LWT - Food Science and Technology 55 (2014) 255e262

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LWT - Food Science and Technology

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

Consumer acceptability and antioxidant potential of probiotic-yogurt

with spices
Vijayalakshmi V. Illupapalayam 1, Stuart C. Smith 1, Shirani Gamlath*,1
School of Exercise and Nutrition Sciences, Faculty of Health, Deakin University, 221, Burwood, Victoria 3125, Australia

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

Article history: The study focuses on developing novel probiotic yogurts containing spices with acceptable sensory
Received 6 December 2012 properties, therapeutic levels of probiotics and with beneficial antioxidant capacity. Eight types of yo-
Received in revised form gurts with added spice oleoresins (cardamom, cinnamon and nutmeg) and probiotics [Lactobacillus ac-
19 September 2013
idophilus strain 5(LA5), or Bifidobacterium animalisssp.Lactis (Bb12)] were produced. Two successive
Accepted 26 September 2013
consumer sensory taste panels (n ¼ 54) using a nine point hedonic scale were conducted to evaluate the
acceptability of the yogurts. Viable counts of probiotics and antioxidant capacity of yogurt samples at 1, 7,
Keywords:
14 and 28 days of storage at 4  C were monitored. The probiotic-yogurt products containing spices
Yogurts
Probiotics
showed good sensory properties, with the best results obtained with cardamom oleoresin (with LA5 or
Spices Bb12). The presence of spice oleoresins (cardamom, nutmeg and cinnamon) did not affect the probiotic
Antioxidant activity population (LA5or Bb12) in yogurt during 4 weeks of refrigerated storage. The antioxidant capacity (with
Flavour LA5or Bb12) over the storage period was also maintained.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Practical application
The development of novel functional products with health
benefits is highly relevant to the food industry. This study produced
consumer-acceptable probiotic-yogurts containing spices with
therapeutic levels of probiotics during storage and with antioxidant
capacity to enhance the health benefits. Consequently, this study
can be used as a reference for yogurt manufacturers looking to
incorporate novel, natural flavourings with functional or health
properties in their products.
2. Introduction
The search for unique food ingredients and flavours with
enhanced health properties is at present one of the key global
market trends (Netzel, Netzel, Tian, Schwartz, & Konczak, 2007).
Spices are a novel source of functional flavouring agents. There is
now mounting scientific evidence of health benefits of spices
including antioxidant, antimicrobial, anti-inflammatory and anti-
carcinogenic properties (Srinivasan, 2004; Tapsell et al., 2006).
The largest published study to date which tested the antioxidant
* Corresponding author. Tel.: þ61 3 92517267.
E-mail address: shirani.gamlath@deakin.edu.au (S. Gamlath).
1
Website: http://www.deakin.edu.au
activity of 1113 food samples found that within the top 50 foods
with antioxidant properties, the top five were spices (Halvorsen
et al., 2006). On the other hand, probiotics which are live benefi-
cial microorganisms, when administered in adequate amounts, also
have been shown to have numerous health benefits (Majeed &
Prakash, 2007). Combinations of probiotics with spices may pro-
vide further therapeutic properties. However since spices are an-
timicrobials, they may affect probiotic viability. In-vitro studies that
tested spices on the growth of selected probiotics showed that
spices significantly enhanced the growth of probiotics while
inhibiting pathogens (Be, Gamlath, & Smith, 2010; Sutherland et al.
2009).
Spices in the form of oleoresins have the full flavour, aroma and
pungency of fresh or dried spices and oleoresins are used at very
low concentrations because they are highly concentrated. They give
more uniform flavour with less variability than their ground spice
counterparts (Krishnan, Bhosale, & Singhal, 2005) without chang-
ing the colour or texture of the final products. Singh, Kapoor, and
Singh (2011) reported that addition of anise oleoresins to plain
yogurt do not have any adverse effect on the physicochemical
properties. They were also reported to possess good antioxidant
properties (Gulcin, Oktay, Kirecci, & Kufreviolu, 2003; Singh,
Kapoor, Singh, & Catalan, 2008). But oleoresins are sensitive to
environmental factors such as oxygen, light and moisture (Krishnan
et al., 2005), however encapsulation overcame this problem.
