International Conference on Biological and Medical Sciences (ICBMS'2014) Jan.

15-16, 2014 Kuala Lumpur (Malaysia)

Effects of Vegetable Milk on Survival of

Probiotics and Rheological and
Physicochemical Properties of Bio-Ice Cream
Fatemeh. Aboulfazli, Ahmad salhin. Baba, and Misni. Misran

other hand, Ice cream is a delicious, wholesome, nutritious

frozen dairy product, which is widely consumed in different
parts of the world. Ice cream has nutritional significance but
encompasses no therapeutic properties [6]. Ice cream is
traditionally made from cow s milk and thus contains about 15
17% (w/w) lactose [7]. The demand for alternatives to cows'
milk is growing due to problems with its fat, cholesterol and
lactose (allergenicity) contents and desire for vegetarian
alternatives. In this regard, soy milk has been recognized as
being nutritionally helpful [8]. Ice cream can be made a
functional food by adding fruits, protein rich ingredients and
also using vegetable milk such as coconut and soy milks and
the addition of probiotics. Moreover, the coconut and soy
milks contain unique nutrient compositions in coconut and soy
milks can support the growth and survival of the lactic acid
bacteria in ice cream and thus could improve the health
benefits and nutritional components and health benefits of
probiotic ice creams. This is because soy milk contains protein
and indigestible fiber and polysaccharide, amino acids, lecithin
and unsaturated fat, minerals and vitamins, as well as bioactive
polyphenols such as isoflavones, phenolic acids, saponins and
tannins [9]. Frequent consumption of soy products also offers
health benefit such as lowering the risk of getting cancers,
diseases associated with cardiovascular, hypercholesterolemia,
diabetes as well as bone and kidney diseases [10]. On the other
hand, coconut (Cocos nucifera) milk is easy to digest and
contains an abundance of nutrients (particularly calcium,
phosphorus and potassium). The coconut milk content
Vitamins (C, E and many B vitamins) and antioxidant
activities. The high oleic and lauric acid content of coconut
milk help in preventing arteriosclerosis and related illness
[11]. Partial replacement of cow milk with vegetable milks
may affect the physical properties of ice creams. Fortification
of yoghurt ice cream with soy protein has been shown to
improve the textural quality of the product including firmness
and viscosity [12]. Lecithin in the soy also acts as emulsifiers
which increase the viscosity and stability of ice cream, refined
texture and extend the melting time [13]. Abdullah et al. [14]
improved the quality of ice cream by using different ratios of
skim milk in soymilk blend and found that large quantity of
skim milk in soymilk decline beany flavour of soy beans and
increased quality of ice cream. Kerdchouay and Surapat [15]

Abstract In this study, non fermented probiotic ice cream

made using soy and coconut milk. The changes in ice cream eating
qualities and survival of added microbes were evaluated. The highest
viscosity and melting resistance and lowest total acceptability and
particle size were found in ice creams containing soy milk. The
viable of Lactobacillus acidophilus reduced by 0.204 and 0.251 log
cycles in soy and coconut milk ice creams throughout 30 days storage
respectively. The counts of Bifidobacterium lactis decreased by 0.01
and 0.091 log cycles in soy and coconut milk ice creams respectively.
The produce of ice cream with vegetable milks developed the growth
and viability of B. lactis and L. acidophilus.
Keywords Ice cream, Soy milk, Coconut milk, Probiotic
bacteria

I. INTRODUCTION

NCREASING consumers interest in functional foods

became evident in recent years because these foods offer
additional health benefits beyond adequate nutrition.
Functional food that contains viable probiotics in particular
addresses the improvement in intestinal micro flora activation
of the immune system, reduction in serum cholesterol and
inhibition of potential pathogens [1]. However, these
probiotics must be present in adequate amount (106107 cfu/g)
at the time of consumption in order to render it being effective
[2]. Fermented yogurt and milk can effectively be used to
deliver probiotic bacteria. The former unfortunately may cause
loss of viable probiotic due to pH reduction and accumulation
of organic acids as a result of fermentation [3]. In this regard
ice cream, due to its neutral pH, may be used to deliver the
probiotics [4]. Partial replacement cow milk with vegetable
milks such as soy milk could improve the pH of probiotic ice
cream resulting increased the survival of probiotics [5]. On the

