Carbohydrate Polymers 136 (2016) 427440

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Development of a fermented ice-cream as inuenced by in situ

exopolysaccharide production: Rheological, molecular,
microstructural and sensory characterization
Enes Dertli a , Omer S. Toker b , M. Zeki Durak b , Mustafa T. Yilmaz b, ,
Nevruz Berna Tatlsu c , Osman Sagdic b , Hasan Cankurt d
a

Bayburt University, Engineering Faculty, Food Engineering Department, 69000 Bayburt, Turkey
Yildiz Technical University, Chemical and Metallurgical Engineering Faculty, Food Engineering Department, 34210 Istanbul, Turkey
c
Istanbul Gelisim University, Vocational School, Food Technology Division, 34310 I stanbul, Turkey
d
Erciyes University, Saye Cikrikcioglu Vocational School, Food Science Division, Kayseri, Turkey
b

a r t i c l e

i n f o

Article history:
Received 18 May 2015
Received in revised form 9 August 2015
Accepted 17 August 2015
Available online 21 August 2015
Keywords:
Exopolysaccharides (EPS)
Ice-cream
Fermentation conditions
Optimization
Rheology
Chemical compounds studied in this article:
Ethanol (PubChem CID: 702)
Trichloroacetic acid (PubChem CID: 6421)
Potassium bromide (PubChem CID:
253877)

a b s t r a c t
This study aimed to investigate the role of in situ exopolysaccharide (EPS) production by EPS+ Streptococcus thermophilus strains on physicochemical, rheological, molecular, microstructural and sensory
properties of ice cream in order to develop a fermented and consequently functional ice-cream in which
no stabilizers would be required in ice-cream production. For this purpose, the effect of EPS producing
strains (control, strain 1, strain 2 and mixture) and fermentation conditions (fermentation temperature;
32, 37 and 42 C and time; 2, 3 and 4 h) on pH, S. thermophilus count, EPS amount, consistency coefcient
(K), and apparent viscosity (50 ) were investigated and optimized using single and multiple response
optimization tools of response surface methodology. Optimization analyses indicated that functional
ice-cream should be fermented with strain 1 or strain mixture at 4042 C for 4 h in order to produce
the most viscous ice-cream with maximum EPS content. Optimization analysis results also revealed that
strain specic conditions appeared to be more effective factor on in situ EPS production amount, K and
50 parameters than did fermentation temperature and time. The rheological analysis of the ice-cream
produced by EPS+ strains revealed its high viscous and pseudoplastic non-Newtonian uid behavior,
which demonstrates potential of S. thermophilus EPS as thickening and gelling agent in dairy industry.
FTIR analysis proved that the EPS in ice-cream corresponded to a typical EPS, as revealed by the presence
of carboxyl, hydroxyl and amide groups with additional -glycosidic linkages. SEM studies demonstrated
that it had a web-like compact microstructure with pores in ice-cream, revealing its application possibility
in dairy products to improve their rheological properties.
2015 Published by Elsevier Ltd.

1. Introduction
Production of exopolysaccharides (EPS) by lactic acid bacteria
(LAB) has gained a special interest over the last decade due to
functional properties of these biopolymers as well as their Generally Recognized as Safe (GRAS) status. The functionality of these
polymers is originated from the structural differences in the sugar
subunits, which is also the reason of the great diversity among
bacterial EPS and novel EPS structures among LAB. But they have
also some technological drawbacks such as low yields, which limits their large-scale production as well as practical applications in

Corresponding author. Tel.: +90 212 383 4575; fax: +90 212 383 4571.
E-mail address: mtyilmaz@yildiz.edu.tr (M.T. Yilmaz).
http://dx.doi.org/10.1016/j.carbpol.2015.08.047
0144-8617/ 2015 Published by Elsevier Ltd.

their puried forms (Dertli et al., 2013; Goh, Nair, & Matia-Merino,
2008). For this reason, in situ EPS production during the production
of fermented milk products such as yoghurt or other food products
is currently the main practical application for EPS from LAB.
One of the main functional roles of EPS is their crucial roles
on physicochemical and rheological properties of fermented dairy
products as a natural bio-thickening agent and stabilizer (Duboc &
Mollet, 2001). Several reports revealed the importance of the use
of ropy cultures and in situ EPS production on physicochemical and
other technological properties of fermented products like yogurt,
sourdough, cheese and ayran (Cerning, 1990; Duboc & Mollet, 2001;
Folkenberg, Dejmek, Skriver, Skov Guldager, & Ipsen, 2006; Goh
et al., 2008; Marshall & Rawson, 1999; Yilmaz et al., 2015). However, in the literature, no fundamental correlation has been built
between the in situ EPS production and rheological/technological

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E. Dertli et al. / Carbohydrate Polymers 136 (2016) 427440

properties of fermented milk products especially ice-cream due to

the complexity of EPS production. Accordingly, some intrinsic factors such as availability of sugar nucleotides as well as complex
genetic mechanism of EPS production and extrinsic factors such
as incubation temperature and time or nutrient availability were
reported to affect in situ EPS production levels and their conformational characteristics (Broadbent, McMahon, Welker, Oberg, &
Moineau, 2003; Duboc & Mollet, 2001; Kleerebezem et al., 1999;
Ruas-Madiedo, Hugenholtz, & Zoon, 2002).
Although ice-cream is a very suitable food system for the
development of new functional foods especially in probiotic industry (Champagne, Gardner, & Roy, 2005), a very limited number
of reports have been identied the functionality of using EPS
producing ropy cultures on ice-cream manufacturing process
(Christiansen, Madeira, & Edelsten, 1999; Goh et al., 2008). Production of ice-cream requires the use of stabilizers like gums,
some galactomannan based hydrocolloids or chemically modied
plant carbohydrates like starch, pectin, guar gum, etc. (De Vuyst
& Vaningelgem, 2009) and glucomannan based salep, which are
indispensable additives in ice-cream production, which implicates
that it would not be possible produce ice-cream at industrial scale
without using any stabilizer. However, it is also known that EPS
show some functional properties of stabilizers, therefore, the stabilizers can be replaced by in situ EPS production by LAB. But,
new studies are denitely required in order to understand the
technological functions of EPS in a real but complex ice-cream
system, which would also require the investigation of cultureincubation conditions affecting the in situ EPS production with
different EPS producer strains as well as the assessment of alterations in rheological, molecular and microstructural as well as
sensory characteristics of fermented ice-cream. Incubation temperature and time are two of the most important parameters
which affect the in situ EPS production levels as well as the resultant rheological characteristics of ice-cream, implicating that these
parameters should be taken under control to have the goal-oriented
results. To achieve this, it is important to calculate/nd optimum
levels of fermentation temperature and time that would result in
maximum/minimum values of response parameters. The best way
to achieve this is to perform an optimization study. Several examples of response surface methodology (RSM) applications which
allows the improvement and optimization of variables determining EPS production conditions have been conducted in order to
achieve the best EPS producing conditions as well as other properties (Pereira Duta, Pessa de Franca, & de Almeida Lopes, 2006;
Yilmaz et al., 2015).
In our study, we aim to develop an EPS based functional icecream by fermentation at the specied fermentation temperatures
and times in strain-specic conditions. Therefore, the aims of this
research were to investigate and optimize the effects of fermentation conditions (incubation temperatures, 3242 C and times;
24 h) on in situ EPS production levels and rheological properties
in/of ice-cream by employing response surface methodology and to
determine the functions of in situ EPS production on physicochemical, rheological, molecular, microstructural and sensory properties
of ice cream. An attempt was also made to correlate the EPS production and other properties to reveal the underlying mechanisms
that would explain the exact reasons of the observed changes in
ice-cream.