Encapsulated spice flavours are prepared by locking spice extracts

0023-6438/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.lwt.2013.09.025
256 V.V. Illupapalayam et al. / LWT - Food Science and Technology 55 (2014) 255e262
into a special matrix resulting in improved shelf life and release the
flavour when dispersed in the hot liquids such as in yogurt prep-
aration (Krishnan et al., 2005).
In recent years, there has been a significant increase in the
popularity of yogurt as a functional food (Granato, Branco, Cruz,
Faria, & Shah, 2010). Yogurt is a conventional food known for its
therapeutic, nutritional and sensory properties (Hekmat, Soltani, &
Reid, 2009) and is most popular and preferred vehicle for probiotic
culture (Cruz, Castro, et al., 2012). Recently, Saxelin et al. (2010)
demonstrated that yogurt is as effective as capsules for the
administration of probiotics. Ejtahed et al. (2011) suggest that
probiotic yogurt may help decrease cardiovascular disease risk
factors in people with Type two diabetes. In fact, the addition of
probiotics in yogurt is a topic of relevance to both manufacturers
and consumers (Cruz et al., 2013). As the popularity of probiotic
yogurt continues to rise, dairy based food manufacturers are
continuously looking into value-added ingredients. Most varieties
of probiotic flavoured yogurts available in the market have added
artificial sweet flavouring agents to enhance the flavours (Dairy
Australia, 2013), but consumers demand flavourful foods with
health benefits that are also natural (Thompson, Lopetcharat, &
Drake, 2007). Therefore, new product ranges and flavour in-
novations are important in encouraging consumer trials and sub-
sequent category growth (Dairy Australia, 2013). Thus the main aim
of the present study was to produce probiotic-yogurt containing
selected spice oleoresins (cardamom, cinnamon and nutmeg) with
acceptable sensory properties and to investigate the effect of these
spices on the viability of the chosen probiotics in yogurt as well as
to investigate the health properties of the developed yogurts by
testing the total antioxidant capacity.
3. Materials and methods
3.1. Selection of spices and probiotics
Three spice oleoresins cardamom and nutmeg (encapsulated),
cinnamon (Nature shop) at the concentration of 0.5 percentage
volume/weight and two probiotic organisms Lactobacillus aci-
dophilus strain 5(LA5), Bifidobacteriumanimalis ssp.Lactis(Bb12)
were selected for this study based on the preliminary studies (Be
et al., 2010; Behrad, Yusof, Goh& Baba, 2009). Due to the unavail-
ability of encapsulated cinnamon oleoresins at the time of study,
oleoresin powder of cinnamon was used.
3.2. Preparation starter culture and probiotics
Freeze-dried (FD) granules of Direct Vat SetÒ cultures (DVSÒ)
Yo-FlexÒ (starter cultures) and FD-DVS probiotics (LA5 and Bb12)
were obtained from Chr. Hansen Pty., Ltd, Australia. The present
study used Yo-Flex-L812 a highly concentrated yogurt starter cul-
ture (Streptococcus thermophilus and Lactobacillus delbrueckii ssp.
bulgaricus) to obtain high cream flavour. As per the recommenda-
tion from the manufacturer, 50 units of FD-DVS culture was used for
each trial and added to 500 ml of sterile milk (preheated to
fermentation temperature at 43  C) as first dilution. From the first
dilution, 0.02 percent is added to 8 L of tempered milk (Chr. Hansen,
2006).
