International Dairy Journal 59 (2016) 72e79

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International Dairy Journal

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

Application of cold- and heat-adapted Lactobacillus acidophilus in the

manufacture of ice cream
Firuze Ergin a, Zeynep Atamer b, Ayse Asci Arslan a, Emine Mine Comak Gocer a,
Muammer Demir a, Meike Samtlebe b, Joerg Hinrichs b, Ahmet Kckcetin a, *
a
b

Department of Food Engineering, Faculty of Engineering, Akdeniz University, Antalya, Turkey

Institute of Food Science and Biotechnology, Department of Soft Matter Science and Dairy Technology, University of Hohenheim, Stuttgart, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 15 September 2015
Received in revised form
15 March 2016
Accepted 20 March 2016
Available online 31 March 2016

Manufacture of ice cream using cold- and heat-adapted Lactobacillus acidophilus was studied. Temperatureetime combinations at 4
C for 18 h and at 45
C for 15 min were set as the adaptation conditions
for below and above the optimum growth temperature (37
C), respectively. Ice cream was produced by
two different methods: method 1, ice cream mix was fermented with cold- and heat-adapted
L. acidophilus prior to freezing; method 2, cold- and heat-adapted L. acidophilus was added to ice
cream mix but the mix was not fermented with L. acidophilus prior to freezing. The lowest reduction ratio
was found in the samples produced by using method 1 and cold-adapted L. acidophilus, adapted at 4
C
for 18 h. L. acidophilus survived at the required levels (>106 cfu g1), with or without an adaptation. The
adaptation conditions improved stability of L. acidophilus in the samples, but the magnitude of
improvement was small.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Consumer interest in different types of probiotic food products
has signicantly increased in recent years due to their nutritional
value and health-promoting properties (Casarotti & Penna, 2015).
Although many scientic studies have demonstrated some benets
to the consumption of probiotic cells, European Food Safety Association (EFSA) rejected nearly every health claim assert by the
probiotics industry due to insufcient proof of their health claims
(Kent & Doherty, 2014; Schmidt, 2013). However, manufacture of
probiotic products continues to expand, as do studies investigating
cell viability and functional properties of probiotic foods (Burgain,
Gaiani, Linder, & Scher, 2011). Among food products, ice cream is
known to be an advantageous vehicle to deliver probiotic bacteria
to human body since it has a neutral pH and high total solid level
providing protection for probiotic cells (Homayouni, Azizi, Ehsani,
Yarmand, & Razavi, 2008). However, cells must survive freezing
as well as frozen storage; temperature changes during freezing and
thawing may cause damage such as reduction or even complete
loss of metabolic activity (Akn, Akn, & Krmac, 2007; Haynes &

* Corresponding author. Tel.: 90 242 3106569.

E-mail address: kucukcetin@akdeniz.edu.tr (A. Kckcetin).
http://dx.doi.org/10.1016/j.idairyj.2016.03.004
0958-6946/ 2016 Elsevier Ltd. All rights reserved.

Playne, 2002; Mohammadi, Mortazavian, Khosrokhavar, & da

Cruz, 2011).
Based on the commonly accepted levels to achieve the claimed
benecial effects of probiotic bacteria, these specic microorganisms must be viable, abundant (ranging from 106 to 109 cfu g1) and
active in the product up to the expiry date (Casarotti & Penna, 2015)
against a number of stresses that may be imposed, such as during
production of the culture, incorporation into milk products, transport and storage, and must also survive transit along the gastrointestinal tract (Corcoran, Stanton, Fitzgerald, & Ross, 2008). As may
be the case for all bacteria, stress can also be dened for probiotics
as a change, whether in the genome, the proteome, or the environment, that produces a decrease in the growth rate or survival
(Vorob'eva, 2004).
In probiotic ice cream production, the main important stress
that probiotics encounter is cold stress (Abghari, Sheikh-Zeinoddin,
& Soleimanian-Zad, 2011; Cruz, Antunes, Sousa, Faria, & Saad,
2009). Due to cold stress, several changes occur in probiotics that
can be summarised as follows: reduction in membrane uidity,
alteration in the level of DNA supercoiling, increase in the rate of
DNA strand breakage, stabilisation of secondary structures of DNA
and RNA and thus changes in replication, transcription and translation. Furthermore, changes in enzyme activity, protein-folding as