Fatemeh. Aboulfazli is with the Institute of Biological Sciences, Faculty of

Science Building, University of Malaya, 50603 Kuala Lumpur, Malaysia
(corresponding author to provide phone: 0060129914192; e-mail:
abolfazli2582@yahoo.com).
Ahmad salhin. Baba, is with the Institute of Biological Sciences, Faculty
of Science Building, University of Malaya, 50603 Kuala Lumpur, Malaysia
(e-mail: ahmad@um.edu.my).
Misni. Misran is with the Department of Chemistry, Faculty of Science
Building, University of Malaya, 50603 Kuala Lumpur, Malaysia (e-mail:
misni@um.edu.my).
1

International Conference on Biological and Medical Sciences (ICBMS'2014) Jan. 15-16, 2014 Kuala Lumpur (Malaysia)

studied the physical and sensory properties of low fat

coconut milk ice cream by adding whey protein concentrate
(WPC) and sugar contents and found the addition of sugar
and replacing skim milk powder with WPC in low fat
coconut milk ice cream increased ice cream mix viscosity
and reduced melting rate of ice cream. The present study was
carried out to investigate the effect of soy and coconut milks
on the survival of probiotic in ice cream during storage and
the rheological and physicochemical properties of non
fermented bio-ice cream.

TABLE II
THE CONTENT OF COMPONENTS USED IN ICE CREAM MIX FORMULATIONS (% BY
WEIGHT)
Ice
Ingredient
crea
Mil
Soy oil
Skim
Sugar
Stabilizer
Vanillin
Water
A
m
k
(Fat =
milk

100%)
powder
Emulsifier
S
55.
9.75
7
17
0.6
0.1
10.25
4
C
55.
7
17
0.6
0.1
20.0
4
A

A. Bacteria strain and preparation of cultures

Bifidobacterium bifidum (Bb-12) and Lactobacillus
acidophilus (La-05) were obtained as pure freeze-dried
probiotic culture from CHR-Hansen (Horsholm, Denmark).
B. Starter culture
Each strain (1 g) was cultured in 100 mL of sterilized
skimmed milk (10 w/v), simplified by the addition of 0.05%
(w/v) L-Cys-HCl, 1% (w/v) yeast extract and 2% (w/v)
glucose. The incubation was carried out under aerobic
condition in a water bath (42C) until pH was reached to 5.0
[16].
C. Culture for inoculation
Inoculation culture for each strain was prepared fresh by
adding 4 mL of starter culture into 100 mL of sterilized
skimmed milk. Incubation was carried out under anaerobic
condition in a water bath (42C) until pH reduced to 5.0 [16].
D. Ice cream production
Fresh pasteurized and homogenized milks (soy and coconut
milk), butter, soy oil and skim milk powder (Dutch lady,
Malaysia), sugar and vanilla was purchased from local grocery
stores. Cremodan SE 734 veg (Danisco AS, Copenhagen,
Denmark) containing mono- and diacyl-glycerols of fatty acid,
cellulose gum, guar gum, carrageenan was used as stabilizer.
Ice cream mix formulated according to some properties of
milks (TABLE I) such that it had 3840% total solids and
10.5% fat for a total batch of 100kg (TABLE II).

T1
T2
A
B

5.99 0.01

0.153 0.002

19 0.02

ilk (S)

ilk (C)

(1)
Samples melting rate were determined as described by
Mahdian et al. [12]. Tempered samples (30 g) were placed on
a 0.2 cm wire mesh screen above a beaker at room temperature
(25C). The weight of the melted material was measured after
20 min and declared as percentage weight melted.
G. Rheological measurements
Rheological measurements of melted ice cream samples
were determined using a Physica MCR 301 rheometer (AntonPaar GmbH, Graz, Austria) with concentric cylinder geometry
and coupled with a circulating cooling bath at 4.00.1C. Ice
creams were left to equilibrate at 4.0C for 15 min. The
samples flow behavior was generated by a linearly increased
shear rate from 19.6 to 67.3 s1 in 20 min and returning to 19.6
s1 over a further 20 min.
The flow behavior and the consistency index were explained
by the Power Law model (equation (2)) of the samples.
Apparent viscosity of samples was estimated as a function of
time under a constant shear rate of 20 s1.