3.3. Formulation of probiotic yogurt with spice oleoresins
As per the manufacturers instruction on yogurt preparation
(Chr. Hansen, 2006) 4 L of milk (local supplier), with the required
fat content (3.9 g/100 ml) was mixed vigorously with 2 percent of
skimmed milk powder (local supplier) and 5 percentage of sucrose
(local supplier) to ensure an efficient mixing. Remaining 4 L of milk
was added and mixed thoroughly. The mix was hydrated for 1 h at
50  C and aliquoted into four equal portions (2 L each). Three
portions were added with either one of the oleoresins of cinnamon
(0.5 percent, volume/weight), encapsulated oleoresins of
cardamom (0.5 percent v/w) or encapsulated oleoresins of nutmeg
(0.5percent v/w) and one portion used without spices to produce
control-probiotic yogurt. The mix was preheated to 85  C for heat
treatment for 30 min. The mix was then cooled to the fermentation
temperature (43  C) in a water bath while stirring slowly with a
hand stirrer and inoculated with the prepared FD-DVS yogurt
starter cultures (0.02 percent v/w). Each 2000 ml was divided into
two equal portions (1000 ml) and each portion was inoculated with
either LA5 (0.02 percent w/v) or Bb12 (0.02 percent w/v). The
inoculated mixtures were incubated at 40  C for fermentation and
terminated when pH reached 4.5 (approximately 6 h), and the
yogurt cups were cooled rapidly in a refrigerator at 4  C. When
thoroughly cooled, the cups were then transferred to refrigerator
and stored below 8  C.
3.4. Sensory evaluation
The sensory evaluation study protocol was submitted to and
approved by the Deakin University Faculty of Health Ethics Sub
Committee (DUHREC-H7603).
3.4.1. Consumer taste panel
The newly developed probiotic-yogurts with and without spices
were evaluated by conducting consumer taste panels using nine
point hedonic scale (Cruz et al., 2010; Gonzalez, Adhikari, & Sancho-
Madriz, 2011) by procedures discussed in Moskowitz, Beckley, and
Anna (2006). A minimum of fifty ratings per product are considered
desirable for the precision of the statistical analysis for consumer
acceptance sensory tests (Moskowitz et al., 2006). Thus 60 partic-
ipants between the ages of 18e60 from Deakin University, Burwood
campus were recruited to take part in consumer taste panel
through poster advertisement. Prospective volunteers (n ¼ 60)
were screened for the frequency of their yogurt consumption and
volunteers liked and were willing to consume yogurt were
recruited. Participants were excluded from the study if they had
any food allergies to limit the potential for any allergic reactions
resulting in 54 participants (35 females and 19 males) for the
sensory panels.
Consumer acceptance of the experimental products was evalu-
ated using a hedonic scale of 1e9 where 1 corresponds with “dislike
extremely” and 9 corresponds “like extremely” in two consumer
panels. The first panel evaluated LA5 yogurt with and without
spices and the second panel evaluated Bb12 yogurt with and
without spices. The sensory testings were conducted in a specially
designed sensory evaluation laboratory illuminated with natural
fluorescent lights. The plain language statements and consent
forms were given to participants and collected prior to testing. No
monetary incentive or rewards were given to consumers for
participation.
The yogurt samples were stored at 4  C and approximately 15 g
of yogurt samples were portioned in uniform plastic cups with lid
labelled with 3-digit random codes just before the arrival of par-
ticipants. Participants were initially asked to complete a brief
questionnaire in order to classify them according to age and gender.
Participants were then presented with four samples along with
water. Consumers were instructed to rinse their mouths with water
between sample testing.
Each consumer evaluated all samples using a balance design to
prevent order and carry over effect. Panellists were instructed to
evaluate each attribute separately to overcome the halo effect.
Panellists evaluated the appearance of the samples first, then,
 V.V. Illupapalayam et al. / LWT - Food Science and Technology 55 (2014) 255e262
aroma, texture and taste respectively. Finally, the overall liking of
each sample was evaluated. Each session took approximately
30 min, with the panels spread over two days.