F. Ergin et al. / International Dairy Journal 59 (2016) 72e79

well as ribosome functions are also observed at low temperatures

(Corcoran et al., 2008).
Numerous strategies have been developed to protect probiotic
bacteria against these stresses, such as prebiotics, microencapsulation and stress adaptation (Akn et al., 2007; Pinto et al., 2012;
Song, Yu, Liu, & Ma, 2014). Regarding adaptation of probiotic bacteria to stress factors, recent studies on increasing the viability of
probiotic bacteria have concentrated on the development of the
mechanisms that bacteria show against stress conditions (Sanders
& Marco, 2010). Probiotic bacteria evolved stress-sensing systems
and defence against stress that allow them to withstand harsh
conditions and sudden environmental changes. These physiological

al, 2007;
changes are called stress adaptation (Streit, Corrieu, & Be
Van de Guchte et al., 2002). When probiotic bacteria are exposed
to a moderate level of stress, i.e., sub-lethal stress, they acquire
increased resistance to a subsequent exposure to a more severe
level of the same stress or other stress(es) (Kim, Perl, Park,
Tandianus, & Dunn, 2001).
The survival of probiotic bacteria against stress is dependent on
the physiological state of the cells, and there is a clear difference
between the exponential and stationary phase cells (Kim et al.,
2002; Saarela et al., 2004). An improvement in the survival of
cells was hypothesised when stress-adapted (Ross, Desmond,

nchez, Ruiz,
Fitzgerald, & Stanton, 2005; Saarela et al., 2004; Sa
Gueimonde, Ruas-Madiedo, & Margolles, 2012; Shah, 2000). The
adaptation of bacteria to cold environments is realised by changes
in the protein synthesis of the cell and the cell membrane fatty acid
composition (Girgis, Smith, Luchansky, & Klaenhammer, 2003).
Bacteria can also adapt to freezing stress in ice cream production
via changes in protein synthesis occurring in their cells during
other stress types such as heat adaptation, explained by the transient synthesis of a set of highly conserved stress proteins. An
example of this is molecular chaperones that confer not only
enhanced resistance to elevated temperatures but also signicant
cross-protection against other stresses such as cold shock (Walker,
Girgis, & Klaenhammer, 1999).
However, there are only a limited number of studies that have
investigated the effect of stress adaptation of probiotic bacteria
on their survival in a real food system such as milk and yoghurt
(Settachaimongkon et al., 2015). Some stress response mechanisms are naturally developed by bacteria for them to be able to
survive under severe conditions (Walker et al., 1999). However, to
our knowledge, no study has been conducted on incorporation of
cold- or heat-adapted probiotic bacteria in ice cream production,
although probiotic characteristics such as bile and acid resistance
can be affected by the method of ice cream processing (Abghari
et al., 2011). In the case of probiotic ice cream production, probiotic bacteria can be incorporated in either fermented or unfermented mix (Homayouni, Azizi, Javadi, Mahdipour, & Ejtahed,
2012). The survival of probiotic bacteria in ice cream may be
improved by the application of an appropriate production technique. Therefore, determination of the inuence of usage of an
ice cream mix, which is fermented either with or without
probiotics, on the survival of probiotic bacteria may play a signicant role.
Our hypothesis was that survival rates would be higher with
cold- and heat-adapted Lactobacillus acidophilus than with nonadapted L. acidophilus when used in manufacture of ice cream. To
increase carriage of probiotic bacteria by ice cream, our approach
was to use cold- and heat-adapted probiotic bacteria in ice cream
production as well as to apply different ice cream production
methods. We therefore manufactured probiotic ice cream using
L. acidophilus that was cold- and heat-adapted to freezing stress at
different growth phases, i.e., mid-exponential phase and stationary
phase.