TABLE I
CHEMICAL PARAMETERS OF SOY AND COCONUT MILK
Total
pH B
Titratable
Fat
Protein
solids
acidity
(g 100 g-1)
(g 100 g-1) B
B
(g 100g(%lactic
1 B
)
acid) B
11 0.07
7 0.01
0.072 0.001
1.4 0.01 2.2 0.02
28 0.08

The milk (soy or coconut milk) was initially heated to 50C

followed by the addition of soy oil respectively, and other
additives. The mixture was mixed at 65C in two stages by a
homogenizer with 16000 rpm for 5 min (IkaHomogenizer T25 basic Ultra Turrax) followed by pasteurization (10 min
heating at 80C followed by cooling to 4C). The mixture was
aged overnight at 4C prior to the addition of each probiotic
culture (4% w/w) followed by immediate freezing in batch ice
cream maker (1.5 L, Baumatic Gelato1SS). The frozen mixture
was packed in 100 mL plastic cups and then stored in a freezer
(20C). Three different batches of ice cream per treatment
were manufactured.
E. Chemical analysis
Milk and ice creams were analyzed for pH by a digital pHmeter and for titratable acid (TA) by titrating a sample (10 g)
with NaOH (0.1 N) using phenolphthalein (0.1 w/v) as an
indicator. The dry matters were measured by drying samples at
1001C for 3.5 h using an air oven [4]. The fat and protein
contents of milk and ice creams were measured by the Soxhlet
and Kjeldahl methods respectively [17]. All chemical analyses
were carried out in triplicate.
F. Physical analysis
The overrun was calculated using the following formula
(equation (1)) [4]:

II. MATERIAL AND METHODS

milkA

2.0 0.02

soy milk (T1), coconut milk (T2).

means values standard deviation.

(2)
2

International Conference on Biological and Medical Sciences (ICBMS'2014) Jan. 15-16, 2014 Kuala Lumpur (Malaysia)

Where: = shear stress (Pa); K = consistency index (Pa sn);

= the shear rate (s1); and n = the flow behavior index [18].
H. Size and zeta potential
The size and zeta potential of the ice creams were evaluated
using a Malvern Nano Series Zeta Sizer (Malvern, UK) at a
constant temperature of 25 C. The zeta potential and size of
ice cream mixes were monitored after aging [19].
I. Optical polarizing microscope imaging (OPM)
The OPM micrographs of ice cream mixes were obtained
using a Leica Polarizing Microscope equipped with a Leica
QWin software. All measurements were carried out at room
temperature (25C) [19]
J. Bacteriological analysis
The viability of probiotics was measured immediately after
inoculating the probiotic cultures and after 1 and 30 days of
frozen storage at 20C. The samples (10 g) were decimally
diluted with sterile peptone water (1 g L-1; Merck). One mL
aliquot dilutions were pour plated in triplicate on MRS agar
for L. acidophilus and MRS agar supplemented with 0.05%
(w/v) L-Cys-HCI (Merck) for B. lactis. The plates were
incubated at 381C for 72 h under aerobic condition with 5%
CO2 (v/v) for L. acidophilus and anaerobic condition
(Anaerocult A) for B. lactis. The bacterial viability was
represented as survival rate [16].
K. Sensory analysis
The ice creams were organoleptically evaluated by sixteen
panelists, using a sensory rating scale of 110 for taste and
flavor, and 15 for consistency and 15 for appearance and
colour [4]. The properties evaluated contained (a) three
characteristics for appearance and colour (no criticism: 5, dull
colour: 41, unnatural colour: 31), (b) seven properties for
taste and flavor (no criticism: 10, cooked flavor: 97, lack of
sweetness and too sweet: 97, lack of flavor: 86, rancid and
oxidized:61, and other: 51) and (c) seven terms describing
texture and body (no criticism: 5, coarse: 41, crumbly: 42,
weak: 41, fluffy: 31,gummy: 41, sandy: 21).
L. Statistics
The viability of the probiotic micro-organisms were
evaluated at three times of storage (0, 1 and 30 days), in
triplicates and the results were expressed as meanS.E.M
(standard mean error) values. The statistical analysis was
performed using SAS statistical software, Version 6.12 edition
[20] followed by Duncans multiple range method for mean