3.5. Enumeration of probiotics
All the chemicals and antibiotics used for the enumeration of
probiotics were obtained from SigmaeAldrich, Australia, unless
stated otherwise. Three replicates plating for enumeration were
done for each sample unless stated otherwise. Each yogurt sample
(1 ml) was mixed thoroughly with 9 ml of buffered peptone water
(Oxoid, UK) and serial dilutions were performed using peptone
water as the diluents.
3.5.1. Enumeration of LA5 (Chr. Hansen, 2007a)
Enumeration of LA5 was carried out using MRS (Oxoid, UK.) agar
with addition of clindamycin stock solution (20 percent) with
milliQ water sterilised by filtration (0.22 mm). Clindamycin was
used as the antibiotic to inhibit the growth of lactic acid bacteria in
the starter culture but allow good growth of LA5. Aliquots of 1 ml of
diluted yogurt were tested using the pour plate method with MRS
agar with addition of 0.05% of clindamycin stock solution per litre of
MRS agar. Samples were incubated anaerobically at 37  C for 48 h in
jars (Oxoid, UK) containing anaerogen sachets (Oxoid, UK) to
determine viable count. The actual viable number of LA5 on the
agar plates was determined by colony counting on days 1, 7, 14
and 28.
cfu=g ¼ cfu=plate  dilution factor
cfu e colony forming unit.
3.5.2. Enumeration of Bb12 (Chr. Hansen, 2007b)
The combination of dicloxacillin, lithium chloride (LiCl) and
cysteine hydrochloride (CyHCL) were shown to inhibit yogurt
starter culture, but allow good growth of Bb12 (Chr. Hansen,
2007b). Dicloxacillin stock solution (10percentage) was prepared
by adding 10 mg of dicloxacillin sodium salt monohydrate with
100 ml distilled water and sterilised by filtration (0.45 mm). LiCl
stock solution (11 percent) was prepared by dissolving 2 g LiCl into
18 g distilled water and sterilising through filtration (0.45 mm).
CyHCl stock solution (10 percent) was made up by dissolving 10 g
CyHCl into 100 ml distilled water and sterilised by autoclaving at
121  C for 15 min. The Bb12 viable cell counts were carried out by
plating diluted yogurt samples using the MRS agar with addition of
0.5 percentage of dicloxacillin stock solution, 1percentageofLiCl
stock solution and 0.5 percentage of CyHCL stock solution per litre
of medium according to the pour plate method. Plates were incu-
bated for 48 h at 37  C in anaerobic conditions similar to LA5. The
actual viable number of Bb12 on the agar plates was determined
similar to LA5 on days 1, 7, 14 and 28.
3.6. pH changes in yoghurt during storage
Yogurt sample (1 g) was mixed with distilled water (1:1)
(Behrad et al., 2009) and the pH was measured using a pH meter
(pHepÒ pH Tester/Hanna instruments), calibrated routinely with
pH 4.0 and 7.0 standard buffers. Yogurt samples were monitored for
pH during the fermentation and during storage for 1, 7, 14 and 28
days at 4  C.
3.7. Antioxidant capacity by 1, 1-diphenyl-2-picrylhydrazyl (DPPH)
Antioxidant capacity of yogurt samples during storage for 1, 7, 14
and 28 days at 4  C were determined with a stable radical substrate,
257
DPPH as described by Behrad et al. (2009) with minor modifica-
tions. Yogurt sample (1 g) was mixed with distilled water (1:1) and
centrifuged at 10,000 rpm for 30 min. To 3 ml of 60 mM DPPH
(SigmaeAldrich, Australia) in ethanol, 250 ml of each yogurt ex-
tracts was added. The mixture was shaken vigorously. Samples
were aliquoted into microplate wells and incubated in the dark.
After 20 min, the absorbance was measured at 517 nm (micro plate
reader-BioTek). The readings were compared with the control,
which contained 250 ml of dH2O instead of the water extracts of
probiotic-yoghurts with or without spices. The percentage of
antioxidant activity was calculated as percentage inhibition of
DPPH radical formation using the following formula.

