass fraction 
decrease (Fig. 2b). These changes are 
characterized by the transition of casein 
and whey fraction into the clot. At the 
heating of skimmed milk, whey proteins 
denature in the presence of acid, deploying 
their polypeptide chains. The complexes of 
whey protein and casein, obtained at 
denaturation, capture dry matters (pectin 
matters) of berry coagulant – they are the 

main components that make up the 
structure of the protein–berry clot which 
was obtained by thermo acid coagulation. 
The coefficient of complexes transition 
was about 25 % (duration of exposure - 3 
min and adding 11 % of berry coagulant 
with pH 2.4). Such parameters are 8.2–
10.8 % higher than those at the adding to 
skimmed milk during the berry coagulant 
processing in amount of 3 % and with the 
active acidity of 2.8...2.6. 
As a result of PBC active acidity analysis, 
the general tendency that PBC pH 
decreased linearly at the increasing of 
berry coagulant amount and decrease of its 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XVI, Issue 1  – 2017 

Olena Grek, Olena Onopriichuk, Tetiana Pshenychna, The rationalization of the parameters of milk proteins’ thermo acid 
coagulation by berry coagulants, Food and Environment Safety, Volume XVI, Issue 1 – 2017, pag. 47 – 53 
 
 

52

pH level was set (Fig. 2c). It was 
determined, that by adding 3 % of BC with 
pH 2.8 and 1 min duration of coagulation, 
the process provided the highest value of 
PBC active acidity at the level 6.02. 
Perhaps it is connected with a shift of the 
isoelectric point of the «casein micelles – 
whey proteins» complex. Coagulation 
begins at higher values of pH – (4.7...4.6). 
After coagulation of denatured whey 
proteins, isoelectric point of micelles is 
changed to the level of pH – 5.2, while 
coagulation of milk proteins at a 
temperature of 75 °C, amount of berry 
coagulant 11 % being introduced, thus 
providing the acidity of coagulation 
environment (skimmed milk + BC) at the 
level of about 6 units of pH. 
The PBC samples which were obtained by 
coagulation with 3 % berry coagulant with 
pH 2.8 and exposure time 3 min (Fig. 2d) 
were characterized by the maximum value 
of water–retaining capacity. This is due to 
the fact that the value of the ionic 
adsorption of proteins varies with the 
change of medium reaction. In the 
isoelectric point, when the protein's degree 
of molecules dissociation is minimal and 
the charge of the protein molecule is close 
to zero then the ability of the protein to 
bind water is the smallest. 
With displacement of pH medium in one 
side or another from the isoelectric point, 
the dissociation of basic or acid groups of 
protein is enhanced, the charge of protein 
molecules and protein hydration increases. 
The interaction between the κ-casein and 
whey proteins (β-laktoglobulin) by using 
remnants H-groups and other hydrophobic 
relations occurs. 
Conducted researches have shown that the 
amount and pH of berry coagulant have 
influence on the yield of protein–berry 
clot. The increasing of hydrogen ions 
concentration shifts the equilibrium 
between dissociated carboxyl and 
phosphate groups at the surface of casein 
micelles and hydrogen ions to the side of 

oversaturated carboxyl and phosphate 
groups. Tht is why the decrease in the 
protein particles negative charge occurs. 
Exception makes the negatively charged 
groups of amino groups (–NH3) which are 
situated on the surface of the protein 
particles. At a certain concentration of 
hydrogen ions (pH 4.6...4.7) the alignment 
of negative and positive charges occurs, in 
other words the isoelectric state comes 
(forces of electrostatic repulsion between 
the particles of protein become weaker and 
the forces of intermolecular interaction 
begin to dominate) in which the 
conformational changes of 
macromolecules protein happens. They 
lose solubility and connect among 
themselves forming a meshy three-
dimensional structure of clot or come in 
the form of flakes. Under the action of 
berry coagulant acids the structure of 
caseinate calcium phosphate complex is 
disturbed: phosphate and organic calcium 
in the form of lactate calcium split off from 
the complex and go into plasma. Calcium 
citrate goes in a more soluble calcium 
lactate as well. The smallest yield – 14.2 % 
was observed at the adding of 3 % of BC 
with the active acidity 2.8 and 1 min 
endurance duration. Thus, the process 
occurs due to the initial denaturation of 
serum proteins which installs itself at the 
surface of casein micelles, increases their 
hydrophilic, thus reducing the degree of 
coagulation. The highest values of active 
acidity – 4.35 were obtained at the adding 
of 11 % of BC with pH 2.4. The smallest 
impact on the PBC proteins water–
retaining capacity (23.51 %) was noticed 
under the following conditions of thermo 
acid coagulation: 3 % of BC with pH 2.8 
and 1 min endurance duration.  
 
4. Conclusions 
 
1. This study presents the findings of an 
experimental investigation into the effect 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XVI, Issue 1  – 2017 

Olena Grek, Olena Onopriichuk, Tetiana Pshenychna, The rationalization of the parameters of milk proteins’ thermo acid 
coagulation by berry coagulants, Food and Environment Safety, Volume XVI, Issue 1 – 2017, pag. 47 – 53 
 
 

53

of quality indicators of protein–berry clots 
in obtaining optimal operating conditions. 
2. According to the above mentioned 
findings, the thermo acid coagulation 
during 1 min by berry coagulant which is 
added in amount of 3 % (pH 2.8) is 
characterized by minimal destabilization 
processes, which results in whey proteins 
not settling-down on the casein micelles 
and transfer in serum. While the increasing 
of the amount to 11 %  decreases the active 
acidity to 2.4 units. pH and endurance 
duration to 3 min increase the degree of 
casein and whey proteins transition into 
protein-berry clot. 
 
5. References 
 
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