samples were produced and investigated as four different treatment groups, and will be referred as following throughout the
manuscript:
(a) Control group: Control ice-cream samples produced by using
non-ropy/EPS strain (EPS S. thermophilus strain; FD-DVS STBO1, CHR HANSEN Co., Ltd, USA),
(b) Strain 1 group: Ice-cream samples produced by using Strain 1
(EPS+ S. thermophilus strain; FD-DVS ST-BODY-3, CHR HANSEN),
(c) Strain 2 group: Ice-cream samples produced by using Strain 2
(EPS+ S. thermophilus strain; FD-DVS ST-BODY-4, CHR HANSEN),
(d) Mixture group: Ice-cream samples produced by using mixture
of Strain 1 and Strain 2.
All strains were activated in sterile, 10% reconstituted skim milk
and stored at 70 C until use. The stock cultures were prepared
at 37 C after 24 h incubation and 1% of the stock cultures were
used for the fermentation of the ice cream mixes depending on the
incubation conditions tested.
2.2. Ice-cream production
Standardized cow milk (pH 6.6, dry matter and fat, 8 and 2%,
respectively) was used for the preparation of ice-cream samples.
In order to prepare the ice-cream mix formulation, the whole milk
was separated into two parts as 20% part of the milk that was used in
regular processing of milk and as the other 80% part of the milk that
was used for inoculation and fermentation. The production steps of
the functional and fermented ice-cream mix as four different treatments are presented as a ow chart in Fig. 1. Each ice cream sample
was produced according to experimental runs (R1R10) each of
which represents different processing conditions; namely, different fermentation conditions (fermentation temperature and time;
Table S1, see Supplementary information).
2.3. Physicochemical and microbiological analysis
The pH values were directly determined in ice-cream mix at
25 C using a pH meter (WTW 315i Set Model, Weilheim, Germany),
following the instructions as outlined (AOAC, 2012). To determine
percent dry matter (g dry matter/100 g sample) content (AOAC,
2012), 3 g sample was dried at 100 C for 3.5 h in a drying oven
(Daihan, WOF sterilizator, South Korea). Protein (g protein/100 g
sample) according to mikro-Kjeldahl method (6.38) and fat
contents (g fat/100 g sample) according to Gerber method were
determined using previously described method (Yney, 1973).
In order to determine increase in volume of ice-cream with
respect to that of ice-cream mix, the overrun values were determined and calculated as outlined (Akn, Akn, & Krmac, 2007)
using the following equation:
Overrun =

weight of unit ice-cream mixweight of equal volume of ice-cream

100
weight of equal volume of ice-cream

For the microbiological analysis of ice-cream samples, serial

dilutions were prepared, plated onto M17 (Oxoid) agar plates and
incubated at 37 C for 48 h for enumeration of S. thermophilus counts
(De Man, Rogosa, & Sharpe, 1960; Terzaghi & Sandine, 1975).
2.4. Isolation and quantication of EPS in ice-cream samples

2. Material and methods

2.1. Bacterial strains and activation conditions
For the preparation of ice-cream samples, EPS+ and EPS Streptococcus thermophilus strains were used. In this study, the ice-cream

Exopolysaccharides were extracted from ice-cream samples

using the method previously described (Yilmaz et al., 2015). Briey,
20 mL of chilled ethanol was added to 20 mL of ice-cream samples
followed by an overnight incubation at 4 C. After centrifugation
at 6000 g for 30 min at 4 C, the pellets containing EPS were resuspended in 5 mL of distilled water with gentle heating (less than

E. Dertli et al. / Carbohydrate Polymers 136 (2016) 427440

429

WHOLE MILK
1 liter (100 %)

MILK FOR FERMENTATION

MILK FOR PROCESSING

(80 % part of whole milk))

(20 % part of whole milk)

HEATING (40 C)

PASTEURIZATION
(85 C for 5 min)

CREAM (70 % fat)

SKIM MILK POWDER
(15 gr)
HEATING (75 C)
COOLING (40 C)
SUGAR AND EMULGATOR
ADDITION (% 25)
INOCULATION OF
EPS+/EPS- CULTURES
PASTEURIZATION (85 C for 5 min)
FERMENTATION (according to incubation conditions outlined in Table S1)

COOLING (40 )

PROCESSED MILK

FERMENTED MILK

MIXING OF PROCESSED MILK (20% part) WITH

FERMENTED MILK (80% part)

FORMATION OF ICE-CREAM MIX

(Ripening at 4 C for 20 h)

HARDENING (by mixing for 30

min in ice-cream machine)

STORAGE (-20 C)
Fig. 1. Flow chart indicating production steps of functional and fermented ice-cream mix/ice-cream samples (strain 1, strain 2 and mixture groups) and control group
ice-cream samples that were inoculated with EPS strain.

50 C) until dissolved. For the removal of proteins, trichloroacetic

acid (TCA) was added at a nal concentration of 20% and the suspension was further incubated for 2 h at 4 C under gentle agitation.
Precipitated proteins were removed by centrifugation at 13,000 g
for 20 min at 4 C and supernatants were collected. Two volumes
of cold ethanol were added to the supernatants for EPS precipitation and the centrifugation process was followed as described

above. The resuspended EPS were dialyzed (12,00014,000 AMU

visking dialysis membrane, Medicell International, UK) against 5 L
of distilled water for 2 days by changing water three times per
day. After isolation of EPS from ice-cream samples, EPS amount in
each sample was determined by following phenol-sulphuric acid
methodology with glucose as a standard (DuBois, Gilles, Hamilton,
Rebers, & Smith, 1956). Standard dilutions of glucose were used in

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E. Dertli et al. / Carbohydrate Polymers 136 (2016) 427440

order to obtain the standard curve for calibration. The EPS content
of each sample was calculated using the glucose standard curve and
expressed as mg/L EPS production per sample. For the isolation and
quantication of EPS from ice cream samples, three independent
tests were performed with three replicates.
2.5. Steady shear rheological analysis
The rheological analysis was conducted in ice cream mix samples. For this purpose, a strain/stress controlled rheometer (Anton
Paar, MRC 302, Austria) equipped with a peltier temperature controller was used for determination of steady shear rheological
characteristics of the ice cream mixes. The measurements were carried out using a parallel plate conguration (plate diameter 35 mm,
angle 4 , gap size 0.5 mm) within the shear rate range of 0.1100 s1
at a constant measurement temperature (5 C) (Yilmaz et al., 2015;
Karasu, Toker, Yilmaz, Karaman, & Dertli, 2015). One milliliter of
the ice cream mix sample was placed between plates and the measurement was started immediately. A total of 30 data points were
recorded at 10 s intervals during the shearing. Each measurement
was repeated three times with two replications (each 1 mL). Apparent viscosity was determined as a function of shear rate at constant
temperature mentioned above (Yilmaz et al., 2015). The shear stress
values versus shear rate were plotted by increasing shear rate.
Obtained data were tted to Ostwald de Waele model using a software (ToolmasterTM , Graz, Austria) and, consistency coefcient (K)
and ow behavior index (n) values were calculated according to the
following model used to describe shear-induced behavior of the ice
cream samples (Yilmaz et al., 2015.
.

 = Kn

(1)

where  is the shear stress (Pa), K is the consistency coefcient

(Pa sn ),  is the shear rate (s1 ) and n is the ow behavior index
(dimensionless).