73

2. Materials and methods

2.1. Bacterial strain, medium, and growth curve
L. acidophilus (DSM20079, Leibniz Institute DSMZ, Braunschweig, Germany) was grown anaerobically at 37
C for 24 h on de
Man, Rogosa, and Sharpe (MRS) broth (Merck KGaA, Darmstadt,
Germany) containing 0.05% cysteine (Merck KGaA) and adjusted to
pH 6.2 with 0.1 N NaOH (Merck KGaA). For production of stock
culture, after incubation the cell suspension was centrifuged
(6000  g, 5
C, 5 min) and the cell pellet resuspended in 5 mL
sterile nutrient-glycerol mixture comprising 0.8 g nutrient broth
(Merck KGaA), 30 mL glycerol (Merck KGaA) and 70 mL pure water,
then aliquoted into 1 mL microcentrifuge tubes (T-6524, SigmaeAldrich Company Ltd., Dorset, UK) and immediately frozen
at 80
C. Subsequently, stock culture was inoculated into MRS
broth (50 mL) to an initial optical density at 600 nm (OD600) of 0.1
and anaerobically incubated overnight at 37
C. To obtain the
growth curve of L. acidophilus, an overnight culture was inoculated
into MRS broth (200 mL) at an initial OD600 of 0.1 and anaerobically
incubated at 37
C for 24 h. Samples were collected hourly to
determine viable L. acidophilus counts OD600. The viable cell counts
were determined by plating on MRS agar (Merck KGaA) (0.05%
cysteine, pH 6.2), and incubating the plates anaerobically at 37
C
for 72 h.
2.2. Cold and heat adaptation conditions
The experimental setup for the cold and heat adaptation treatments is illustrated in Fig. 1. L. acidophilus in both mid-exponential
and stationary growth phases in MRS broth was cold- and heatadapted to stress faced during freezing in ice cream production
and storage at 20
C for 30 d. For cold adaptations, timeetemperature combinations at 4, 10 and 20
C for 0.5, 1, 2, 3, 4, 5, 6,
7, 18, 24 h were tested; for heat adaptations the timeetemperature
combinations were 45, 50 and 55
C for 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 18,
24 h. The experiments were conducted for both phases (midexponential and stationary) of growth. Following the adaptation
treatments, aliquots of cold- and heat-adapted L. acidophilus were
divided into four portions. One of these was plated on MRS agar and
anaerobically incubated at 37
C for 72 h to determine the initial
viable cell count; the remaining three were stored at 20
C. Aliquots of non-adapted L. acidophilus (control) at both phases of
growth were also prepared and all portions including the control
were stored at 20
C. Aliquots were removed at 1, 15 and 30 d
storage and viable cell counts determined immediately after
thawing at room temperature (within a few minutes). Taking the
average viable counts obtained for the samples after storage (1, 15
and 30 d) and according to the highest survival rate of the cold- and
heat-adapted L. acidophilus, adaptation conditions were set for the
L. acidophilus used in the ice cream productions. The adaptation of
L. acidophilus during ice cream production was realised by means of
adaptation of L. acidophilus in reconstituted milk, unlike the experiments conducted for determining the adaptation conditions.
Adaptation means in this study that improvement of probiotic
bacterial viability against the stresses faced during freezing in ice
cream production and storage at 20
C.
2.3. Preparation of L. acidophilus for the manufacture of ice cream
An overnight L. acidophilus culture was inoculated into MRS
broth (450 mL) at an initial OD600 of 0.1. After anaerobic incubation
at 37
C for 7 h (mid-exponential phase, OD600 approximately 0.5),
the cells were collected by centrifugation (6000  g, 5
C, 5 min)
and resuspended in reconstituted skim milk (11%, w/v), then used

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F. Ergin et al. / International Dairy Journal 59 (2016) 72e79

Fig. 1. Schematic illustration of the experimental setup for the adaptation tests.

in ice cream production at a concentration of approximately 3% (w/

v), which resulted in colony counts higher than 108 cfu mL1 in the
ice cream mix. The prepared samples were placed in a water bath
(JSRC-22C, JS Research Inc., Gongju-City, Korea), adjusted to the
predetermined cold (4
C for 18 h) and heat (45
C for 15 min)
adaptation conditions. For the production of the control ice cream
samples, L. acidophilus was subjected to the same procedure
without adaptation treatment.
2.4. Manufacture of probiotic ice cream with cold- and heatadapted L. acidophilus
In 3 kg of ice cream formulation 323 g skim milk powder, 540 g
sucrose, 103 g butter, 17 g stabiliser, and 2017 g water were used.
Probiotic ice cream mix composition was 3% (w/w) fat, 10% (w/w)
milk solids nonfat, 18% (w/w) sugar, 0.5% (w/w) stabiliser and 68.5%
(w/w) water. The mixture was pasteurised at 75
C for 5 min and
cooled to 20
C. The mixture was homogenised using a mechanical
mixer during pasteurisation. The cooled mix was ripened overnight
at 4
C. Probiotic ice cream was produced by using L. acidophilus
DSM 20079, which was cold- and heat-adapted at specied temperatureetime conditions. Two different production methods were
applied: method 1, ice cream mix was fermented at 37
C by coldand heat-adapted L. acidophilus until pH decreased to 5.5 prior to
freezing; method 2, cold- and heat-adapted L. acidophilus were
added to the ice cream mix prior to freezing and the mix was not
fermented. Control probiotic ice cream samples were also produced
by using non-adapted L. acidophilus at mid-exponential phase of
growth for both manufacturing methods. The probiotic ice cream
samples were produced by using a batch freezing machine with a
10 kg capacity (M10C, Mehen Food Machine Manufacture Co. Ltd.,
Nanjing, China). The samples were packaged in 200 mL cups and
stored at 20
C for 90 d.

