International Dairy Journal 144 (2023) 105701

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International Dairy Journal

journal homepage: www.elsevier.com/locate/idairyj

Properties of yoghurt treated with microbial transglutaminase

and exopolysaccharides

e
St n Marhons a, *, Ivana Hyr
slova

pa  b, Veronika Stetsenko a, Eva Jablonska
 c,
Martin Veselý d, Hana Míchova

 c, Ladislav Curda a

, Ji
rí Stĕtina a

a
Department of Dairy, Fat and Cosmetics, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, Prague, Czech Republic
b
Department of Microbiology and Technology, Dairy Research Institute Ltd., Prague, Czech Republic
c
Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, Prague, Czech
Republic
d
Department of Organic Technology, Faculty of Chemical Technology, University of Chemistry and Technology, Prague, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history: The focus of our study was to observe the simultaneous effects of microbial transglutaminase (MTG)
Received 21 November 2022 treatment and exopolysaccharides (EPS) on the rheological and microstructural properties of a yoghurt.
Received in revised form Transglutaminase was applied to milk before fermentation and simultaneously with a yoghurt culture.
2 May 2023
Yoghurts were fermented in short-term (42
C, 6e8 h) and long-term (30
C, 16e18 h) fermentations
Accepted 3 May 2023
with a EPS-producing culture that produces EPS and a non-EPS-producing that does not. Treatment with
Available online 12 May 2023
transglutaminase caused an increase in gel strength and viscosity, especially in the case of simultaneous
application of MTG and culture. Furthermore, the presence of EPS enhanced these properties. New three-
dimensional structures of proteins decreased the size of pores between protein chains, which led to
improved water binding properties. Syneresis was further reduced by the presence of EPS. Treatment of
yoghurts with MTG decreased the extensive ropiness of EPS yoghurts and contributed to a higher
acceptability of the texture.
© 2023 Elsevier Ltd. All rights reserved.

1. Introduction
In recent decades, a wide range of substances and additives have
been used in food production (e.g., to improve rheological prop-
erties, taste, flavour, overall appearance). However, these sub-
stances faced resistance from a substantial proportion of the
consumers. Therefore, the effort of the food manufactures is to
avoid the use of these substances and find other methods. One of
the ways is modification of the product by enzymatic treatment,
which is being used extensively (de Go es-Favoni & Bueno, 2014).
In the dairy industry, microbial transglutaminase (EC 2.3.2.13)
(MTG), which catalyses an acyl transfer reaction between g-car-
boxyamide of peptide- or protein-bound glutamine and a primary
amine of lysine forming isopeptide bonds, has been widely tested
(Romeih & Walker, 2017). The most susceptible proteins of the
native casein fraction in micelles are k-caseins, which are located
on the surface of the casein micelle. One molecule of k-casein
contains four potentially available glutamine residues (Christensen,
* Corresponding author.

Marhons).
E-mail address: marhonss@vscht.cz (S.
Sørensen, Højrup, Petersen, & Rasmussen, 1996; Tang, Yang, Chen,
Wu, & Peng, 2005). By cross-linking casein fractions, new bonds
are formed within the protein chains. Newly formed milk protein
polymers affect the textural and rheological properties of fer-
mented milk products. Cross-linking of milk proteins by MTG leads
to the formation of narrow pores between the peptide chains,
where water is retained. New peptide structures and water reten-
tion also cause higher firmness and viscosity of yoghurts (Lorenzen,
Neve, Mautner, & Schlimme, 2002). Most studies confirmed the
positive impact of MTG on the rheological properties of yoghurt,
including syneresis. Therefore, the application of the trans-
glutaminase can be used instead of increasing milk dry matter by
the addition of skim milk powder or using the hydrocolloids (Guyot
& Kulozik, 2011; Romeih, Abdel-Hamid, & Awad, 2014; Ziarno &
Zare˛ ba, 2020).
Exopolysaccharides (EPS) that are produced by various strains of
yogurt cultures have similar effects to MTG on the textural prop-
erties of fermented dairy products, including increased firmness
and viscosity and decreased syneresis (Mende, Rohm, & Jaros, 2016;
Tiwari, Kavitake, Devi, & Shetty, 2021). Exopolysaccharides are
produced by bacteria, in capsular or free forms. Capsular EPS
remain attached to the bacterial cell wall after synthesis and free

https://doi.org/10.1016/j.idairyj.2023.105701
0958-6946/© 2023 Elsevier Ltd. All rights reserved.