2010; Ylmaz, Karaman, Cankurt, Kayacier, & Sagdic, 2011). Basically, 100 mL ice-cream samples were presented and served at
certain intervals in odorless, randomly coded glass beakers of
100 mL capacity covered with glass dishes. Sensory evaluation was
performed in a room with appropriate temperature (25 C) in open
sitting. The panelists had access to tap water to cleanse their palates
before proceeding to the next sample. Sensory analyses of the icecream samples were carried out by ten selected staff and graduate
students of Food Engineering department at Yldz Technical University, comprised of ve females and ve males. Each panelist was
trained before evaluation in order to familiarize with the sensory
analysis, samples and methodology. All coded ice-cream samples
were evaluated for color, mouth-solubility, icy texture, melting
resistance, acidity, cream taste, foreign taste and general acceptance properties in a scale ranging from 1 to 9 points where 1
reected a very low in terms of disliking and 9 a very high score
in terms of liking. Panelists evaluated all (10) samples in two sessions (ve at each session) consecutively in two days. The manner
in which the treatment combinations were divided between the
sessions and the order in which the samples were presented was
randomized to minimize the carryover effects (Muir & Hunter,
1991; Karaman, Yilmaz, & Kayacier, 2011).
2.9. Data analysis, modeling and optimization
Response surface methodology was separately used for each
treatment to analyze changes in the amount of EPS production in
ice-cream samples and in rheological parameters of ice-cream mix
samples as a function of incubation temperature and time. For this
purpose, a three level, two variable central composite design was
used. The two factors, their levels and experimental design in terms
of coded and uncoded (actual values) can be seen (Table S1, see
Supplementary information). The model used was as following:
Y = 0 +

2.6. Molecular characterization of EPS by ATR-FTIR spectroscopy

ATR-FTIR (attenuated total reectance-Fourier transform
infrared spectroscopy) spectra of the EPS samples isolated from
strain 1 and strain 2 were measured with a Bruker Tensor 27
spectrometer (Bremen-Germany) equipped with a DLaTGS detector and a single-reection diamond ATR sampling accessory. For
instrument control and data acquisition, OPUS program Version
7.2 for Windows software from Bruker GMBH was also employed.
The spectra were recorded in the region of 4000400 cm1 with
16 scans at 4 cm1 resolution and processed by the spectrometer
software for detection of various functional groups due to their
vibration characteristics. Specic peaks at specic regions reected
the presence of specic functional groups within the EPS samples.
2.7. Scanning electron microscopy
Microstructure of the ice cream samples was analyzed by
scanning electron microscopy (SEM) using previously described
methodology (Yilmaz et al., 2015). Samples were examined under
high vacuum in a eld emission scanning electron microscope (JSM5510, JEOL Ltd., Tokyo, Japan) with a working distance of 8 mm.
Secondary electron images were acquired at an accelerating voltage of 5 kV. For processing of the images, SEM Control User Interface
(version 5.21; JEOL Ltd.) was used.
2.8. Sensory analysis
The sensory analysis of ice-cream samples were determined
based on protocols described before (Soukoulis, Lyroni, & Tzia,

N

i=1

i xi +

N

i=1

ii xi 2 +

 
i=

ij xi xj ,

(2)

1 j=i+1

i<j
where Y is the corresponding predicted response value, is the
error term, 0 is the intercept term, i is the linear term, ii
is the quadratic term, ij is the interaction term, and Xi and Xj
are the coded levels of the independent variables. The regression coefcients of linear, quadratic and interaction terms were
determined using Design Expert package software for each output
parameter.
The best tting models were determined using multiple linear regressions with backward elimination regression (BER) where
insignicant model parameters were removed from the models and
only the variables signicant at P < 0.01, P < 0.05 and P < 0.1 levels
were selected for the model construction using BER procedure.
In this study, both single and multiple response optimization procedures were followed. Single response optimization is
useful for nding the optimum values of factors for only one
response. But, for industrial applications, optimization should be
synchronously performed for all the responses involved since all
responses are correlatively changed. In other words, it is not possible to think a response would change alone; namely independent
of other responses. Moreover, a competition occurs between these
responses in many cases; namely, improving one response may
lead another response to deteriorate, further complicating the situation. In order to overcome this problem, multiple responses are
solved through use of a desirability function which combines all the
responses into one measurement (Myers & Montgomery, 1995).
The computational work including designation of experimental points, randomization, analysis of variance, tting of the

E. Dertli et al. / Carbohydrate Polymers 136 (2016) 427440

second-order polynomial models and graphical representations as

well as optimization was performed using a statistical package,
Design-Expert version 7.0 (Stat-Ease Inc., Minneapolis, USA). SPSS
Statistics (17.0) (SPSS Statistics 17.0, Armonk, NY, USA) was used to
conduct an ANOVA to show the differences between experimental
runs and between different treatment groups at a condence level
of 0.05. Bivariate correlations between pH, rheological parameters,
microbial counts (S. thermophilus counts), and amounts of EPS were
analyzed by Pearsons test using Minitab 14.0 software.

3. Results and discussion

3.1. Differences between treatment groups at each run
Table 1 shows the physicochemical properties of ice cream
samples produced with non-EPS producing strain (control group),
strain 1, strain 2 and strain mixture (strain 1 + strain 2) and
signicant (P < 0.05) differences between the treatments and experimental runs for each physicochemical parameter can be seen. The
dry matter contents of control group were generally higher than
those of strain 1 and strain 2 treatment groups (Table 1).
This can be rstly attributed to neutral or charged groups like
carboxyl groups present in EPS, serving as binding sites for divalent cations (Wang et al., 2010) and hydroxyl groups that were
the preferred groups for the occulation process (Guo et al., 2009),
revealing structure function relationship of EPS (Sutherland, 1994).
Accordingly, in this study, ATR-FTIR analysis revealed the said functional groups of EPS including hydroxyl and carboxyl groups, as will
be discussed later. It should also be especially noted here that the
remarkable effect on water absorption was also ascribed to high
molecular mass of polysaccharides; more particularly, heteropolymeric EPS isolated from S. thermophilus strains with a molecular
mass of at least 2.106 AMU (De Vuyst & Vaningelgem, 2009). The
dry matter content of ice-cream samples ranged between 27.12%
and 35.50% depending on the conditions tested which were similar to the previous observations (Akaln, Karagzl, & nal, 2008;
Akn et al., 2007; Gven & Karaca, 2002). Similar trend was also
observed on the fat and protein contents of ice-cream samples that
were found to be higher in mixture group. The reason could be
ascribed again to the contribution of EPS since dry matter are primarily composed of fat and protein. The highest and the lowest
fat contents were found to be 4.05% and 4.70%, respectively, for
all ice cream samples tested, which reveals that the fat content
of ice-cream samples were relatively lower than those reported in
the previous studies (Homayouni, Azizi, Ehsani, Yarmand, & Razavi,
2008) and close to that of the low fat ice cream (Baer, Wolkow, &
Kasperson, 1997). On the other hand, strain selection as a function of EPS production signicantly (P < 0.05) affected the protein
content of ice-cream samples (Table 1). It should be noted here that
production of ice-cream with strain mixture resulted in higher protein levels in ice-cream samples than that with other treatments;
namely, strain 1, strain 2 and control groups, as well. The relative
differences in protein levels might have resulted from different levels of protein adsorption in ice-cream mix due to the EPS production
and strain specic metabolic properties (Bolliger, Goff, & Tharp,
2000). Another explanation for the higher protein levels in treatments as compared to control group might be the potential effect
of EPS on hydration of proteins as well as its own hydration, which
might have probably resulted in less free water molecules in the
ice-cream samples and, thus which relatively increased the apparent concentration of proteins (Duboc & Mollet, 2001). We should
also note that the protein-polysaccharide interaction determines
the physicochemical and stabilization properties of dairy products
such as ice-cream (Ye, 2008) and consequently their physicochemical properties.