2.5. Analyses for the ice cream mixes and ice creams
The pH values of the mixes and ice creams were measured using
a pH-meter (Thermo Scientic Orion 2-Star, Bremen, Germany).
Percentage of titratable acidity for the samples was measured by
the method of Bradley et al. (1992). The viscosity value of the mix
used in the ice cream production was measured using a Brookeld
viscometer (model DV-II; Brookeld Engineering Laboratories,
Inc., Stoughton, MA), with spindle 2, and speed at 1 rpm, following
the method described by El-Rahman, Madkor, Ibrahim, and Kilara
(1997) after 24 h of ripening at 4
C. Increase in volume (overrun)
was determined using a 100 mL cup and Eq. (1) described by
Voulasiki and Zerridis (1990):

% Overrun

Weight of ice cream mix  Weight of ice cream

Weight of ice cream
 100
(1)

The ice cream samples were analysed for dry matter content by
drying samples at 102 2
C for 3.5 h using an air oven, for fat
content using the Gerber method and for protein content by the
Kjeldahl method (Anonymous, 2003). Melting rate was estimated
according to El-Nagar, Clowes, Tudoric, Kuri, and Brennan (2002)
with slight modications. For the measurement of melting rate,
100 g of tempered samples were left to melt (at a constant temperature 15.5 0.3
C) on a 2.5 mm wire mesh screen above a
beaker. The weight of drip was measured over a 60 min period.
Firmness analysis was conducted using a texture analyser (TA-XT2,
Stable Microsystems, Godalming, Surrey, UK) tted with a 5 mm
diameter stainless steel probe, setup to record the force used to

F. Ergin et al. / International Dairy Journal 59 (2016) 72e79

penetrate the sample to a depth of 25 mm at a speed of 1 mm s1.

Firmness was measured as the peak compression force (N) during
the penetration of the ice creams (El-Nagar et al., 2002).
2.6. Enumeration of survival of L. acidophilus
The counts of viable L. acidophilus were determined directly
after ripening of the mix at 4
C for 24 h and during storage of the
ice cream samples. One gram of sample was diluted with 9 mL of
sterile Ringer solution (1/4 strength) (Merck KGaA) and mixed
uniformly with a vortex mixer (Classic, Velp Scientica, Usmate,
Italy) (Anonymous, 2001). Subsequent serial dilutions were made
and viable cell numbers enumerated using the pour plate technique. The counts of viable L. acidophilus were enumerated on MRS
agar incubated aerobically at 37
C for 72 h (Anonymous, 1997).
2.7. Statistical analyses
In this study, all measurements were performed in duplicate. All
statistical calculations were performed using SAS Statistical Software (release for Windows, SAS Institute Inc., Cary, NC, USA). The
Duncan's multiple range test was conducted to detect differences
among the treatment means.
3. Results and discussion
3.1. Determination of cold and heat adaptation conditions
L. acidophilus was incubated in MRS broth to determine the
times taken to reach mid-exponential and stationary phases of
growth. Mid-exponential and stationary phases of L. acidophilus
were found to be reached after approximately 7 h (OD600 0.5) and
14 h (OD600 1.0) of anaerobic incubation at 37
C, respectively. In
line with the hypothesis that the survival of cold- and heat-adapted
L. acidophilus is higher than that of non-adapted L. acidophilus in ice
cream samples, cold and heat adaptation conditions were determined in the beginning of the experiments according to the highest
survival rate of L. acidophilus in MRS broth stored for 30 d at 20
C.
The experiments were conducted using L. acidophilus in both midexponential and stationary growth phases. The samples were
analysed after 1, 15 and 30 d storage at 20
C. Fig. 2 shows an
example of the obtained results for the counts of viable
L. acidophilus adapted under cold adaptation conditions at 4, 10 and
20
C for the selected adaptation times (1, 18 and 24 h) and for both
growth phases. The counts of viable L. acidophilus ranged from 8.53
to 9.46 log cfu mL1 after 1 d stress exposure time, whereas after
30 d they ranged from 4.03 to 9.21 log cfu mL1. At an adaptation
temperature of 4
C, 1.81 log cfu mL1 reduction was observed in
the counts of viable L. acidophilus in its mid-exponential phase after
1 h of adaptation time; however, lower reductions, 0.20 and
0.32 log cfu mL1, were detected after longer adaptation times of 18
and 24 h, respectively (Fig. 2a, after 30 d exposure).
Some of the results for heat adaptation conditions at 45, 50 and
55
C for the selected adaptation times (15 min, 18 and 24 h) are
given in Fig. 3. Long adaptation times (18 and 24 h) at high temperatures (50 and 55
C) resulted in complete loss of the viability of
the cells. At high temperatures, the best temperatureetime combination for the adaptation of the cells was found to be at 45
C for
15 min. When all the results for the examined adaptation times
(0e24 h) were considered (data not shown), after 30 d of exposure
time a mean of 1.94 log cfu mL1 reduction in the viability of nonadapted L. acidophilus was observed. These observed reductions in
the viable cell counts are illustrated in Fig. 3 for cold- and heatadapted L. acidophilus in MRS broth.