, V. Stetsenko et al.
S. Marhons, I. Hyr
slova
EPS are released from the cell wall to medium (Mende et al., 2016).
Different types of lactic acid bacteria synthesise EPS with different
compositions, chain lengths, degrees of branching, and charge. This
variability is mostly species-specific but also strain-specific, and the
composition of EPS may vary depending on fermentation condi-
tions (S
anchez, Martínez, Jime nez-Díaz, & Rodríguez, 2006).
Descriptions of the individual effects of MTG treatment and EPS
on the rheological properties of dairy products has already been
reported by Şanli, Şenel, Sezgin, and Benli (2014). However, there is
insufficient information on the combined effects of MTG treatment
and EPS. Furthermore, effects of short- and long-term fermentation
of yoghurts treated with microbial transglutaminase have not been
compared. Therefore, the focus of our study was to observe the
effect of microbial transglutaminase treatment of yoghurt at
various stages of the production process with or without EPS-
producing bacterial strains to achieve improved gel properties
and lower the syneresis of the yoghurt. We focused on determining
the effect of various enzyme concentrations (0, 0.1, 0.5 and 1.0 U g1
of protein) on the microbiological and functional properties of
yoghurt, with and without inactivation during the production
process. Furthermore, the influence of duration, temperature of
fermentation (short-term and long-term fermentation), and a
comparison of cultures producing and not-producing exopoly-
saccharides used in MTG treated and non-treated yoghurts were
evaluated.
Fig. 1. Flowchart of yoghurt samples preparation; MTG, microbial transglutaminase.
2
International Dairy Journal 144 (2023) 105701
2. Materials and methods
2.1. Yoghurt preparation
All yoghurt samples were made from UHT milk with 1.5 g fat
100 g1 milk (Mle  k
arna Pragolaktos a.s., Czech Republic). The
content of dry matter in the milk was increased by adding skimmed
milk powder at a level of 50 g L1 (Moravia Lacto a.s., Czech Re-
public). The milk was then mixed thoroughly and pasteurised at
85
C after reaching temperature for 10 min in a water bath. The pH
of the milk was 6.4 ± 0.05 after pasteurisation. The composition of
milk for yoghurt production was analysed with a Milkoscan IR
analyser (Foss, Denmark) that was calibrated on cow milk with
composition ranges (100 g1 milk): fat 2.8e5.5 g, protein 3.2e3.7 g,
lactose 4.6e5.3 g and dry matter 11.8e14.3 g. Calibration was per-
formed according to ISO 8196-2:2009jIDF 128-2:2009 (ISO, 2009).
Average composition of the milk used for yoghurt manufacture
(100 g1 milk): dry matter 13.95 ± 0.80 g; fat 1.50 ± 0.04 g; protein
5.12 ± 0.03 g and lactose 7.73 ± 0.09 g.
For enzymatic treatment a microbial transglutaminase SAP-
RONA Texture LX2 (C&P Group, Germany) with declared activity
100 U g1 containing lactose and 1.1% (w/w) of protein was used.
Transglutaminase was applied at doses of 0.1, 0.5 and 1.0 U g1 of
protein was applied in two ways (Fig. 1). First, MTG was applied to
milk before pasteurisation at 30
C for 2 h. In the second case, MTG

, V. Stetsenko et al.
S. Marhons, I. Hyr
slova
was applied after pasteurisation simultaneously with the yoghurt
culture. Milk was inoculated with 1.0 mL 100 mL1 of yoghurt
culture for short-term fermentation and 0.1 mL 100 mL1 for long-
term fermentation. The yoghurts were fermented at 42
C for 6e8 h
(short-term) and at 30
C for 16e18 h (long-term) in a 100 mL cup.
Yoghurt without MTG was used as a control sample.
2.2. Yoghurt bacteria
For yoghurt production, cultures from the Culture Collection of
Dairy Microorganisms (CCDM) (Laktoflora®, Czech Republic) were
used: yoghurt culture CCDM 22 RX (non-EPS-producing) and a
mixed culture of Streptococcus thermophilus CCDM 144 and Lacto-
bacillus delbrueckii subsp. bulgaricus CCDM 767 (EPS-producing)
with proven production of exopolysaccharides. The mixed culture
for yoghurt production was prepared in a ratio of 2:1.
2.3. Effect of MTG on the viability of yoghurt bacteria
The cell counts of yoghurt bacteria were determined according
to ISO 7889:2003 (ISO, 2003). Cell counts of L. delbrueckii subsp.
bulgaricus were determined on MRS agar (Thermo Fisher Scientific,
USA) by anaerobic cultivation at 37
C for 72 h and cell counts of S.
thermophilus were determined by aerobic cultivation at 37
C for
48 h. Yoghurt pH measurements were performed with a WTW
3310 pH meter (WTW, Germany) with a SenTix 41 (WTW, Ger-
many) combined electrode.
2.4. Degree of polymerisation
The procedure to evaluate the degree of polymerisation of the
yoghurt casein fraction was performed by modifying the method by
Bo€nisch, Lauber, and Kulozik (2004). Yoghurt samples were neu-
tralised to pH 7 with 1 M sodium hydroxide. To remove fat, the
samples were centrifuged at 2370  g for 30 min in a Universal 320
R centrifuge (Hettich Zentrifugen, Germany) at 22 ± 2
C. After
Fig. 2. Size-exclusion chromatogram of untreated and treated (short-term fermentation, 0.5 U g1) yoghurts
simultaneously with culture , molecular mass standard (DTT, dithiothreitol).
3
International Dairy Journal 144 (2023) 105701
centrifugation, the cream was removed from the sample surface.
The sample preparation continued with acid precipitation of the
casein fraction using acetic acid (concentration 10 mL 100 mL1) at
pH 4.6. After precipitation, the samples were centrifuged at
2370  g for 30 min at room temperature. The supernatant was
collected, and the sediment of the casein fraction was washed with
demineralised water and centrifuged (twice in total). The washed
sediment (2.25 g) was transferred to a PottereElvehjem glass
homogeniser, which was then filled with demineralised water to a
total weight of 15 g. The entire volume was homogenised and then
pH value of the solution was adjusted to 6.8 using 1 M sodium hy-
droxide. The caseinate obtained was mixed with sample buffer
(containing the mobile phase with the addition of 1 g L1 DTT) in a
1:1 ratio, left overnight in a refrigerator (4
C) and filtered with a
Watrex 0.45 mm MCE syringe filter prior to analysis.
The determination was processed on an AKTA € Pure (GE
Healthcare, USA) protein preparative purification chromatography
system (size-exclusion) with a Superdex 200 Increase 10/300 col-
umn (GE Healthcare). For the separation, the flow rate of the mobile
phase was 0.50 mL min1 (0.1 M sodium phosphate buffer pH 6.8,
containing 6 M urea, 0.1 M sodium chloride, and 0.1 g 100 g1
CHAPS), with 50 mL of sample injection. Protein fractions were
determined using a UV detector at 280 nm. Measured data were
evaluated using the UNICORN 7.0 program (GE Healthcare). Peak
areas in the retention times range of 14e21 min were evaluated
from the chromatogram as oligomers according to Raak, Abbate,
Lederer, Rohm, and Jaros (2018). Monomers were evaluated as
peak areas at retention times of 24e26 min by performing the trial
with not-treated milk by transglutaminase (Fig. 2). The degree of
polymerisation was calculated according to the following equation
(1).
Aoligomers
Degree of polymerisation ð%Þ ¼ $100 (1)
Atotal
, no microbial transglutaminase , microbial transglutaminase