comparison. The mean values and the standard error were

calculated from the data obtained with triplicate trials. The
criterion for statistical significance was p < 0.05 [21].
III. RESULTS AND DISCUSSION
A. Physicochemical properties
The physicochemical compositions of the probiotic icecreams, soy and coconut milks are shown in TABLES III. The
highest pH and lowest TA and overrun values were in ice
creams containing soy milk.
The melting behavior was a more function of type of milk
used than type of probiotic added. Soy milk based ice creams
showed slower melting time than ice cream made with coconut
milk. Soy protein, which contains lecithin acts as an emulsifier
and thus provides protection for the membrane proteins against
damage due to freezing. In addition lecithin also assists in
good air distribution and fat structure in the ice cream thereby
increasing the time of melting of the ice cream [22]. The lower
melting time of coconut milk ice creams was due to the poor
emulsifying properties of the coconut proteins. The differences
in melting resistance of ice cream treatments may also be
influenced amongst others by the difference in freezing points
and viscosity [6].
B. Rheological measurements
The apparent viscosity, consistency index and flow behavior
index of the probiotic ice cream produced with different milks
are shown in TABLE IV. The apparent viscosity of the ice
cream decreased with an increase in the shear rate, as
illustrated by the non-Newtonian fluid behavior (Fig 1). This
decrease in viscosity is partly because of the aggregation of fat
globules, which decrease in size during shearing, thus the
reduction in the viscosity of ice cream [18]. Such a decrease in
the apparent viscosity with the increase in shear rate is a
common factor of milk products of similar texture such as
frozen yogurt [23]. Ice cream made with soy milk tended to
have a higher apparent viscosity than ice cream made with
coconut milks (TABLE IV). The differences can be attributed
to the type of their proteins [24]. Soy proteins are able to form
a stable network like a gel structure because of their molecular
properties which can bind with water molecules [25], which
present greater resistance to flow. The formation of gel by
globular proteins is a complex process, and it may involve
reactions such as molecular unfolding, dissociation

TABLE III

PHYSICOCHEMICAL PARAMETERS OF MILKS AND PROBIOTIC ICE CREAMS MADE USING SOY AND COCONUT MILK
Titratable
Fat (g100g-1)
Protein
Overrun
Melting rate (%
B
acidity
(g 100 g-1) B
melted after 20 min)
(%lactic acid) B
S
11.0 0.07
7.0 0.01
0.072 0.001
1.4 0.01
2.2 0.02
C
12.0 0.08
6.76 0.01
0.162 0.004
3.4 0.02
3.4 0.02
a
a
b
a
a
d
SL
39.2 0.12
7.14 0.02
0.072 0.006
10.4 0.04
2.47 0.03
11.57 1.67
60.6888.1b
SB
40.8 0.11a
7.15 0.01a
0.072 0.004b
10.3 0.05a
2.51 0.02a
12.5 0.1c
61.31210b
CL
39.9 0.12 a
6.71 0.01b
0.117 0.003a
10.3 0.02a
2.13 0.02a
17.71.03b
81.0289.3a
CB
38.8 0.10a
6.72 0.03b
0.126 0.009a
10.4 0.02a
2.32 0.04a
18.36 1.56a
83.4411.4a
A
S: soy milk; C: coconut milk. Ice creams inoculated with Lactobacullus acidophilus (La-05) made using soy milk (SL) or coconut
milk (CL); ice creams inoculated with Bifidobacterium bifidum (Bb-12) made using soy milk (SB) or coconut milk (CB).
B
means values standard deviation.
a-c
Values in the same column followed by different letters were significantly different (p < 0.05).
Ice
creamA

Total solids
(g 100 g-1)B

pH B

International Conference on Biological and Medical Sciences (ICBMS'2014) Jan. 15-16, 2014 Kuala Lumpur (Malaysia)

association, and aggregation [26].