75

The lowest decrease in the viability was obtained for cold

adaptation at 4
C for 18 h and for heat adaptation at 45
C for
15 min. A decrease in viable L. acidophilus counts from 9.41 to
9.21 log cfu mL1 (approximately 2.1% reduction) and from 9.50 to
8.45 log cfu mL1 (approximately 11.1% reduction) under cold
adaptation (4
C; 18 h stress exposure) and heat adaptation (45
C;
15 min stress exposure) conditions was observed, respectively.
These were the lowest reductions obtained for all the temperatureetime combinations for cold and heat adaptation. The coldadapted (4
C for 18 h, mid-exponential phase) and heat-adapted
(45
C for 15 min, mid-exponential phase) L. acidophilus survived
up to 10-fold and 2-fold better than non-adapted L. acidophilus
when stored to at 20
C for 30 din MRS broth, respectively. The
same adaptation conditions were also applied to L. acidophilus
suspended in reconstituted skim milk and the results showed that
there were no signicant differences between the counts of
L. acidophilus suspended in MRS broth and reconstituted skim milk
(data not shown).
These conditions were therefore set as the cold and heat
adaptation conditions for L. acidophilus in MRS broth, which were
then used in the production of probiotic ice cream. L. acidophilus in
its mid-exponential phase was used for the further experiments,
since it was observed to be more stable than that in the stationary
phase for the set adaptation temperatureetime combinations in
which the lowest decrease in the viability was detected.
It should be also noted that the stress responses of the nonadapted L. acidophilus from the mid-exponential and stationary
phases did not show differences. Parente et al. (2010), in their
screening study for stress tolerance in Lactobacillus plantarum,
Lactobacillus pentosus and Lactobacillus paraplantarum, also
concluded that exponential phase cells were more tolerant than
stationary phase cells. However, for some of the strains, Parente
et al. (2010) also reported no signicant differences between the
two growth phases of the cells. An increased survival for exponentially growing cells of Lactobacillus paracasei exposed to heat
and salt stresses was also shown by Desmond, Stanton, Fitzgerald,
Collins, and Ross (2001). A good explanation for this observed
difference between the two phases of cells was given by Kim et al.
(2002): each cell in an exponential phase culture is in different
stage of their cell cycle due to the random nature of cell growth,
therefore the cells in exponential phase are physiologically heterogeneous groups of cells, which is in contrast to the situation at
stationary phase. Wouters et al. (1999) reported approximately
100-fold increased survival for Lactococcus lactis MG1363 exposed
to cold stress at 10
C for 4 h (mid-exponential phase) prior to
freezing at 20
C for 24 h, while Walker et al. (1999) showed that
when Lactobacillus johnsonii VPI11088 was subjected to 55
C for
30 min before frozen storage at 20
C for 7 d, its viability increased
20% in comparison with the control cells.
3.2. Physicochemical properties of the ice cream mixes and
probiotic ice cream
The pH, titratable acidity and viscosity values of ice cream mixes
varied from 5.50 to 6.48, from 0.19 to 0.42% and from 7.07 to
13.1 Pa s, respectively. These parameters were found to be inuenced signicantly (P < 0.001) by the manufacturing method
(Table 1). The titratable acidity and viscosity values increased as the
pH value decreased, which is due to the fermentation step applied
to the ice cream mix. This result was similar to those reported by
Salem, Fathi, and Awad (2005). During fermentation, probiotic
bacteria could produce exopolysaccharides that could result in an
increase in the viscosity of the products (Patel, Michaud, Singhania,
Soccol, & Pandey, 2010). Furthermore, increase in the viscosity can
be explained with the increased voluminosity of the dispersed