, V. Stetsenko et al.
S. Marhons, I. Hyr
slova
where Aoligomers is the peak area of oligomers and Atotal is the total
peak area of oligomers and monomers of casein.
The molecular mass of the polymers was estimated by
comparing with the Protein Standard Mix 15e600 kDa (Sigma-
Aldrich, USA).
2.5. Gel strength
The gel strength of the yoghurts was determined using a Vis-
cotester iQ rheometer (Thermo Haake, Germany) with a FL22 vane
geometry at 5
C. Torque data were expressed as apparent stress by
multiplying by the factor 5642  104 Pa N1 m1 stated by the
manufacturer for the given geometry dimensions (Krulis & Rohm,
2004).The determination of the time dependence of apparent
stress at 100 rpm was performed. Local maximum of the apparent
stress at the beginning of the measurement was indicated as gel
strength. The evaluation of the gel strength of the yoghurt was
performed one day after manufacture and after 21 days of storage
at 5
C by two independent trials (each with five measurements).
2.6. Syneresis
The syneresis was determined as water loss in 15 mL centrifuge
tubes at 5
C. The tubes were centrifuged at 3000  g using an EBA
200 centrifuge (Hettich GmbH & Co. KG, Germany) for 3 min. The
evaluation of syneresis was determined as the amount of whey
released relative to the total amount of fermented product.
2.7. Determination of microstructure
The microstructure was determined by scanning electron mi-
croscopy (SEM) and confocal laser scanning microscopy (CLSM). For
SEM, samples were prepared according to Brodziak et al. (2021).
Each yoghurt sample was mixed with an equal weight of Noble agar
solution (2.5 g 100 g1) in a microwell plate and stored in the
refrigerator to solidify. After cooling, samples were cut into
2  2  2 mm cubes with a razor blade and immersed in a 4 mL
100 mL1 glutaraldehyde solution in 1.0 M phosphate buffer (pH
7.0) overnight. The fixed samples were washed twice with phos-
phate buffer (pH 7.0) and dehydrated by a graded ethanol series of
30, 50, 70, 90 and 99.8 mL 100 mL1 for 20 min intervals. After
dehydration, the samples were dried using the Leica EM CPD300
critical point dryer (Leica Microsystems, Germany). Parameters
were set according to the CPD300 auto Program protocol “Human
blood cells” described in the application booklet provided by the
manufacturer. The sample morphology was investigated using a
FEG electron gun FIB-SEM TESCAN LYRA3GMU (Tescan, Czech Re-
public) at an acceleration voltage of 5 kV. Prior to SEM measure-
ment, the sample was placed on a carbon conductive tape and
coated with a 10 nm thin gold layer in a sputter coater, Quorum
Q150R S (Leica Microsystems, Germany).
For CLSM, samples were placed in 96-well glass bottom black
plate wells with high-performance cover glass (Cellvis, Canada) and
stained with 20 mL of 0.01 mg mL1 FastGreen FCF (Sigma-Aldrich,
USA). The plates were observed with an inverted confocal micro-
scope with a spinning disc Olympus IX81F-ZDC2 (Olympus, Japan)
equipped with Andor IQ software (Andor, UK) using an objective
Clara 100 with oil immersion. Representative areas were recorded
as series of z-stacks with a z-step selected according to Nyquist
sampling (0.2 mm) in a far-red channel (excitation/emission of 640/
715 nm). The z-stacks were processed using IMARIS x64 8.0.0
software (Bitplane, UK) using maximum intensity projection.
4
International Dairy Journal 144 (2023) 105701
2.8. Sensory analysis
Sensory evaluation was performed by an eleven-member panel
composed of staff and students of the Department of Dairy, Fats and
Cosmetics UCT Prague. The following descriptors were rated by
seven-point scales: viscosity during stirring, ropiness, overall
texture acceptance, intensity of yoghurt aroma, overall taste and
odour acceptance. Results were expressed as median ratings. The
sensory panel noted any yoghurt defects identified. Rating scales
were from 1 to 7 and the respective extremes defined as follows:
viscosity and ropiness, very low to very high; texture acceptance,
very bad, unacceptable to outstanding; aroma and taste intensity,
imperceptible to very strong; taste and odour acceptance, very bad,
unacceptable to outstanding. Additionally, a ranking test was per-
formed on selected product combinations in terms of overall
product preference according to ISO 8587:2006 (ISO, 2006).
2.9. Statistical analysis
Data were statistically analysed with the Microsoft Excel
desktop application (Microsoft Corporation, USA) using the two-
way ANOVA method to compare significant differences among
data sets and Student's t test to compare significant differences
between individual samples. The significance level of the statistical
analysis was kept at 0.05. For the determination of statistically
significant differences between sensory evaluation samples, a
KruskaleWallis test was also used with a set level of significance
0.05. The KruskaleWallis test was performed using Minitab 17.0
software (Minitab LLC, USA). The overall product preference was
evaluated using the Friedman ranking test performed by the
Microsoft Excel. Average and standard deviation values are deter-
mined from two independent trials: 2 measurements for total
bacteria cell counts, pH, degree of polymerisation; 3 measurements
for syneresis; 5 measurements for the gel strength.
3. Results and discussion
3.1. Effect of MTG on the viability of yoghurt bacteria
Several studies point to a slightly negative effect of MTG on the
growth and viability of yoghurt bacteria S. thermophilus and L. del-
brueckii subsp. bulgaricus. Neve, Lorenzen, Mautner, Schlimme, and
Heller (2001) reported a decrease in the number of colonies of
L. delbrueckii subsp. bulgaricus in yoghurt samples treated with MTG
preincubation at 40
C for 2 h with thermal inactivation of enzyme. A
possible reason for the decrease in the cell counts of lactobacilli is
the reduction in the availability of nitrogen sources, low molecular
weight proteins, peptides and simple amino acids. In contrast, a
recent study of Dinkci (2012) did not prove a significant effect of
MTG concentration (0.74; 1.29 and 1.85 U g1 protein) on the
viability of cultures during 14 days of storage.
Observations of the growth of yoghurt bacteria (Table 1)
revealed that the enzyme dosage and the method of applying MTG
did not have a significant effect on the growth of yoghurt bacteria
(P > 0.05). Lower counts after storage were recorded only in the
case of simultaneous addition of enzyme and culture, specifically
non-EPS-producing culture with 1 U g1 of protein and EPS-
producing culture with 0.1 U g1 of protein for short-term
fermentation and non-EPS-producing with 0.1 U g1 of protein
and EPS-producing culture with 0.5 U g1 of protein for long-term
fermentation. The effect of transglutaminase treatment on bacterial
growth was not conclusive. Values of pH of all yoghurts ranged
from 4.06 to 4.61 after manufacture and it was reduced to
4.01e4.48 after 21 days of storage. Dependence of pH on MTG
treatment was not proven as statistically significant (P > 0.05) for