Percentage inhibition ¼ Absorbancecontrol  Absorbancesample =

Absorbancecontrol  100
4. Statistical analysis
All data analyses were performed using the Statistical Package
for Social Science Software 18 (SPSS version 18.0). A one-way
analysis of variance (ANOVA) was used to evaluate the difference
between the yogurts with respect to the five sensory attributes.
Least Significant Difference (LSD) was used to compare the mean
value between samples (Gonzalez et al., 2011) and data was sig-
nificant if the p-value was found to be < 0.05. Results were pre-
sented as the mean and the standard error of the mean (SE).
One way ANOVA with post-hoc mean separation using LSD was
carried out for statistical analysis of the treatment and storage ef-
fect on the viable microorganisms (Hemsworth, Hekmat, & Reid,
2011), pH and antioxidant capacity (Ali, 2010) over the 28 day
period of storage. Results were presented as the mean and the
standard error of the mean (SE). Data was significant if the p-
value was found to be < 0.05.
5. Results and discussion
The study successfully developed combined spice and probiotic-
yogurts using strains of LA5and Bb12 with good sensory properties.
Probiotic-yogurts with various flavours such as peach, blackberry
and strawberry have been developed by some researchers
(Adhikari, Mustapha, & Grün, 2003; Cruz et al., 2010; Gonzalez
et al., 2011). However, yogurt products with probiotics and spice
oleoresins have never been previously developed. Availability and
cost effective nature of spices make these suitable food additives for
enhancing the functionality of commercial yogurts.
5.1. Consumer sensory testing
In the present study yogurt products were evaluated for
appearance, flavour, texture and overall acceptability since yogurt
is characterized as a food product with a refreshing flavour, a
smooth viscous gel, and a slight sour taste (Hekmat & Reid, 2006).
The results for the sensory evaluation of the yogurt products are
shown in Tables 1 and 2.
One of the main limitations of consumer sensory tests is the
relatively small number of participants in the evaluation
(Moskowitz et al., 2006). However the present study recruited
participants sufficient to limit any homogeneity. A total of 54 par-
ticipants evaluated the yoghurt product in 2 consumer sensory
taste panels (65 percent of female and 35 percent of male). Among
them 81 percent were in the 18e30 year age group, 15 percent in
the 31e50 year age group and 4 percent in the 51e60 year age
group.
258 V.V. Illupapalayam et al. / LWT - Food Science and Technology 55 (2014) 255e262

Table 1 probiotic-yogurt. Similar results were observed with nutmeg

The mean scores for the sensory characteristics of LA5 probiotic-yogurts with yogurt containing LA5 (p ¼ 0.005) or Bb12 (p ¼ 0.015). This suggests
different spices.
that encapsulated oleoresins were better accepted by the con-
Sensory characteristics Samples Mean scoresA  Std. error sumers for both varieties of yoghurt product. However cinnamon-
Appearance Control 5.46  0.281b yogurt (oleoresin) with LA5 or Bb-12 was not significantly
Cardamom 6.57  0.256a different from control probiotic-yogurt.
Nutmeg 6.46  0.232a In respect to texture, all yoghurt products received scores above
Cinnamon 5.33  0.281b
5 in the nine point hedonic scale, which indicates consumers liked
Aroma Control 5.17  0.302b
Cardamom 6.11  0.288a the products and spice oleoresins did not affect the textural prop-
Nutmeg 6.30  0.266a erties of the yogurt products. In both cardamom yogurt containing
Cinnamon 5.28  0.279b LA5 (p ¼ 0.010) or Bb12 (p ¼ 0.005) had significantly higher mean
Texture Control 5.50  0.267b
score than the control probiotic-yogurt suggesting that cardamom
Cardamom 6.44  0.233a
Nutmeg 5.87  0.286b
had a positive effect on the texture of yogurt. The texture of cin-
Cinnamon 5.59  0.243b namon and nutmeg containing yogurts with LA5 or Bb12 were not
Taste Control 5.15  0.274b significantly different from the control probiotic-yogurt.
