Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
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
428
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 =
429
WHOLE MILK
1 liter (100 %)
HEATING (40 C)
PASTEURIZATION
(85 C for 5 min)
COOLING (40 )
PROCESSED MILK
FERMENTED MILK
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.
430
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)
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 +
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
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
432
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
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
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
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
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.
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.
434
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).
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
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
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
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
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
D Gal (1 6) D Gal
[4]
[5]
[6]
[7]
438
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
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