, V. Stetsenko et al.
S. Marhons, I. Hyr
slova International Dairy Journal 144 (2023) 105701

Table 1
Total yoghurt bacteria counts log (cfu g1) and pH values of short and long-term fermented yoghurts after manufacture and after 21 days of storage.a

Conditions and culture MTG (U g1 protein) After manufacture After 21 days of storage

log (cfu g1) pH yoghurt log (cfu g1) pH yoghurt

Short-term fermentation
Control
Non-EPS-producing 8.68 ± 0.07a,A 4.29 ± 0.18a,A 8.47 ± 0.26a,A 4.24 ± 0.37a,A
EPS-producing 8.63 ± 0.44a,A 4.44 ± 0.32a,A 8.48 ± 0.55a,A 4.16 ± 0.04a,A

Pre-incubation with MTG

Non-EPS-producing 0.1 8.68 ± 0.07a,A 4.06 ± 0.09a,A 8.52 ± 0.19a,A 4.01 ± 0.06a,A
0.5 8.84 ± 0.04a,A 4.09 ± 0.04a,A 8.65 ± 0.07a,A 4.10 ± 0.07a,A
1.0 8.84 ± 0.13a,A 4.50 ± 0.55a,A 8.63 ± 0.12a,A 4.05 ± 0.04a,A
EPS-producing 0.1 8.36 ± 0.68a,A 4.40 ± 0.04a,A 8.23 ± 0.50a,A 4.21 ± 0.11a,A
0.5 8.30 ± 1.02a,A 4.27 ± 0.06a,A 8.16 ± 0.42a,A 4.22 ± 0.08a,A
1.0 8.27 ± 0.02b,A 4.28 ± 0.07a,A 8.09 ± 0.27a,A 4.21 ± 0.11a,A

Simultaneous addition of MTG with culture

Non-EPS-producing 0.1 8.88 ± 0.10a,A 4.18 ± 0.06a,A 8.78 ± 0.04a,A 4.19 ± 0.10a,A
0.5 8.75 ± 0.03a,A 4.31 ± 0.23a,A 8.63 ± 0.14a,A 4.34 ± 0.46a,A
1.0 8.65 ± 0.11a,c,A 4.19 ± 0.08a,A 8.01 ± 0.89a,B 4.04 ± 0.04a,A
EPS-producing 0.1 8.66 ± 0.08a,c,A 4.29 ± 0.03a,A 8.36 ± 0.05a,B 4.47 ± 0.11a,A
0.5 8.68 ± 0.07a,A 4.61 ± 0.71a,A 8.47 ± 0.26a,A 4.25 ± 0.13a,A
1.0 8.68 ± 0.07a,A 4.28 ± 0.05a,A 8.52 ± 0.19a,A 4.23 ± 0.02a,A
Long-term fermentation
Control
Non-EPS-producing 8.41 ± 0.28a,A 4.27 ± 0.21a,A 8.44 ± 0.22a,A 4.48 ± 0.04a,A
EPS-producing 8.99 ± 0.00a,A 4.44 ± 0.09a,b,A 8.75 ± 0.47a,A 4.23 ± 0.14a,A

Pre-incubation with MTG

Non-EPS-producing 0.1 8.31 ± 0.28a,A 4.13 ± 0.01a,A 8.75 ± 0.06a,A 4.07 ± 0.03b,c,A
0.5 8.76 ± 0.20a,A 4.15 ± 0.04a,A 8.68 ± 0.06a,A 4.17 ± 0.16a,c,A
1.0 8.48 ± 0.34a,A 4.14 ± 0.04a,A 8.82 ± 0.08a,A 4.14 ± 0.10b,c,A
EPS-producing 0.1 8.90 ± 0.01b,A 4.35 ± 0.10a,b,A 8.81 ± 0.33a,A 4.18 ± 0.06a,A
0.5 8.96 ± 0.02a,c,A 4.30 ± 0.18a,b,A 8.90 ± 0.36a,A 4.21 ± 0.06a,A
1.0 8.65 ± 0.52a,b,A 4.17 ± 0.01a,A 8.24 ± 0.26a,A 4.17 ± 0.06a,A