The lower apparent viscosity of ice cream made using
coconut milk was due to the poor emulsifying properties of
coconut proteins [27]. No significant differences in the
apparent viscosity were among ice creams incubated with La05 and Bb-12. The Power Law model was used to establish
consistency index (K) and flow behavior index (n) (TABLE
IV). The flow behavior index (n) showed a shear thinning
behavior (n<1) ranging from 0.604 to 0.647 (TABLE IV)
which is accordance with several other studies [18, 23]. Soy
milk gives ice cream the highest consistency index (K) and
lowest flow behavior index (n) which again demonstrates that
the soy milk increased the resistance to structural breakdown
due to aggregation of soy proteins which resulted in gel
formation and subsequent increase in water retention [22]

evidence to support finding from the rheological studies that

increased viscosity of ice creams containing soy milk was
caused by the change in microstructure. This is reflected by the
largest size for ice creams containing coconut milk in contrast
to the smallest droplet size for ice creams containing soy milk
(TABLE V). Taken as a whole, samples containing coconut
milk showed larger droplet sizes than others because of poor
emulsifying properties of coconut proteins adsorbed at the oil
water interface [27]. No significant differences in the apparent
viscosity were among ice creams incubated with La-05 and
Bb-12.

TABLE IV
RHEOLOGICAL PARAMETERS OF PROBIOTIC ICE CREAMS MADE USING SOY OR
COCONUT MILKS OBTAINED USING LAW MODEL
Ice
Apparent
K(Pa sn)b
nb
R2c
creamsa viscosity
(mPa s)b
SL
3510.89b 1.0990.01a
0.612 0.01a
0.998
SB
3851.04a 1.1820.01a
0.604 0.01a
0.997
CL
3151.05c 0.8170.03b
0.647 0.02a
0.993
CB
2931.12d 0.7850.02b
0.633 0.01a
0.994
a

K = consistency index; n = flow behavior index; ice creams inoculated with

Lactobacullus acidophilus (La-05) made using soy milk (SL) or coconut
milk (CL); ice creams inoculated with Bifidobacterium bifidum (Bb-12)
made using soy milk (SB) or coconut milk (CB).
b
Mean values standard deviation. Values with different letters in the
same column are significantly different (P < 0.05) (Tukey test).
c
Coefficient of determination

Fig.2. Micrographs (50 magnification) of ice cream samples with

different milk. Ice creams inoculated with Lactobacullus acidophilus
(La-05) made using soy milk (SL) or coconut milk (CL); ice creams
inoculated with Bifidobacterium bifidum (Bb-12) made using soy
milk (SB) or coconut milk (CB).
TABLE V
ZETA POTENTIAL AND FAT PARTICLE DIAMETER (DM) OF ICE CREAMS
MADE USING VEGETABLE MILKS

Samples A
SL
SB
CL
CB

Particle
size (nm)
3707 b
3080 b
5335 a
5963a

Zeta
potential (mV)
-38.5a
-38.27a
-42.47b
-41.14b

ice creams inoculated with Lactobacullus acidophilus (La-05)

made using soy milk (SL) or coconut milk (CL); ice creams inoculated
with Bifidobacterium bifidum (Bb-12) made using soy milk (SB) or
coconut milk (CB).

Fig.1. Effect of shear rate on the apparent viscosity of soy or

coconut milk ice creams. Note: ice cream (La-05) made with soy milk
(SL), with coconut milk (CL); ice cream (Bb-12) made with soy milk
(SB), with coconut milk (CB).

D. Viability of probiotic bacteria in ice cream

Fig 3 shows the changes in bacterial counts in nonfermented ice creams made using vegetable milks. The
survival rate of probiotic bacteria in ice creams after 30 days
tend to be higher in Bb-12 (7.767 and 7.371 log cfu g_1 for soy
and coconut ice cream respectively) than in the presence of
La-05 (7.847 and 6.870 log cfu g_1 for soy and coconut ice
cream respectively) (TABLE VI). However, the reduction in
viable bacterial counts due to freezing is associated with freeze
injury on these cells. In addition the mechanical stresses

C. Effect of milk replacement on droplets suspension

The microscopic structure of ice cream showed that ice
creams containing coconut milk had larger fat globule sizes
than others. The coconut proteins have less surface active [27]
and this could be a factor on the tendency to create small
droplets or to prevent droplet aggregation during or after
homogenization[28]. As a result the reduction in the fat
particle diameters is an increase in K value and thus develope
the product stability [29]. The micrographs provide further
4