431

As can be seen from Table 1, the highest dry matter, fat and
protein contents were generally determined in the experimental
runs, R2, R3, R7 and R10 where fermentation temperature were
32, 37 and 42 C while fermentation time was 4 h. This result led
us think that fermentation time was more prevalent factor on the
proximate condition than fermentation temperature.
Overrun is another physicochemical characteristic of ice-cream
which can be dened as the percent of ice-cream expansion due
to the incorporation of air into matrixes of the ice-cream mix during the freezing process. In our study, depending on the conditions
tested, the overrun values were determined to range between 22.54
and 33.33% (Table 1), which; however, relatively low in comparison to those reported in the literature, probably due to the use
of a batch type freezer (Akaln et al., 2008). On the other hand,
there were signicant (P < 0.05) differences between the treatment
groups with respect to overrun values, which revealed the fact that
production of ice-cream with the strain inoculation increased the
overrun values; furthermore, this increase was more prominent in
strain 1 samples. Accordingly, it was reported that EPS produced
by three ropy LAB strains signicantly increased % overrun of icecream (Goh et al., 2008). These results should be important for dairy
industry given that overrun affects stability, texture, melting rate
and sensory properties of ice-cream and that higher overrun values are preferred due to inhibition of ice crystals, enhancing the
melting resistance and improving of foam stability during the storage period (Flores & Goff, 1999; Sofjan & Hartel, 2004; Soukoulis,
Chandrinos, & Tzia, 2008).
From Table 1, the highest overrun values were determined in R3,
R7 and R 10 where fermentation temperature were 32, 37 and 42 C
while fermentation time was 4 h, as reported above. This result
also revealed that maximum overrun values could be obtained in
prolonged fermentation time irrespective of fermentation temperature. Possible explanation can be done based on stimulation effect
of EPS due to the formation of a matrix to retain more oxygen to
increase the overrun (Hong & Marshall, 2001) as all as foam stabilization properties of EPS as a polysaccharide, which led more
air adsorption during fermentation period (Stanley, Goff & Smith,
1996). In addition, lower pH values at these runs (Table 2) might be
responsible for higher overrun values, as previously reported after
longer incubation (Allen, Dickinson, & Murray, 2006; Goh et al.,
2008). Overall, the fermentation period was the most effective variable affecting the overrun values of ice cream samples.
Table 2 shows differences between the treatment groups with
respect to pH, M17 number (S. thermophilus count), EPS amount
and rheological parameters (K, n and 50 ). As can be seen, there
were signicant (P < 0.05) differences between the treatments at
each experimental run in terms of these parameters. The pH values of ice-cream samples were determined to range from 5.11 to
5.44, 4.95 to 5.53, 5.07 to 6.12 and 4.98 to 5.67 for control, strain
1, strain 2 and mixture groups, respectively. These results were
similar to previous observations where pH values were reported
to be around 5.3 to 6.5 (Akaln et al., 2008; Goh et al., 2008;
Karaca, Gven, Yasar, Kaya, & Kahyaoglu, 2009). These pH values
were also suggested for a mild nal product (Cruz, Antunes, Sousa,
Faria, & Saad, 2009). On the other hand, at some runs, relatively
low pH values such as 4.95 and 4.98 were observed in our study,
which was also in accordance with previous studies in which a
nal pH of 4.74.8 was reached during probiotic ice-cream production (Cruz et al., 2009). Table 2 shows also the differences between
the treatments and experimental runs in terms of S. thermophilus
count values. These values were determined to change between
5.937.04 log CFU/mL, 4.748.33 log CFU/mL, 5.457.80 log CFU/mL
and 6.708.26 log CFU/mL for control, strain 1, strain 2 and mixture
groups, respectively. Similar numbers were reported in previous
observations (Akn et al., 2007; Davidson, Duncan, Hackney, Eigel,
& Boling, 2000). In this study, the lower numbers of S. thermophilus

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E. Dertli et al. / Carbohydrate Polymers 136 (2016) 427440

Table 1
Differences between treatment groups at each run with respect to physicochemical parameters.
Runs

Treatments
Control

Treatments
Strain 1

Strain 2

Mixture

29.69Bg
29.78Cg
31.93Bc
30.59Ae
32.16Cab
32.07Cb
32.23Ba
29.76Cg
30.14Df
31.76Dd

27.12Ce
28.98Dd
29.98Ccd
29.61Bcd
30.33Dc
30.11Dc
31.48Cb
29.49Ccd
31.47Cb
32.69Ca

30.33ABf
31.91Ade
32.08Bd
30.96Aef
33.95Abc
33.29Bc
33.82Abc
31.96Ade
34.69Aab
35.50Aa

Control

Dry matter (%)

30.91Ac
31.30Bc
34.68Aa
29.69Bd
32.84Bb
33.49Ab
31.35Cc
31.25Bc
33.14Bb
34.55Ba

R1
R2
R3
R4
R5
R6
R7
R8
R9
R10

1.52Ba
1.49Bab
1.11Ccd
1.00Cde
1.14Ccd
1.60Ba
1.19Cbcd
0.88Cde
0.70De
1.37Cabc

Strain 2

Mixture

Fat (%)
4.10Ac
4.05Bc
4.20Bbc
4.10Bc
4.05Bc
4.40Aabc
4.40Babc
4.35Babc
4.60Aab
4.70Aa

Protein (%)
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10

Strain 1

4.30Aab
4.70Aa
4.70Aa
4.20Bb
4.40Aab
4.50Aab
4.40Bab
4.40Bab
4.10Cb
4.30Bab

4.30Aabc
4.10Bbc
4.10Bbc
4.50Aab
4.30ABabc
4.10Bbc
4.70Aa
4.05Cc
4.25BCbc
4.30Babc

4.05Ad
4.10Bcd
4.05Bd
4.50Aab
4.40Aabc
4.35Abcd
4.70Aa
4.70Aa
4.50ABab
4.70Aa

Overrun (%)
2.71Aa
1.55Bc
1.49Cc
1.63Bc
1.67Bc
1.15Cd
2.26Bb
1.00BCd
1.77Cc
1.13Dd

1.81Bb
1.59Bbc
2.26Ba
1.60Bbc
1.38BCbcd
1.28Ccd
2.26Ba
1.09Bd
2.27Ba
1.79Bb

2.82Acd
2.62Af
2.87Ac
2.61Af
2.69Aef
2.73Ade
3.01Aa
2.90Abc
2.84Ac
2.99Aab

26.49Cc
30.04Bb
30.34Db
24.64Cd
30.51Bb
30.70Bb
33.28Aa
22.54De
23.37Ce
32.70ABa

29.35A
32.65Ab
32.99Aa
30.73Ag
31.15Af
31.82Ad
32.90Ba
30.41Ah
31.46Ae
32.19Bc

29.59Acd
30.38Bbc
31.71Ca
27.58Be
29.64Ccd
29.36Cd
30.31Dbc
27.82Ce
30.60Bb
32.46Ba

27.87Bd
28.26Ccd
32.10Bb
22.80De
27.90Dd
29.36Cc
31.59Cb
28.70Bcd
31.08Ab
33.33Aa

A-D
Different superscript uppercase letters show differences between the treatments (P < 0.05). ah Different superscript lowercase letters show differences between the runs
(P < 0.05).

were generally detected in control group than in the other treatment groups, which suggests the potential preservative role of
EPS on the bacterial cells during the freezing process as decline
of bacterial counts in ice-cream was reported to be related with
their death during freezing process (Akaln et al., 2008; Looijesteijn,
Trapet, de Vries, Abee, & Hugenholtz, 2001). Accordingly, our ndings conrmed the previous knowledge in which EPS functioned as

a potential preservative for the bacterial cells under frozen conditions.