Simultaneous addition of MTG with culture

Non-EPS-producing 0.1 8.44 ± 0.02a,A 4.31 ± 0.25a,A 8.66 ± 0.02a,B 4.43 ± 0.44a,c,A
0.5 8.25 ± 0.28a,A 4.64 ± 0.69a,A 8.54 ± 0.30a,A 4.05 ± 0.05b,c,A
1.0 8.45 ± 0.54a,A 4.44 ± 0.44a,A 8.31 ± 0.32a,A 4.23 ± 0.30a,c,A
EPS-producing 0.1 8.89 ± 0.05a,b,A 4.36 ± 0.13a,b,A 8.82 ± 0.16a,A 4.35 ± 0.06a,A
0.5 9.02 ± 0.13a,b,A 4.28 ± 0.01b,A 8.56 ± 0.04a,B 4.20 ± 0.05a,A
1.0 8.92 ± 0.01b,c,A 4.32 ± 0.04b,A 8.26 ± 1.25a,A 4.24 ± 0.04a,A
a
Cultures are: non-EPS-producing, yoghurt culture CCDM RX 22; EPS-producing, a mixed culture of Streptococcus thermophilus CCDM 144 and Lactobacillus delbrueckii
subsp. bulgaricus CCDM 767. Abbreviation: MTG, microbial transglutaminase. Values are the mean ± standard deviation of 2 independent trials (each trial, 2 measurements);
values with different lowercase superscript letters in a column are significantly different (P < 0.05) within the culture type and fermentation type (short- and long-term);
values with different superscript uppercase letters indicate significantly different (P < 0.05) values of the given parameter one day after manufacture and after storage
within the culture type and fermentation type (short- and long-term).

both types of application: preincubation, simultaneously with
culture. The same was reported by Neve et al. (2001) when the
value of pH during fermentation did not result in a significant
change of the acidification activity in the enzyme-treated milk at
the end of fermentation. Also, dependence of pH on fermentation
type (short-long-term) after manufacture was not statistically sig-
, Piot,
nificant (P > 0.05). On the contrary, Laligant, Famelart, Brule
and Paquet (2003) reported a lower pH value in the case of long-
term fermentation at 30
C for 18 h. Swelam, Rashed, and
Khames (2019) reported higher pH values of yoghurts fermented
by culture producing EPS in comparison with control yoghurt cul-
ture. On the other hand, the difference between cultures used was
not statistically significant in this study, probably due to using
different EPS producing culture type. Comparing the pH before and
after 21 days of storage did not reveal a statistically significant
difference between yoghurt samples (P > 0.05).
3.2. Degree of polymerisation
The extent of the MTG reaction was evaluated by determining
the degree of polymerisation (DP) of the proteins by size-
exclusion chromatography (Fig. 2). From the catalysed reaction,
oligomers with an approximate molecular mass up to 670 kDa
were formed. The influence of yoghurt culture on DP was not
statistically significant (P > 0.05) for both short-term and long-
term fermentations (Fig. 3). It can be assumed from this finding
that the exopolysaccharides produced by a EPS-producing culture
did not affect the protein polymer content. However, in both fer-
mentations (short and long-term), a higher DP was recorded for
the simultaneous application of MTG. In these samples MTG
remained active, so the crosslinking of proteins could continue
during fermentation. An increase in DP of milk proteins was
measured with a higher MTG dose in both fermentations (short-
and long-term). The highest DP of short-term fermentation (43%)
was observed using 1 U g1 of MTG simultaneously added with
culture. The MTG gradually loses its activity at a pH of <5.0 (Ando
et al., 1989).
The growth of yoghurt bacteria is faster at temperatures above
40
C and more lactic acid is produced which leads to decrease of
pH value than at temperature 30
C (Laligant et al., 2003; Nguyen,
Ong, Kentish, & Gras, 2014). Therefore, polymerisation reaction
could continue for a longer period and application of 1 U g1 of

5

, V. Stetsenko et al.
S. Marhons, I. Hyr
slova International Dairy Journal 144 (2023) 105701

Fig. 3. Degree of polymerisation of yoghurts treated with microbial transglutaminase (MTG) (Control, samples without MTG) manufactured by short-term fermentation (A) and
long-term fermentation (B) one day after manufacture: ( ), non-EPS-producing yoghurt culture CCDM RX 22; ( ), EPS-producing mixed culture of Streptococcus thermophilus CCDM
144 and Lactobacillus delbrueckii subsp. bulgaricus CCDM 767. Values with different letters above the column are significantly different (P < 0.05) within the type of culture used. Bars
represent the average value of two independent trials (each trial 5 measurements); error bars represent the standard deviations.