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F. Ergin et al. / International Dairy Journal 59 (2016) 72e79

Fig. 2. Survival of Lactobacillus acidophilus adapted under low temperature conditions, 4
C (a, d), 10
C (b, e) and 20
C (c, f) for mid-exponential phase (a, b, c) and stationary phase
(d, e, f) after 1, 15 and 30 days of exposure at 20
C. Adaptation times were:
, non;
, 1 h;
, 18 h;
, 24 h.

particles during acidication of milk (Aboulfazli, Baba, & Misran,

2015; Alvarez, Wolters, Vodovotz, & Ji, 2005).
As the pH of milk decreases, especially in the pH range 5.5e5.0,
considerable changes in physicochemical properties of casein micelles such as dissociation of caseins and increase in voluminosity
of casein micelles are observed (Lucey & Singh, 1998). It was also
observed that the viscosity of ice cream mix was signicantly
(P < 0.05) affected by cold and heat adaptation treatments. The
viscosity values of ice cream mixes prepared using cold- and heatadapted L. acidophilus were lower compared with those prepared
using non-adapted L. acidophilus. The reason for this could be that
during the adaptation bacteria reduce its metabolic activity to stay
alive longer (McDougald, Rice, Weichart, & Kjelleberg, 1998).
Probiotic ice cream with pH ranging from 5.52 to 6.47 and
titratable acidity ranging from 0.13 to 0.35% was obtained. The results showed that pH and titratable acidity values of the ice cream
samples were signicantly (P < 0.001) inuenced by the
manufacturing methods, which could be due to the fermentation
process (Table 1).
The physicochemical characteristics (total solids, fat and protein
contents) of probiotic ice cream samples were not inuenced by the
adaptation treatment and the manufacturing method. The total
solid, fat and protein contents of the probiotic ice cream samples
ranged from 27.4 0.2 to 28.8 0.8%, from 2.78 0.13 to

2.93 0.25% and from 3.69 0.09 to 4.01 0.01%, respectively.

Similar observations were also reported by Vijayageetha, Begum,
and Reddy (2011) and Akaln and Erisir (2008) in their studies
conducted with L. acidophilus and Bidobacterium animalis strains.
The mean rmness, overrun and melting rate values of ice
creams varied from 5.70 0.32 to 8.63 0.48 N, from 57.4 2.2 to
66.7 2.2% and from 14.3 0.5 to 16.6 0.6 g 60 min1, respectively. The rmness and melting rate values of ice cream samples
decreased signicantly (P < 0.001) as the mix was fermented prior
to freezing, whereas an increase was observed in the measured
overrun values. These properties were not affected by the adaptation treatment on L. acidophilus (Table 1).
3.3. Survival of L. acidophilus
The changes in the viable counts of L. acidophilus in milk before
cold and heat adaptation and in ice cream mixes and in ice cream
samples after cold and heat adaptation during storage are presented in Fig. 4. There was a log decrease in L. acidophilus survival
ranging from 0.14 to 0.39 after freezing. Turgut and Cakmakci
(2009) showed that after storage of ice cream at 20
C for 90 d,
the numbers of viable L. acidophilus and Bidobacterium bidum
decreased by 0.38 and 0.26 log units, respectively, as compared
with the levels at the rst day of the storage. A decline in bacterial

F. Ergin et al. / International Dairy Journal 59 (2016) 72e79

77

Fig. 3. Survival of Lactobacillus acidophilus adapted under high temperature conditions, 45
C (a, d), 50
C (b, e) and 55
C (c, f) for mid-exponential phase (a, b, c) and stationary
phase (d, e, f) after 1, 15 and 30 d exposure at 20
C. Adaptation times were:
, none;
, 15 min;
, 18 h;
, 24 h.