In situ EPS production levels in treatment groups at each run;
namely different fermentation conditions are also shown in Table 2.
These values ranged between 3.6423.53 mg/L, 0.8614.57 mg/L
and 1.7517.93 mg/L in strain 1, strain 2 and mixture groups,
respectively. In comparison to strain 1 and strain 2 groups, the

Table 2
Differences between treatment groups at each run with respect to pH, S. thermophilus count, EPS amount values and rheological parameters.
Runs

Treatments
Control

Treatments
Strain 1

Strain 2

Mixture

5.35De
5.41Cb
5.44Ba
5.40Ac
5.21Bg
5.21Bg
5.11Ai
5.35Ad
5.25Bf
5.15Bh

0.03Dh
0.24Cg
0.42Df
0.14Cgh
1.10Dde
1.11Bd
2.94Cb
1.02Ce
1.57Dc
4.01Da

Strain 2

Mixture

6.12Aa
5.86Ab
5.79Ac
5.27Cf
5.29Ae
5.25Ag
5.11Ai
5.31Bd
5.18Ch
5.07Bj

5.67Ba
5.45Bb
5.12Dg
5.39Bc
5.17Ce
5.12Cf
4.98Bi
5.18Cd
5.05Dh
4.98Ci

5.93Be
6.12Bcde
6.02Dde
6.11Ccde
6.28Cbc
6.18Dbcd
6.38Cb
6.09Dcde
6.26Cbcd
7.04Da

4.74Dd
4.74Cd
6.70Cc
7.72ABb
8.30Aa
8.33Aa
8.32Aa
8.14Aab
8.16Aab
8.07Bab

5.45Cd
6.13Bc
7.33Ab
7.40Bb
7.80Ba
7.68Ca
7.72Ba
7.38Cb
7.33Bb
7.77Ca

0.39Af
0.60Aef
0.78Ce
0.47Bf
1.88Cd
1.83Bd
6.36Bb
1.80Bd
5.99Cc
12.77Ca

0.23Bh
0.58Bg
3.12Af
1.50Agh
4.09Bde
6.95Ad
6.95Bb
3.85Ae
15.74Ac
24.07Aa

0.91Ab
0.60Be
0.85Ac
0.82Bc
0.95Aa
0.91Ab
0.73Ad
0.94Aa
0.82Ac
0.62Ae

Strain 1

Strain 2

Mixture

3.19B
7.52Ae
7.24Cf
0.86Cj
5.33Cg
4.44Ch
11.43Bc
9.92Ad
11.77Cb
14.57Ca

1.75Cj
4.61Ci
13.19Ac
6.06Bh
9.75Be
11.49Bd
7.01Cg
9.42Bf
14.06Ab
17.93Aa

EPS amount (mg/L)

6.70Ad
6.75Ad
7.07Bc
7.88Ab
8.26Aa
8.24Ba
8.25Aa
7.88Bb
8.08Aab
8.21Aa

Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd

3.64Ag
6.54Bfg
8.11Bf
9.45Aef
16.45Abc
17.24Ab
23.53Aa
9.13Cf
13.12Bcd
16.53Bbc

50 (Pa sn )

n
0.11Cf
0.20Cf
2.22Be
0.36BCf
7.20Ac
7.82Ac
11.92Ab
3.57Ad
8.66Bc
15.87Ba

Control

S. thermophilus count (log CFU/mL)

5.53Ca
5.25Dd
5.20Cf
5.05Dg
5.02Dh
5.02Dh
4.95Ci
5.35Ab
5.31Ac
5.24Ae

K (Pa sn )
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10

Treatments
Strain 1

pH
R1
R2
R3
R4
R5
R6
R7
R8
R9
R10

Control

0.73Bc
0.98Aa
0.68Bc
0.96Ab
0.37Ce
0.36Ce
0.26Cf
0.53Cd
0.35Be
0.22Cf

0.47Ca
0.27Cd
0.28Ccd
0.38Cb
0.33Cbc
0.35Cb
0.17De
0.35Db
0.18Ce
0.09Df

0.67Bb
0.49Be
0.66Bc
0.79Bc
0.62Ba
0.50Bb
0.52Bd
0.64Ba
0.34Bc
0.25Be

0.03Cg
0.05Cg
0.13Ce
0.07Cf
0.87Bb
0.77Bd
1.01Ba
0.81Bc
0.83Bc
0.86Bb

0.04BCg
0.31Af
0.71Ba
0.31Bf
0.63Cc
0.63Cc
0.66Cb
0.58Ce
0.61Cd
0.70Ca

0.05Bde
0.03Cf
0.05Dd
0.04Cef
0.13Dc
0.13Dc
0.23Db
0.13Dc
0.22Db
0.33Da

0.07Ag
0.30Bg
0.85Ae
0.73Af
0.95Ab
0.97Ad
1.03Aa
0.93Ac
1.15Aa
1.19Aa

A-D
Different superscript uppercase letters show differences between the treatments (P < 0.05). aj Different superscript lowercase letters show differences between the runs
(P < 0.05).

EPS amount and M17 enumeration (S. thermophilus count) analysis were conducted in ice-cream samples while pH and rheological analyses were conducted in ice-cream
mix samples.

R2 values (determination coefcients) for the Ostwald de Waele parameters were determined to change between 0.84 and 0.99.

E. Dertli et al. / Carbohydrate Polymers 136 (2016) 427440

433

Table 3
Pearson correlation coefcients (r) between different parameters for each treatment group .
Treatments

Parameters

Control

K
50
M17
EPS
pH
K
50
M17
EPS
pH
K
50
M17
EPS
pH
K
50
M17
EPS
pH

Strain 1

Strain 2

Mixture

1.000
0.750**
0.918***

0.834***
1.000
0.697**
0.665**
0.842***
0.351
1.000
0.955***
0.450
0.820***
0.620*
1.000
0.697**
0.555*
0.822***
0.679*

50

M17

1.000
0.569*

0.859***

1.000

0.694**

1.000

1.000

1.000
0.762***
0.694**
0.513

1.000
0.765**
0.594*

1.000
0.745**

1.000

1.000
0.567*
0.819***
0.739**

1.000
0.346
0.884***

1.000
0.550*

1.000

1.000
0.871***
0.834***
0.953***

1.000
0.550*
0.762***

1.000
0.816***

1.000

EPS

pH

K, 50 , M17 (S. thermophilus count), EPS amount and pH values were used to perform the Pearson correlation analysis.
***
P < 0.01.
**
P < 0.05.
*
P < 0.1.

K: consistency coefcient, 50 : apparent viscosity at 50 s1 , M17: count of S. thermophilus strains in each ice-cream samples enumerated on M17 agar, EPS: amounts of
EPS produced by different strains.

Correlation analysis was not performed since there was no EPS production in control ice-cream samples.

EPS production amount were lower at different runs (Table 2),

which reveals dependency of EPS production to the availability
of carbon/nitrogen sources that can be depleted by the strains in
ice-cream. In addition, EPS amounts at each run were different,
which can be attributed to the biosynthesis mechanism of EPS by S.
thermophilus since this bacterium produces heteropolymeric type
EPS, which may show genetic instability depending on fermentation conditions (Broadbent et al., 2003; De Vuyst & Degeest, 1999;
Duboc & Mollet, 2001).
Rheological properties of ice-cream mix samples are presented
in Fig. S1 (see Supplementary information) which demonstrates
share rate data versus share stress data for treatment groups at
each experimental run. As seen from Fig. S1 (see Supplementary information), shear stress values decreased as shear rate
values increased, indicating that ice-cream mix samples showed
non-Newtonian shear-thinning (pseudoplastic) behavior at all
treatment groups and experimental runs. This was also clear from
ow behavior index (n) values all lower than unity (Table 2). Similar behavior was reported for ice-cream mixes in different studies
(Dogan, Kayacier, Toker, Yilmaz, Karaman, 2013; Toker et al., 2013).
Accordingly, these data were well tted to Ostwald de Waele
model and model parameters were calculated for different treatment groups at each run. These results revealed that fermentation
of ice-cream did not change the ow behavior of ice-cream, leading to a conclusion that fermented and symbiotic ice-cream is also
a product showing non-Newtonian pseudoplastic behavior. Pseudoplasticity of biogums like EPS is of great importance to dairy
industry as it enhances sensory qualities such as avor release and
mouthfeel in food products and guarantees good mixability and
pourability (Sajna et al., 2013).
Table 2 shows the model parameters; namely consistency coefcient (K), ow behavior index (n) values as well as apparent
viscosity values (50 ) measured at 50 s1 , the shear rate in mouth.
The determination coefcient values (R2 ) for Ostwald de Waele
model of ice-cream mix samples were close to 1 showing that this
model was appropriate to determine the ow behavior of ice-cream
mixes. As for the differences between the treatment groups at each