protein with culture resulted in almost 50% DP in the case of long-
term fermentation. The highest polymerisation value obtained in
this study was similar to results of Bo €nisch et al. (2004) who
recorded 46% protein polymerisation with a higher dose of MTG
(3 U g1), but with a lower incubation temperature of 40
C. In
another study of Bo €nisch, Lauber, and Kulozik (2007), the authors
recorded approximately 50% DP of caseinate and a 30% DP of
micellar casein using the same incubation conditions. In our study,
a statistically significant difference (P < 0.05) between methods of
MTG application was demonstrated in the case of long-term
fermentation.
3.3. Syneresis
The whey release on the top of yoghurt products can be a
negative aspect to consumer preferences. Most studies point to a
reduction in syneresis of yoghurts after MTG treatment. Treatment
of milk with transglutaminase led to the formation of a modified
protein structure with narrower pores. In these pores, water was
retained, which led to decreasing syneresis of the product
(Domagała, Wszołek, Tamime, & Kupiec-Teahan, 2013; García-
Gomez, Romero-Rodríguez, Va zquez-Ode riz, Mun
~ oz-Ferreiro, &
zquez, 2018; Pakseresht, Mazaheri Tehrani, & Razavi, 2017).
Va

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Fig. 4. Syneresis of yoghurts treated with microbial transglutaminase (MTG; 0.1, 0.5 and 1 U g1 of protein; Control, samples without MTG) manufactured by short-term
fermentation with ( ) non-EPS-producing yoghurt culture CCDM RX 22 and ( ) EPS-producing mixed culture of Streptococcus thermophilus CCDM 144 and Lactobacillus del-
brueckii subsp. bulgaricus CCDM 767 one day after manufacture (A) and after 21 days of storage (B). Values with different letters above the column are significantly different
(P < 0.05) within the type of culture used. Bars represent the average value of two independent trials (each trial 3 measurements); error bars represent the standard deviations.

In our study, a decrease in the amount of whey released was
identical to that in the publications described. All untreated yoghurts
expressed a higher level of syneresis than almost all treated samples.
A comparison of cultures showed a difference in favour of a mixed
EPS-producing culture that produces exopolysaccharides, with
significantly lower (P < 0.05) syneresis, compared with non-EPS-
producing yoghurts for both types of fermentation. Exopoly-
saccharides modified the creation of a protein network during gel-
ling by increasing the viscosity of the aqueous phase, probably
leading to the creation of new structures where water (whey) per-
sists and is retained. The influence of EPS on structural modification
was also dependent on its charge and molecular weight (Mende
et al., 2016). The results of short-term fermentation (Fig. 4)
revealed a statistically significant effect of MTG on syneresis in the
case of non-EPS-producing culture (P < 0.05), where syneresis
decreased with higher enzyme dose. However, in the case of EPS-
producing culture, this dependence was not proven (P > 0.05). The
presence of EPS with MTG treatment after long-term and short-term

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Fig. 5. Syneresis of yoghurts treated with microbial transglutaminase (MTG; 0.1, 0.5 and 1 U g1 of protein; Control, samples without MTG) manufactured by long-term
fermentation with ( ) non-EPS-producing yoghurt culture CCDM RX 22 and ( ) EPS-producing mixed culture of Streptococcus thermophilus CCDM 144 and Lactobacillus del-
brueckii subsp. bulgaricus CCDM 767 one day after manufacture (A) and after 21 days of storage (B). Values with different letters above the column are significantly different
(P < 0.05) within the type of culture used. Bars represent the average value of two independent trials (each trial 3 measurements); error bars represent the standard deviations.

fermentation caused a significant decrease in syneresis below 5% in 3.4. Gel strength

all treatments. The method of simultaneous application of MTG and
culture caused a more extensive reduction in syneresis than pre-
incubation, probably due to the presence of an active enzyme during
fermentation, which led to a higher DP (Fig. 3) in the case of short-
term fermentation. Long-term fermentation (Fig. 5) led to lower
syneresis of all treated yoghurts compared with short-term yo-
ghurts. The influence of storage on the syneresis of non-EPS-
producing fermented yoghurts was not statistically significant
(P > 0.05).
The gel strength of unstirred yoghurt according measurement
conditions was evaluated. Gel strength values of the short-term
fermented yoghurts are shown in Fig. 6 and for the long-term
fermented yoghurts in Fig. 7. All of the following comments are
based on testing the statistically significant differences using the
two-way ANOVA method within the MTG application method,
fermentation type (short- and long-term), the effect of the used
culture and the effect of storage. All preincubated yoghurt

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Fig. 6. Gel strength treated of yoghurts treated with microbial transglutaminase (MTG; 0.1, 0.5 and 1 U g1 of protein; Control, samples without MTG) manufactured by short-term
fermentation with ( ) non-EPS-producing yoghurt culture CCDM RX 22 and ( ) EPS-producing mixed culture of Streptococcus thermophilus CCDM 144 and Lactobacillus delbrueckii
subsp. bulgaricus CCDM 767 one day after manufacture (A) and after 21 days of storage (B). Values with different letters above the column are significantly different (P < 0.05) within
the type of culture used. Bars represent the average value of two independent trials (each trial 5 measurements); error bars represent the standard deviations.

samples with MTG (short-term and long-term fermentation)
expressed an increase in the gel strength compared with control
samples. However, increasing the dose of MTG no longer had an
obvious effect (P > 0.05), and the samples showed similar prop-
erties. The reason could be the polymerisation of k-casein by the
action of a lower MTG level, mainly on the surface of the casein
micelles (Huppertz & de Kruif, 2007), where small changes in the
properties of the gel occurred (Pakseresht et al., 2017). A higher
degree of polymerisation can, on the other hand, reduce the
stiffness of the gel and the viscosity of the coagulum (Jaros,
€tzold, Schwarzenbolz, & Rohm, 2006) probably due to an in-
Pa
crease in the cohesion of the micelles.
In contrast, MTG dose had a significant effect (P < 0.05) where
MTG was added simultaneously with non-EPS-producing culture in
the case of the short-term fermentation and also in the case of the
long-term fermentation for both culture types used. The explana-
tion may be that during acidification, when colloidal calcium
phosphate present in casein micelles is firstly solubilised and then
proteins of casein fraction dissociate (Dalgleish, Alexander, &
Corredig, 2005), the casein fractions within the micelle were also
more extensively cross-linked; this would have had a greater effect
on the gel strength, particularly if there was sufficient casein
polymerisation. However, independent trials differed in the final
pH values (Table 1) of the yoghurts, thus the gel strength values