International Conference on Biological and Medical Sciences (ICBMS'2014) Jan. 15-16, 2014 Kuala Lumpur (Malaysia)

E. Sensory evaluation
TABLE VII shows the organoleptic property scores in nonfermented ice creams made using vegetable milks (TABLE
VII). The lowest total acceptability was seen in soy milk ice
cream which could be explained by the soy milk woody or
beany off flavours [14]. The presence of either La-05 or Bb-12
had no significant influence on sensory properties of ice
creams.

associated with the mixing and freezing process which

incorporate oxygen into the mixture may be responsible in
further reduction in bacterial count [16]. The survival of both
probiotics in ice cream was high (p<0.01) in the presence of
soy and coconut milks. This could be explained by the high pH
of soy and coconut ice creams which is known to be conducive
to probiotic survival since these organisms are susceptible to
inactivation when stored in acidic conditions [30]. Heenan et
al. [5] demonstrated that the survival of probiotics increased in
the frozen soy dessert due to the prevailing neutral pH. The
highest survival of both probiotics was in soy milk ice cream.
It is probably due to the soy milk proteins which provide
physical protection against freezing damage by encapsulating
probiotics by form a stable network looks like a gel structure
[25]. Keerati-u-rai and Corredig [11] mentioned soy proteins
adsorb at the interface of oil droplets, with surface loads
varying between 2 and 4 mg m 2 and a layer thickness
between 30 and 40 nm. So probably, these proteins can cover
probiotics as a capsule. The high survival rate of L.
Acidophilus cells during the frozen storage in other studies
was attributed to the protection provided to the cells by the
solid ingredients and the high fat content of the ice cream in
the form of emulsion [31].

TABLE VII
ORGANOLEPTIC PROPERTY SCORES OF ICE CREAMS WITH DIFFERENT MILKSA
Ice
Colour and
Body and
Flavor and
Total
creamsB
Appearance
Texture
Taste
(120)
(15)
(15)
(110)
SL
3.12 0.05a
3.0 0.04b
5.080.04b
11.2 0.06b
SB
3.17 0.03b
3.2 0.02b
5.1 0.07b
11.25 0.04b
a
a
a
CL
3.25 0.03
3.21 0.03
6.5 0.03
12.96 0.05a
CB
3.20 0.02a
3.01 0.02a
6.3 0.04a
12.98 0.02a
a
ice creams inoculated with Lactobacullus acidophilus (La-05) made using soy
milk
(SL) or coconut milk (CL); ice creams inoculated with Bifidobacterium
bifidum (Bb-12) made using soy milk (SB) or coconut milk (CB).
a-b
Values with different letters in the same column are significantly different (P<
0.05) (Tukey test).

ACKNOWLEDGMENT
We acknowledge the financial support of University of
Malaya Research Grant (PV113-2012A).
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Fig. 3. Viable counts of L. acidophilus (L) and B. lactis (B) in icecream with different kind of milk during 30 days storage at _20 C.
Note: ice cream (La-05) with soy milk (SL), with coconut milk (CL);
ice cream (Bb-12) with soy milk (SB), with coconut milk (CB)
TABLE VI
COUNTS OF PROBIOTIC BACTERIA IN MIXTURES AND ICE CREAMS
Ice
Mixture
Ice cream 30 days
Survival
creamsa
(Log10
(Log10 cfu/g)
Rate (%)
cfu/g)
SL
7.857 0.15
7.847 0.01
99.87 a
SB
7.858 0.03
7.767 0.04
98.83 a
CL
7.121 0.04
6.870 0.06
96.48 b
CB
7.745 0.04
7.371 0.26
95.17 b
a
ice creams inoculated with Lactobacullus acidophilus (La-05) made using
soy milk (SL) or coconut milk (CL); ice creams inoculated with Bifidobacterium
bifidum (Bb-12) made using soy milk (SB) or coconut milk (CB).
a-b
Values with different letters in the same column are significantly different
(P< 0.05) (Tukey test).

International Conference on Biological and Medical Sciences (ICBMS'2014) Jan. 15-16, 2014 Kuala Lumpur (Malaysia)

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[12]

[13]

[14]

[15]

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[23]

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