run in terms of rheological properties, production of ice cream

samples with different EPS producing strains under different fermentation conditions was also observed to signicantly (P < 0.05)
affect the rheological model parameters; namely, consistency coefcient (K), ow behavior index (n) and apparent viscosity (50 )
(Table 2). From Table 2, it is clear that production of ice-cream
with EPS producing strains resulted in a remarkable increase in K
values in comparison to control sample. Accordingly, some differences were found between viscosity values of ice-cream samples
fermented with ropy and non-ropy cultures at high share rates (Goh
et al., 2008). Additionally, Goh et al. (2008) showed that EPS can
actually be used as a stabilizer replacer with important rheological characteristics, showing the importance of producing ice-cream
as a fermented product with ropy cultures. However, an inverse
phenomenon was the case for n values which were found to be
generally the lowest in control samples, which should expected
since there are generally negative correlations between K and n
values in food systems. Regarding 50 values, the highest values
were observed in mixture group followed by strain 1 and even control samples; however, the lowest ones were observed in strain 2.
Similarly, for example at R10, the highest K and 50 values, but the
lowest n values were observed both in control and the other groups
although ice-cream samples were not produced with EPS+ strains in
control group in which there was no EPS production. These results
revealed that more than one factor should have played a role on the
rheological properties; therefore, the observed viscosity increase
in ice-cream samples should be attributed not only to EPS production by EPS+ strains, but also to other factors such as kinetics
of microbial activity and resultant pH change that can play a crucial role on rheological properties of ice-cream. Still, the effect of
EPS produced by strain 1, strain 2 and strain mixture on viscosity increase should not be overlooked given that EPS was regarded
as a bio-thickening agent, inuencing the rheological properties
(Duboc & Mollet, 2001). As a conclusion, these results led us think
that it would not be enough to explain the changes in rheological
properties of the functional and fermented ice-cream based on only
EPS or pH or any other factor. Therefore, correlation analyses were

434

E. Dertli et al. / Carbohydrate Polymers 136 (2016) 427440

Fig. 2. Effect of fermentation temperature and time on amounts of EPS production, K, and 50 values at different treatment groups; namely, control, strain1, strain 2 and
mixture (admixture of strain 1 and strain 2).

conducted, which revealed the effects of parameters or parameter

combinations that would play an effective role on the rheological
properties.
Table 3 shows that there were signicant (P < 0.01; 0.05; 0.1) correlations between the effective parameters (responses); namely, K,
50 , S. thermophilus counts, EPS amount and pH that would inuence each other, leading to the resultant rheological properties.
The positive correlations between S. thermophilus number and EPS
amount for each treatment group treated with EPS+ strains conrmed the assumption that there might be a potential preservative
role of EPS on the bacterial cells during the freezing process (Akaln
et al., 2008; Looijesteijn et al., 2001). However, from the other side
of view, the interaction between EPS and S. thermophilus count
was thought to be bidirectional. Namely, higher bacterial proliferation during prolonged fermentation time was also thought to
inuence EPS production, which was in accord with previously
reports on other dairy products (de Kruif & Tuinier, 2001; Hassan,
Ipsen, Janzen, & Qvist, 2003b), supporting the relationship between

bacterial growth and EPS production levels (Broadbent et al., 2003;

De Vuyst & Degeest, 1999).
There are also other positive correlations between the parameters; however, a noteworthy point that should be pointed out
here was that there were always negative correlations between
pH values and all the remaining parameters (Table 3). Final pH of
ice cream mix were reported not to affect the bacterial counts in
frozen ice-cream (Davidson et al., 2000) whereas we found negative correlations between pH and S. thermophilus count, which was
also reported by Akaln and Erisir (2008) who correlated reduction
in pH with proliferation of probiotic bacteria in ice-cream mix.
From Table 3, pH values were also seen to be negatively correlated EPS amount. This was in accordance with the information
by Broadbent et al. (2003) who reported that there can be a bidirectional interaction between pH and in situ EPS. Decrease in pH
values could be attributed to the fact that EPS production is an
energy intensive process (Broadbent et al., 2003), resulting in an
increase in acidity of ice-cream mixes. A negative correlation was

E. Dertli et al. / Carbohydrate Polymers 136 (2016) 427440

also the case between pH and K/50 values, indicating that viscosity of ice-cream mixes increased as pH values increased. However,
this should not necessarily mean that ice-cream viscosity would
only dependent on pH under strain-specic conditions in this study.
From Table 3, there was no correlation between pH and K/50 values
for strain 1 group, which would support this assumption. Moreover,
it was clear from Table 2 that K values of strain 1, strain 2 and mixture groups were remarkably higher than those of control group in
which there was no EPS production. These results revealed that rheological properties of ice-cream mixes were dependent on not only
pH values, but also on EPS production amount. Accordingly, there
were positive correlations between EPS amount and the rheological
parameters; K, 50 values (Table 3).
3.2. Effect of fermentation temperature and time
Table S2 (see Supplementary information) shows the F values of model parameters showing the effects of fermentation
temperature and time on pH, S. thermophilus count, EPS amount

435

and rheological parameters (K, n and 50 ). As can be seen, the R2

(coefcient of determination) values for the parameters were generally higher than 0.90, suggesting that the models generated were
enough to predict the studied properties of ice-cream. From Table
S2 (see Supplementary information), it is clear that fermentation
temperature and time had signicant (P < 0.01, 0.05, 0.10) effects
on these parameters. In addition some interaction and quadratic
effects were found to be signicant (P < 0.01, 0.05, 0.10) on these
parameters. The magnitudes of F values of quadratic effects of the
fermentation temperature were higher than those of fermentation
time, which revealed that effect of fermentation temperature on
pH, S. thermophilus count, EPS amount and rheological parameters
was relatively higher than that of fermentation time.
Fig. 2 illustrates three-dimensional response surface graphs
where direction of the effects of fermentation temperature and
time on EPS amount, K and 50 values along with secondorder regression model equations predicting effects of incubation
temperature and time on the parameters of concern. As can be seen,
for all treatment groups, EPS amount (except for control group), K

Table 4
Single and multiple response optimizations for calculation of maximum and minimum values of response parameters.
Treatments

Control

Response parameters

K (Pa s )
50 (Pa s)
EPS (mg/L)
pH
M17 (log CFU/mL)

Strain 1

K (Pa sn )
50 (Pa s)
EPS (mg/L)
pH
M17 (log CFU/mL)

Strain 2

K (Pa sn )
50 (Pa s)
EPS (mg/L)
pH
M17 (log CFU/mL)

Mixture

K (Pa sn )
50 (Pa s)
EPS (mg/L)
pH
M17 (log CFU/mL)

SRO

MRO

Fermentation conditions Response values

Fermentation conditions Response values

T ( C)

T ( C)

32.91
42.00
32.18
41.25
#
#
41.91
32.00
32.00
42.00
32.63
41.94
32.00
42.00
32.00
38.54
37.20
32.00
32.03
40.60
32.94
42.00
32.19
41.79
32.00
42.00
39.76
32.00
32.00
40.42
32.05
42.00
32.00
41.77
32.00
42.00
41.70
32.00
32.03
39.98

t (h)
2.07
4.00
2.07
3.81
#
#
3.97
2.01
2.00
4.00
2.02
4.00
2.00
4.00
2.00
3.98
3.71
2.00
2.08
3.97
2.82
4.00
2.05
3.98
2.00
4.00
3.68
2.00
2.00
3.80
2.89
4.00
2.00
3.24
2.00
4.00
3.76
2.00
2.07
3.93

Min

Max

t (h)

0.01
3.92

32.00
42.00

Min

2.17
4.00

0.01

Max

0.01
3.92

Desirability
0.912
0.960

0.01
1.01
#
#

1.11
#
#

5.08

5.36
5.36

5.07

6.02

6.02
6.89

0.01
16.82

6.89
32.00
38.84

2.00
4.00

0.05

0.01
12.60

0.978
0.942

0.05
0.71

0.71

3.66

3.66
23.62

23.53
5.48

4.95
5.48

4.98

4.59

4.52
8.52

0.08
12.72

8.77
32.00
41.86

2.00
4.00

0.03

0.70
12.51

0.971
0.967

0.03
0.33

0.34

1.36

1.36
13.89

13.81

5.05

6.07
6.07

5.08

5.52

5.52
7.85

0.09
22.42
0.25

7.61
32.00
42.00

2.00
4.00

0.01
22.42

0.955
0.963

0.25
1.19

2.40

1.38
2.40

16.65
4.97
5.66
6.66

16.65
5.66
4.93
6.63

8.41

8.21

SRO (single response optimization) was performed to calculate optimum fermentation temperature (T) and time (t) values by separately considering each response
parameter.