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Fig. 7. Gel strength treated of yoghurts treated with microbial transglutaminase (MTG; 0.1, 0.5 and 1 U g1 of protein; Control, samples without MTG) manufactured by long-term
fermentation with ( ) non-EPS-producing yoghurt culture CCDM RX 22 and ( ) EPS-producing mixed culture of Streptococcus thermophilus CCDM 144 and Lactobacillus delbrueckii
subsp. bulgaricus CCDM 767 one day after manufacture (A) and after 21 days of storage (B). Values with different letters above the column are significantly different (P < 0.05) within
the type of culture used. Bars represent the average value of two independent trials (each trial 5 measurements); error bars represent the standard deviations.

were deviated because the rheological properties of acid coagu-
lated gels is dependent on the pH (Renan et al., 2009). Simulta-
neously treated yoghurts with 0.1 U g1 MTG achieved lower values
of the gel strength than control samples in the case of the long-term
fermentation. It appears that with this method of fermentation and
the activity of MTG, a small degree of protein polymerisation may
have had a disruptive effect on gel formation. However, there is not
much information about this finding and this should be the focus of
further research.
Results further confirmed the observed influence of EPS on the
rheology of fermented dairy products described in most other
studies (Şanli et al., 2014; Tiwari et al., 2021). The samples fer-
mented by EPS-producing culture, independent of the action of
MTG, had a significantly higher gel strength which corresponded
10
with the significantly lower syneresis (P < 0.05) of the yoghurts
fermented by EPS-producing culture. The presence of the EPS did
not affect (P > 0.05) the MTG preincubation treatment in the case of
the short- and long-term fermentation. On the other hand, the
difference was found comparing the short- and long-term
fermentation with the simultaneous application of MTG and EPS-
producing culture where the long-term fermented yoghurts
showed increasing gel strength values with higher MTG dose
(P < 0.05). Nevertheless, in the case of the short-term fermentation
the increasing gel strength was not observed within the higher
MTG dose.
From the point of view of the fermentation conditions, signifi-
cantly higher values of the gel strength (P < 0.05) were recorded
after short-term fermentation. This may have been caused by a

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higher incubation temperature that promotes a more rapid creation
of the protein network during fermentation, leading to the for-
mation of narrower pores and a stiffer structure (Haque,
Richardson, & Morris, 2001; Khanal & Lucey, 2018; Nguyen et al.,
2014). Similar results were achieved in our study by visualisation
of microstructure in Section 3.5. Generally, MTG treatment was not
dependent on the yoghurt fermentation conditions (P > 0.05).
Overall, From the results obtained after 21 days of yoghurt
storage, a good stability of all MTG treated yoghurts was observed
as the gel strength values did not significantly changed (P > 0.05)
within short-term and long-term fermentations. Insignificant dif-
ferences between the individual samples of the simultaneous MTG
application with culture after storage could be caused by the re-
sidual activity of the MTG in yoghurt despite low pH value and
storage temperature.
3.5. Microstructure
The method of sample preparation used for microstructure
determination by SEM according to Brodziak et al. (2021) may have
an effect on the true microstructure of the yoghurt by agar used in
procedure. However, this method was also published previously by
Sandoval-Castilla, Lobato-Calleros, Aguirre-Mandujano, and
Vernon-Carter (2004) and also similar by Fang and Guo (2019),
Wang, Wang, Wang, and Guo (2017), and Zhang, McCarthy, Wang,
Liu, and Guo (2015) for yoghurt microstructure visualisation. Our
samples analysed by this method showed differences between
treated and untreated samples even though it may not be the true
microstructure of yoghurt as before sample preparation. Samples
without transglutaminase treatment contained larger pores, rep-
resented as dark spots on the images (CLSM, Fig. 8A,C; SEM,
Fig. 9A,C). The presence of larger pores could be correlated with
significantly higher syneresis of untreated yoghurt samples. In
contrast, images in Fig. 8B,D (CLSM) and Fig. 9B,D (SEM) represent
samples treated with 1 U g1 of MTG protein applied simulta-
neously with culture. The microstructure of these samples was
denser with smaller pores as a result of the high protein polymer
content. Smaller pores in the yoghurt microstructure were also
observed in the study of Şanli et al. (2014), where the authors also
used the same amount of MTG (1 U g1). The denser structure of the
proteins also increased the gel strength. Globular structures visible
in the images in Fig. 9 may represent milk fat globules. From the
images obtained from CLSM and SEM, the formation of new protein
bonds by MTG action was clear.

Fig. 8. Confocal laser scanning microscopy images of yoghurt samples: A, yoghurt fermented by non-EPS-producing culture CCDM RX 22 without MTG addition; B, yoghurt fer-
mented by non-EPS-producing culture CCDM RX 22 treated by 1 U g1 of MTG simultaneously with culture; C, yoghurt fermented by a mixed EPS-producing culture of Streptococcus
thermophilus CCDM 144 and Lactobacillus delbrueckii subsp. bulgaricus CCDM 767 without MTG addition; D, yoghurt fermented by a mixed EPS-producing culture of Streptococcus
thermophilus CCDM 144 and Lactobacillus delbrueckii subsp. bulgaricus CCDM 767 treated by 1 U g1 of MTG simultaneously with culture. Red structures and proteins stained with
FastGreen dye. Dark areas represent empty pores.