Table 1
Effect of the manufacturing method of ice cream and the adaptation treatment of Lactobacillus acidophilus on the physicochemical properties of ice cream mixes and ice
creams.a
Ice cream mix
pH

Ice cream

Titratable acidity (%) Viscosity (Pa s) pH

Manufacturing method of ice cream

0.42
Method 1
5.52 0.0a
Method 2
6.44 0.0a
0.20
Adaptation treatment of L. acidophilus
Non-adapted
5.96 0.40a 0.30
4
C for 18 h
5.99 0.50a 0.31
45
C for 15 min 5.97 0.50a 0.31

0.00a
0.00b

12.6.0 1.5a
8.1 1.1b

0.10a
0.10a
0.20a

11.2 4.0a
9.9 3.9b
9.9 4.2b

5.53 0.02b
6.43 0.04a

Titratable acidity (%) Overrun (%)

Firmness (N) Melting rate (g 60 min1)

0.34 0.02a
0.13 0.00b

65.98 2.18a 6.09 0.73b

58.93 3.19b 8.35 0.71a

14.41 0.29b
16.42 0.25a

5.98 0.43ba 0.24 0.15a

6.00 0.48a 0.24 0.15a
5.97 0.45b 0.23 0.15a

63.58 3.56a 6.93 1.39a

61.78 4.31a 6.79 1.07a
62.02 5.14a 7.05 1.46a

15.34 1.02a
15.38 0.99a
15.53 1.11a

a
Method 1, Ice cream mix was fermented with cold- and heat-adapted L. acidophilus prior to freezing; method 2, Cold- and heat-adapted L. acidophilus was added to the ice
cream mix prior to freezing and the mix was not fermented. Values are expressed mean standard deviation, different superscript letters after values indicate signicant
differences using Duncan's multiple range test (P < 0.05).

viable counts after freezing could be a result of cold shock during

freezing, mechanical stress and osmotic pressure (Cruz et al., 2009).
Mean viable log counts of L. acidophilus in the samples were found
to be between 7.69 and 8.25 log units at the end of 90 d storage.
Nousia, Androulakis, and Fletouris (2011) conducted their studies in
an ice cream mix inoculated with either freeze-dried or activated
cultures of L. acidophilus LMGP-21381; a signicant decrease in the
viable L. acidophilus counts due to freezing process was reported,

but during frozen storage at 25
C for 45 weeks no signicant

change in the viability was observed.
In this study, when compared with the L. acidophilus counts in
the ice cream mix, during 90 d of storage the lowest reduction in
viable L. acidophilus counts was determined in the probiotic ice
cream samples produced by using cold- and heat-adapted
L. acidophilus and the rst manufacturing method. However, the
highest reduction was observed in the samples manufactured using

F. Ergin et al. / International Dairy Journal 59 (2016) 72e79

Survival of L. acidophilus (log cfu g- )

10.5
10
9.5
9
8.5
8
7.5
7
1

Milk

Ice cream mix

30
60
Ice cream, storage time (days)

90

C

Fig. 4. Survival of non-adapted, heat adapted (45

for 15 min, mid-exponential
phase) and cold adapted (4
C for 18 h, mid-exponential phase) Lactobacillus acidophilus in milk, ice cream mixes and probiotic ice cream samples. Ice creams were
produced by: -, non-adapted, method 1; , heat-adapted, method 1; , cold-adapted
method 1; , non-adapted, method 2; , heat-adapted, method 2; , cold-adapted
method 2. The viable counts of L. acidophilus in milk before stress adaptation (Milk)
and stress-adapted and non-adapted in ice cream mix (Ice cream mix) are shown. The
bars represent mean values, and the error bars represent standard deviation of the
mean.

non-adapted L. acidophilus and the second manufacturing method.

Although Maus and Ingham (2003) reported that a fermentation
process might override the positive effect coming from the stress
adaptation of the cells, it is important to know that the responses of
probiotic bacteria to stress are dependent on the type of stresses
exposed as well as the strain (Maus & Ingham, 2003; Mozzetti et al.,
2013; Saarela et al., 2004; Settachaimongkon et al., 2015).
The results showed that reduction of viable L. acidophilus counts
in probiotic ice cream were inuenced signicantly (P < 0.001) by
the manufacturing method and the adaptation treatment. When
the counts of L. acidophilus in the ice cream samples for each
storage day was compared with the counts in the ice cream mix, the
viable counts of L. acidophilus in the ice cream samples produced
using the rst manufacturing method were found to be higher than
the counts obtained for the samples manufactured according to the
second method (Fig. 4). This could be due to the fermentation
step applied in method 1, which may provide protection to the
probiotic cells against cold stress. Streit, Delettre, Corrieu, and Beal
(2008) also reported an increased resistance to cold stress in the
cells of Lactobacillus delbrueckii subp. bulgaricus, when cells were
adapted to acid stress. This was explained as a cross-protection
Table 2
Effect of the manufacturing method of ice cream, adaptation treatment of Lactobacillus acidophilus and the storage time on reduction of Lactobacillus acidophilus
counts in probiotic ice cream.a
Parameter