MRO (multiple response optimization) was performed to calculate optimum T and t values by simultaneously considering all response parameters.

Optimum levels of fermentation conditions that would result in minimum K, 50 , EPS amount, and M17 values, but maximum pH values were calculated due to negative
correlations between pH and the other parameters (Table 3).
Optimum levels of fermentation conditions that would result in maximum K,  , EPS amount, and M17 values, but minimum pH values were calculated due to negative
50
correlations between pH and the other parameters (Table 3).
#
EPS amount parameter was not included in MRO performed for control treatment.

436

E. Dertli et al. / Carbohydrate Polymers 136 (2016) 427440

and 50 values remarkably increased as fermentation temperature

and time values increased. In other words, for all the treatment
groups, the higher fermentation temperature/the longer fermentation time, the more EPS production (except for control group) in
ice-creams and the more viscous ice-cream mixes.
The effective role of fermentation temperature and time on
EPS production in this study were also observed in previous
observations in which these two parameters were reported to be
crucial extrinsic factors determining the EPS production levels of
LAB (Broadbent et al., 2003; Petry, Furlan, Crepeau, Cerning, &
Desmazeaud, 2000; Tallon, Bressollier, & Urdaci, 2003). Effect of
incubation temperature to increase EPS production may be related
with the biosynthesis mechanism and requirement for EPS production in different conditions (Kojic et al., 1992; Sutherland, 1972).
The observed increase in K (consistency) and 50 (apparent
viscosity) values of ice-cream samples may be related with formation of stronger gels due to increased hydrophobic interactions
between proteins as temperature increased and with further formation of intermolecular cross-links within the protein network
with prolonged fermentation period, as suggested for other dairy
products (Haque, Richardson, & Morris, 2001; Ozdemir & Kilic,
2004; Shaker, Abu-Jdayil, Jumah & Ibrahim, 2002). Another and
probably more remarkable contribution to increase in consistency
and apparent viscosity values of ice-cream samples was thought
to come from faster biosynthesis of EPS; furthermore, increased
molar mass of synthesized polysaccharide under more privileged
conditions of biosynthesis. This suggestion was in accord with the
nding reported by Degeest and De Vuyst (1999) who determined
that the amount of EPS-polymer dry mass considerably increased
until 12th hour of fermentation period.
3.3. Optimization of fermentation conditions
Table 4 shows single (SRO) and multiple response optimizations
(MRO) for calculation optimum levels of fermentation temperature and time that would result in maximum/minimum values of
response parameters. It can be concluded based on these results
that the ice-cream should be fermented with strain 1 or strain
mixture at 4042 C for 4 h in order to produce the most viscous
ice-cream with maximum EPS amount. Given the great differences
between the treatment groups in terms of these parameters at same
optimum fermentation conditions, the effect of strain specic conditions appeared to be the much more efcient factor on these
parameters than did fermentation conditions.
3.4. Characterization of functional groups of EPS and molecular
structure
The major functional groups of the EPS from strain 1 and strain
2 were determined by ATR-FTIR spectroscopy, indicating a typical
pattern of absorption for a polysaccharide (Fig. 3). It was reported
that Fourier Transform-Infrared Spectroscopy could offer a useful
tool in terms of monitoring structural changes that would occur in
biopolymers (Wilson, Goodfellow, & Belton, 1988). In this study,
the FTIR spectra of EPS produced by S. thermophilus strains (strain
1 and strain 2) were analyzed and absorption bands were assigned
to reveal the typical polymeric structure of carbohydrate (Fig. 3).
The broad stretched peaks at 3287.19 and 3304.78 cm1 for strain 1
and strain 2, respectively, were ascribed to the stretching vibration
of hydroxyl groups (Nwodo & Okoh, 2013). In this region (Fig. 3),
the absorption can be seen as rounded feature, which is typical
of hydroxyl groups (Howe, Ishida, & Clark, 2002), which would
suggest that the materials analyzed were a polysaccharide. This
region represents the characteristic absorption band of carbohydrate rings, which was reported to be responsible for the water
solubility of EPS (Lim et al., 2005; Karbowiak, Ferret, Debeaufort,

Fig. 3. ATR-FTIR spectra of the isolated EPS produced by (a) strain 1 and (b) strain 2
(Streptococcus thermophilus) in the range of 4004000 cm1 .

Voilley, & Cayot, 2011). The FTIR spectra of the EPS also revealed two
weak C H stretching peaks at 2932.99 and 2947.52 cm1 for strain
1 and strain 2, respectively, which were attributed to an asymmetrical C H stretching vibration of the aliphatic CH2 group, which
indicates the presence of organic substances such as proteins, sugars etc. (Kavita, Singh, Mishra, & Jha, 2014).
There was no peak around 17001775 cm1 , putting forward
that glucuronic acid and diacyl ester were not present in the EPS
produced by S. thermophilus strains (Trabelsi, Slima, Chaabane,
Riadh, 2015). The absorption band found in the region between
1650 and 1540 cm1 usually represents the stretching vibrations of
enol and N H bending of amides II of proteins (Singh et al., 2011;
Allen et al., 2004; Lin et al., 2005). Therefore, absorption observed at
1664 cm1 for our EPS probably corresponded to the amide I > C O
stretch (Trabelsi et al., 2015) and C N bending of protein and peptide amines (Wang et al., 2010). And the peaks at 1570.81 and
1570.34 cm1 were ascribed to N H bending of amides II of protein (Lin et al., 2005). The peaks at 1389.13 and 1399.71 cm1 could
be assigned to >C O stretch of the COO groups and C O bond
from COO groups (Wang et al., 2010; Haxaire, Marechal, Milas, &
Rinaudo, 2003).
The ngerprint region where bands characterize the molecule
as a whole is assigned to be the wave number region from
1200 to 800 cm1 , which can be used to characterize differ
ent polysaccharides (Cern et al., 2003; Copkov
et al., 2006).
The exopolysaccharides produced by S. thermophilus strains in
this study differ from the EPS produced by Leuconostoc sp. CFR
2181 and algal polysaccharide by having an additional peak at
around 1240 cm1 region due to the presence of o-acetyl ester
(Kazy, Sar, Singh, Sen, & Souza, 2004) and from the EPS produced
by Pseudozyma sp. NII 08165 by having an additional peaks at
around 1078, 1070 and 1035 cm1 regions due to the presence of