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Fig. 9. Scanning electron microscopy images of yoghurt samples: A, yoghurt fermented by non-EPS-producing culture CCDM RX 22 without MTG addition; B, yoghurt fermented by
non-EPS-producing culture CCDM RX 22 treated by 1 U g1 simultaneously with culture; C, yoghurt fermented by a mixed EPS-producing culture of Streptococcus thermophilus
CCDM 144 and Lactobacillus delbrueckii subsp. bulgaricus CCDM 767 without MTG addition; D, yoghurt fermented by a mixed EPS-producing culture of Streptococcus thermophilus
CCDM 144 and Lactobacillus delbrueckii subsp. bulgaricus CCDM 767 treated by 1 U g1 simultaneously with culture. White fibres are proteins and globular structures are milk fat
globules. Dark areas are empty pores.

3.6. Sensory analysis EPS-producing cultures. The presence of exopolysaccharides also

Regarding the influence of MTG on the sensory properties (taste
and odour) of fermented milk products, the opinion of the scientific
population is not completely uniform, particularly the influence of
MTG on bacterial growth (Section 3.1). The growth of yoghurt
bacteria was closely related to the levels of sensory active sub-
stances. Several studies have recorded an increase in the textural
properties of yogurts (firmness, viscosity) with no significant effect
on the typical taste and smell after MTG treatment (Pakseresht
et al., 2017; Şanli et al., 2014). However, by sensory evaluation of
MTG-treated yoghurts, Lorenzen et al. (2002) recorded a lower
yoghurt-specific taste and smell than control samples.
In our study, the sensory properties of yoghurts were evaluated
one day after manufacture (Fig. 10). Yoghurts fermented with EPS-
producing culture showed high ropiness, which was evaluated by
the sensory panel as a rather negative property. As a result of MTG
treatment, the ropiness of most of the treated samples decreased
during short-term fermentation, especially in the case of simul-
taneous enzyme application. The reduction in MTG-induced
ropiness caused an improvement in the acceptance of texture.
The difference between short- and long-term fermentation was
statistically significant (P < 0.05) for the non-EPS-producing and
contributed to the higher viscosity of the yoghurts. The most
preferred samples evaluated by ranking test were MTG supple-
mented yoghurts treated simultaneously with culture by a dose of
0.1 U g1 for non-EPS-producing and 0.5 U g1 for EPS-producing
cultures in short-term fermentation. By comparing the methods of
applying MTG in yoghurt production, the sensory profiles showed
that long-term fermented non-EPS-producing yoghurts prepared
by preincubation of MTG contributed to a higher acceptability of
the texture compared with the simultaneous application of the
enzyme with the culture. Furthermore, the samples treated with
MTG together with the culture resulted in the lumpiness of the
yoghurts, which negatively affected the final evaluation. These
products contained MTG with residual activity, which could
contribute to the formation of coarser particles perceived on the
tongue. Statistically significant differences between samples
(P < 0.05) were detected for both yoghurt cultures in the case of
long-term fermentation. Generally, MTG treatment increased the
observed sensory parameters of almost all samples fermented
with non-EPS-producing yoghurt culture. The best rated samples
in ranking test for long-term fermentation were yoghurts treated
by preincubation of MTG with a dose 0.1 U g1 for both yoghurt
cultures.

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Fig. 10. Sensory profiles of short-term (A, B) and long-term (C, D) fermented yoghurts after manufacture: A and C, yoghurts fermented by culture CCDM RX 22; B and D, yoghurts
fermented by mixed culture of Streptococcus thermophilus CCDM 144 and Lactobacillus delbrueckii subsp. bulgaricus CCDM 767: , without MTG; , 0.1 U g1 preincubation;
, 0.5 U g1 preincubation; , 1 U g1 preincubation; , 0.1 U g1 simultaneously with culture; , 0.5 U g1 simultaneously with culture; , 1 U g1 simultaneously
with culture. Results are expressed as median ratings. Definition of intensity scales for 1 to 7 are: viscosity and ropiness, from very low to very high; texture acceptance, very bad,
unacceptable to outstanding; aroma and taste intensity, imperceptible to very strong; taste and odour acceptance, very bad, unacceptable to outstanding.

4. Conclusion Declaration of competing interest

Treatment of milk proteins with microbial transglutaminase None.

and the application of cultures producing exopolysaccharides in
the manufacture of yoghurt could be a useful way to improve the
properties of fermented milk products without the use of addi-
tional components. While the effect of MTG dose on the gel
strength of preincubated yoghurts was not statistically signifi-
cant, in the case of the application of the enzyme simultaneously
with culture, the gel strength increased significantly with an in-
crease in the dose of the enzyme. Yoghurts prepared by
fermentation with exopolysaccharide producing culture were
negatively perceived due to an excessive ropy texture. The
application of MTG reduced ropiness and had a positive effect on
overall acceptance of texture.
These results revealed optimal conditions for sensory and gel
properties, in an MTG dose range of 0.1e0.5 U g1 protein with
enzyme inactivation occurring before fermentation, especially
during long-term fermentation. EPS-producing cultures contrib-
uted to the increase in the gel strength and could be more effective
in improving sensory properties with a lower production of EPS.
The formation of new milk protein structures by MTG treatment in
the presence of EPS significantly decreased yoghurt syneresis,
which may be useful in the development of fermented milk prod-
ucts with a lower content of milk dry matter.
Acknowledgements
This research was supported by the Ministry of Agriculture of
the Czech Republic with the project number: QK1910024. M. V.
acknowledges the European Structural and Investment Funds, OP
RDE-funded project ‘CHEMFELLS IV’ (No. CZ.02.2.69/0.0/0.0/
20_079/0017899) for support.
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