Reduction of L. acidophilus counts (log cfu g1)

Manufacturing method of ice cream

Method 1
0.24
Method 2
0.43
Adaptation treatment of L. acidophilus
Non-adapted
0.48
4
C for 18 h
0.23
45
C for 15 min
0.30
Storage period of ice cream (d)
1
0.27
30
0.32
60
0.35
90
0.40

0.12b
0.19a
0.17a
0.07c
0.20b
0.15b
0.19ba
0.19a
0.21a

a
Method 1, ice cream mix was fermented with cold- and heat-adapted
L. acidophilus prior to freezing; method 2, cold- and heat-adapted L. acidophilus
was added to the ice cream mix prior to freezing and the mix was not fermented.
Values are expressed mean standard deviation, different superscript letters after
values indicate signicant differences using Duncan's multiple range test (P < 0.05).

Reduction of L. acidophilus counts (%)

78

10
9
8
7
6
5
4
3
2
1
0
1

30

60

90

Storage (days)
Fig. 5. The percentage reduction for L. acidophilus counts in probiotic ice cream
samples according to ice cream mixes during storage. Ice creams were produced by: A
and :, non-adapted, methods 1 and 2, respectively; - and , heat-adapted (45
C for
15 min, mid-exponential phase), methods 1 and 2, respectively; and C, cold adapted
(4
C for 18 h, mid-exponential phase), methods 1 and 2, respectively.

phenomenon, since the resistance to a given stress (e.g., cold

stress) was improved by applying a different stress beforehand
(e.g., acid stress). In studies conducted using cells of Lactobacillus
rhamnosus GG, similar observations were also made (Ampatzoglou,
Schurr, Deepika, Baipong, & Charalampopoulos, 2010). Furthermore, it should be noted that the results showed that adaptation of
L. acidophilus revealed higher viable counts in the ice cream samples. The reduction of viable L. acidophilus counts in all samples
increased signicantly (P < 0.01) as the storage period extended
(Table 2, Fig. 5). These results are in agreement with those of
Walker et al. (1999) and Akaln and Erisir (2008), who reported that
the viable counts of L. acidophilus decreased at the end of 90 d
frozen storage. Although there was a decrease in the number of
viable cells, all the produced ice cream samples may be considered
as a probiotic food even after 90 d storage, since the remained
number of viable cells was above 107 log cfu g1.
4. Conclusion
The investigations in this work showed that a fermentation step
before freezing in method 1, unlike method 2, did make a difference
regarding the physicochemical properties of the ice cream samples.
To conclude, this study showed that cold and heat adaptation
treatments as well as a fermentation step in ice cream processing
can improve the survival of L. acidophilus DMS 20079 in ice cream.
The ndings support the hypothesis that the survival was better
when L. acidophilus DMS 20079 was adapted. The numbers of viable
L. acidophilus in all ice cream samples were equal or above
107 cfu g1 at the end of 90 d storage. In this study, when the results
of probiotic viability considered, a positive impact on the viability
has been observed as cold- and heat-adapted L. acidophilus were
used in ice cream manufacture. Although the magnitude of this
effect was below our expectations, the study highlights which
adaptation conditions are denitely not suitable.
However, one has to keep in mind that these observations are
valid only for the particular strain of L. acidophilus studied.
Although the strain that we selected for this study was found to be
resistant against the main stresses (freezing and frozen storage)
faced in ice cream production, which was found to be valid for both
conditions of the strain (temperature stress-adapted or nonadapted), selection of a sensitive strain could lead to different results. Furthermore, similar to the studies conducted on the adaptation to freezing stress, further research on adaptation to the

F. Ergin et al. / International Dairy Journal 59 (2016) 72e79

stresses encountered during gastrointestinal model system application, such as acid and bile stresses, is of signicant importance
due to the fact that adaptation of the cells to one stress could result
in improved resistance not just to that stress, but also to different
stresses.
Acknowledgements
This cooperative project between the Akdeniz University and
the University of Hohenheim was supported by the Scientic
and Technological Research Council of Turkey (TUBITAK, Project
number: 112 O 890), the Alexander von Humboldt Foundation
(Project number: DEU/1116797) and the Scientic Research Projects
Coordination Unit of Akdeniz University (Project number:
2013.01.0102.006).
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