E. Dertli et al. / Carbohydrate Polymers 136 (2016) 427440

monosaccharide constituents of pectic and hemicellulosic polysaccharides (Sajna et al., 2013). The strong absorption peaks at 1003.25
and 1006.00 cm1 indicated the presence of a polysaccharide (Jin
et al., 2010), which evidenced the presence of presence of carboxyl
groups serving as binding sites for divalent cations (Wang et al.,
2010) and hydroxyl groups that were the preferred groups for the
occulation process (Guo et al., 2009). In the anomeric region, the
existence of absorption bands around at 800 cm1 revealed the
possible presence of -glycosidic linkages while absence of those
at 870890 cm1 suggested that there could be no -glycosidic
linkages in the EPS produced by S. thermophilus (Tao, Biao, Yu, &
Ning, 2008). The absorption bands observed at 743.60 cm1 and
744.20 cm1 probably corresponded to C O C bending vibration
(Sekkal & Legrand, 1993).
As a conclusion, in Fig. 3, the absorption peaks within the
12001000 cm1 region were attributed to vibrations of a broad
stretch of C O and C O C glycosidic bands, which revealed
the presence of carbohydrates (Zhang et al., 2013; Bremer &
Geesey, 1991; De Vuyst & Vaningelgem, 2009) that would be sugar
monomers such as glucose, galactose and rhamnose in the EPS
produced by S. thermophilus. Accordingly, in our case, the presence of these monomers were also conrmed by De Vuyst and
Vaningelgem (2009) who invented that the EPS by S. thermophilus
ST 111 is a polymer of heptasaccharide ([1][7]) units composed of
galactose and rhamnose in a 5:2 ratio, for instance, having preferably the following structure:

[1]



[2]



437

EPS string or network were presented in control group ice-cream

sample, which was also consistent with previous observations in
other dairy products (Hassan et al., 2003a; Yilmaz et al., 2015).
The SEM images in this study conrmed the multiple response
optimization (MRO) which calculated that minimum EPS amount
would be obtained at around 3233 C for 2 h of fermentation while
maximum EPS amount could be achieved at around 4042 C for
4 h of fermentation (Table 4). Accordingly, from Fig. 4a and b, it is
seen that EPS network does not clearly appear at SEM images for
R1 where fermentation temperature and time values were 32 C
and 2 h, respectively (Table S1, see Supplementary information). At
SEM images for R5 where these values were 37 C and 3 h, intensity of EPS networks was moderate. However, at SEM images for
R10 where these values were 42 C and 4 h, EPS networks could be
clearly seen.
As a conclusion, these images conrmed the effects of EPS on
ice-cream network system and allowed a direct microscopic observation showing the formation of EPS network with milk proteins
during ice-cream fermentation process.
3.6. Sensory properties
Table S3 (see Supplementary information) shows differences between the treatment groups with respect to color,

D Gal (1 6) D Gal

2) L Rha (1 2) D Gal (1 3) D Gal (1 3) D Gal (1 3) L Rha (1





 

 

 

 
[3]

[4]

wherein N is between 800 and 5000, preferably between 814 and

4886, or wherein N is at least 1000, 1500, 2000, 2500, 3000, 3500,
4000, 4500, 5000, 5500, 6000, 6500 or at least 7000 or any number
in-between these numbers.
As far as the molecular dimension of the aforementioned EPS
were concern, at least 60%, 65% or 80%, or more preferably at
least 85% or 90% or 95% of the exopolysaccharide was suggested
to have a molecular mass of at least 106 AMU or at least 2.106 AMU,
more preferably at least 2.5 times 106 AMU, 3.106 AMU, 3.5 times
106 AMU, most preferably at least 4.106 AMU, 5.106 AMU, 6.106 ,
7.106 , 8.106 AMU or more. Given the reports, it is possible to say
that the EPS in our study would probably have a molecular mass of
more than 2.106 AMU. Given that the structure would have a strong
impact on rheological properties, such a great molecular mass of
EPS could easily explain the considerable increase of the viscosity
and remarkable changes observed in the rheological properties of
the strain 1, strain 2 and mixture groups treated with EPS+ strains
as compared to those of control group in this study (Fig. 2, Table 4).
3.5. Microstructural characterization
SEM images showed the effect of in situ EPS production on
microstructural properties of ice cream samples where interactions
of EPS with other milk components were analyzed (Fig. 4a and b).
As can be seen, the presence of EPS resulted in formation of web
and pore-like structures due to EPS network formed within proteins
present in ice-cream samples, which was in accord with previous
reports on other dairy products (Hassan, Frank, & Elsoda, 2003a;
Hassan et al., 2003b). In contrast to the presence of EPS laments
in ice-cream samples produced with EPS+ cultures (strain1, strain2
and mixture treatment groups), a compact structure without any

[5]

[6]

[7]

mouth-solubility, icy texture, melting resistance, acidity, cream

taste, foreign taste and general acceptance. As can be seen, there
were signicant (P < 0.05) differences between the treatments at
each experimental run in terms of these parameters. Mixture group
received generally the highest scores by panelists in terms of color,
mouth solubility, icy texture and general acceptance. Strain 1 group
received the highest scores with respect to icy texture and cream
taste. Although panelists perceived a little acidity and foreign taste
from the ice-cream samples produced with EPS+ strains, this was
also the case for control ice-cream samples at some experimental
runs.
In contrast to previous reports on negative role of some stabilizers on taste perception of dairy products (Koksoy & Kilic, 2004), the
production of ice-cream samples with EPS which is a natural stabilizer, did not result in undesirable sensory characteristics in this
study. From the sensory analysis results, it was clear that EPS presence in ice-cream resulted in better product characteristics in terms
of color, mouth solubility, icy texture and cream taste. In other
words, the panelists found the ice-cream with EPS more soluble in
mouth, low icy and creamier with better color characteristics, suggesting that the strain specic effects could positively determine
the sensory characteristics. Given that stabilizers are indispensable
ingredients in dairy products of high rheology (Tasneem, Siddique,
Ahmad && Farooq, 2014); however, our functional and fermented
ice-cream product could be produced without using any stabilizer,
these should be very important for dairy industry which would
probably aim to save costs for stabilizers that are readily used in
the production of ice-cream.

438

E. Dertli et al. / Carbohydrate Polymers 136 (2016) 427440

Fig. 4. (a) Scanning electron micrographs for different treatment groups; namely, control and strain1 at 1st, 5th, and 10th runs . White eps shows the network strands
suggesting EPS structure. Refer to Table S1 (see Supplementary information) to see the experimental runs. (b) Scanning electron micrographs for different treatment groups;
namely, strain 2 and Mixture (admixture of strain 1 and strain 2) at 1st, 5th, and 10th runs . White eps shows the network strands suggesting EPS structure. Refer to
Table S1 (see Supplementary information) to see the experimental runs.

4. Conclusions
The main aim of this study was to produce a functional ice-cream
without using any stabilizer on the one hand, but maintaining
desired rheological and technological properties on the other hand.
The results of this study revealed the possibility to develop a fermented functional ice cream using S. thermophilus strains with
different EPS production characteristics without any stabilizer. The
technological characteristics of fermented ice cream samples relied
on the fermentation temperature and time depending on strain
specic conditions in which fermentation temperature appeared
to be highly effective and as a result of in situ EPS production,
these technological characteristics were improved. Optimization
analysis revealed that the functional ice-cream should be fermented with strain 1 or strain mixture at 4042 C for 4 h in
order to produce the most viscous ice-cream with maximum EPS
content. In addition, the results showed that higher viscosity of

ice-cream was related with both pH and in situ EPS production in

ice-cream.
Specically, the rheological analysis of the fermented icecream samples demonstrated the potential of EPS from S.
thermophilus as thickening and gelling agent forming web-like
compact microstructure within the ice cream supporting the functionality of EPS of LAB in dairy industry. Importantly, it should be
noted that fermented ice-cream could be produced without using
any stabilizer, which should be very important for dairy industry
which would probably aim to save costs for stabilizers given that
stabilizers are indispensable ingredients in dairy products of high
rheology.
Overall, the results of this study showed the functional role of
in situ EPS production on technological functions of fermented ice
cream and further studies are required in order to show the bilateral
effects of EPS and LAB cultures including probiotic strains in ice
cream as promising product for future industrial applications in

E. Dertli et al. / Carbohydrate Polymers 136 (2016) 427440

terms of developing novel functional foods, particularly in the dairy

industry.
Acknowledgements
This work was supported by The Scientic and Technological
Research Council of Turkey (TOVAG, project no: 112O169).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carbpol.2015.08.047.
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