Cardamom 6.33  0.306a The results in Tables 1 and 2 indicate that the overall taste scores
Nutmeg 5.30  0.254b
were comparable when assessed according to the hedonic scale. In
Cinnamon 5.06  0.271b
Overall Control 5.22  0.254b the products with LA5, there was a statistically significant differ-
Cardamom 6.22  0.281a ence between the yogurt products (p ¼ 0.003) with cardamom
Nutmeg 5.39  0.258b compared with the others which indicates that the taste of the
Cinnamon 4.89  0.252b cardamom-yogurt was better liked by the consumers, compared to
Means within each sensory attribute with common alphabet superscripts are not other yogurts. Both nutmeg and cinnamon-yogurt were not
significantly different (p < 0.05). significantly different from the control probiotic-yogurt. In case of
A
e 9 point hedonic scale was used in consumer taste panel. (1 ¼ “dislike extremely”
products containing Bb12 there was no significant difference be-
and 9 ¼ “like extremely).
tween the mean score of taste for any yogurt. These results indicate
that spice oleoresins did not significantly affect the taste of the
The mean scores for the appearance for LA5 yogurt with yogurt.
cardamom (p ¼ 0.003) and nutmeg (p ¼ 0.008) were significantly In case of overall acceptability, cardamom-yogurts containing
higher than the control-yogurt. Table 2 indicates that the mean LA5 was significantly different (p ¼ 0.007) from the other yogurts.
scores for the appearance of Bb12 yogurt with cardamom and The mean scores for the nutmeg and cinnamon yogurts were not
nutmeg were within the acceptable range, but there was no sig- significantly different from the control. In products containing
nificant difference between the types of yogurt. In case of both Bb12, there was no significant difference between nutmeg and
yogurt products (LA5 or Bb12), cinnamon treated yogurts had a cinnamon yogurt and the control, but cardamom-yogurt was
mean score below the other spice yogurt, but there was no signif- significantly different suggesting that compared to other types,
icant difference between control and cinnamon-yogurts. cardamom-yogurt was well accepted by the consumers.
The results in Tables 1 and 2 indicate that the mean scores for Appearance appears to be the important factor for consumer
the aroma of both cardamom yogurts containing LA5 (p ¼ 0.20) or acceptability. In the present study, yogurts containing natural col-
Bb12 (p ¼ 0.001) were significantly higher compared to control ours (cardamom and nutmeg) scored significantly higher than the
brown coloured yogurt (cinnamon), indicating preference of this
group by consumers.
In the present study, overall yogurt containing, cardamom and
Table 2
The mean scores for the sensory characteristics of Bb12 probiotic-yogurts with nutmeg were highly accepted by consumers in both probiotics with
different spices. higher preference towards cardamom. Cinnamon-yogurt scored
less in both products (LA5 or Bb12), but it was not significantly
Sensory characteristics Samples Mean scoresA  Std. error
different with the control-yogurt, which may indicate the possi-
Appearance Control 6.24  0.230a bility of using spices in the form of oleoresin.
Cardamom 6.93  0.230a
Nutmeg 6.83  0.194a
The predominance of female participants (65 percent) and
Cinnamon 6.09  0.313a participants in the 18e30 year age group (81 percent) may have
Aroma Control 5.50  0.267b been a limitation on the results of this study, as these percentages
Cardamom 6.67  0.258a indicated that the demographics were inconsistent with the wider
Nutmeg 6.37  0.226a
population. However, the data suggests the possibility of producing
Cinnamon 5.81  0.254b
Texture Control 5.78  0.332b commercially acceptable products containing these spice when
Cardamom 6.91  0.250a compared with a recent study conducted on consumer accept-
Nutmeg 6.46  0.237b ability with three commercially available yogurt products (Cruz,
Cinnamon 6.22  0.288b Cadena, 2012; Cruz, Castro, et al., 2012) for appearance (6.74e
Taste Control 5.15  0.354a
Cardamom 6.13  0.340a
7.02), aroma (6.02e6.28), taste (4.38e5.07), texture (5.93e6.07)
Nutmeg 5.96  0.415a and overall liking (5.52e5.71) based on 9 point hedonic scale.
Cinnamon 5.35  0.327a However, there are no consumer acceptability studies available on
Overall Control 5.09  0.348b spice added yogurts for a better comparison of our data.
Cardamom 6.20  0.341a
Nutmeg 5.56  0.298b
Cinnamon 5.02  0.318b 5.2. Survival of probiotics

Means within each sensory attribute with common alphabet superscripts are not
significantly different (p < 0.05).
The survival of probiotics in yogurt during storage until con-
A
e 9 point hedonic scale was used in consumer taste panel. (1 ¼ “dislike extremely” sumption is an important consideration. To achieve optimal po-
and 9 ¼ “like extremely). tential beneficial therapeutic effects, the number of probiotics in a
 V.V. Illupapalayam et al. / LWT - Food Science and Technology 55 (2014) 255e262
product at the time of consumption should at least meet a sug-
gested “therapeutic minimum” 105e106 cfu/g or ml of the final
product for functionality (Criscio et al., 2010; Cruz, Cadena, 2012;
Cruz, Castro, et al., 2012; Granato et al., 2010). These minimal
amounts then translate to a daily serve or dose of 108 cfu/g for
presentation to the gut for any functional benefit. The mean viable
CFU at day 1, 7, 14 and at the end of shelf-life (day 28) in the spice
added probiotics yogurt and control probiotic yogurts are shown in
Fig. 1.
Although the same volume of cfu/ml of LA5 and Bb12 was added
at the time of yogurt production viable cfu for the two probiotics at
the day 1 of storage was quite different. In general, LA5 commenced
with a high viable count (107 cfu/g) in yogurts with or without
spices whereas the viable number of Bb12 added to the yogurt
appeared less (106 cfu/g). This may indicate freeze drying may have
killed more Bb12 culture than LA5 prior to manufacture of the
yogurt.
Even though, there was variability observed in the viability of
the two probiotics in the present study, after 4 weeks of storage, all
the spice added probiotic-yogurts (both LA5 and Bb12) contained
an acceptable level (between 5  107 e 108 cfu/g for LA5 and 106e
107 cfu/g for Bb12) of commercial probiotics and in most cases
higher than the control. These results are comparable to Behrad
et al. (2009) that concluded that LA5 and Bb12 were maintained
Fig. 1. Comparisons of viability of probiotics in yogurt with spices and control yogurt.
(The error bars represents standard error of means). ae Comparisons of viability of LA5 in
yogurts with spices and control-yogurt during refrigerated (4  C) storage. b e Compar-
isons of viability of Bb12 in yogurts with spices and control-yogurt during refrigerated
(4  C) storage.
259
in spice (cinnamon and liquorice) yogurt for 28 days storage at the
level between 106 e 107 cfu/ml. The results are also comparable to
daily serving sizes of approx. 200 ml of probiotic yogurt that
maintain viable levels of approximately 108 cfu/g or more (Cruz
et al., 2010; Granato et al., 2010).
5.2.1. Survival of LA 5 in yogurts
The presence of spices such as cardamom or nutmeg do not
affect the viable count of LA5 on day 1 compared to control
probiotic-yogurt. But the presence of cinnamon resulted in a lower
count of viable LA5 in cinnamon (p ¼ 0.001) yogurt on day1 of
storage compared to control and other two spice yogurts. The
viable count of LA5 increased from day 1 to day 14 of storage for all
yogurts with the lowest rate in the control-yogurt. The viable LA5
count however reduced from day 14 to day 28 of storage for all
yogurts with the fastest rate occurring in control-yogurt. The viable
LA5 count on day 28 of storage for cardamom-yogurt (5.7  107 cfu/
g) was not significantly different from control-yogurt. At day 28,
nutmeg with a 1.7  108 cfu/g (p ¼ 0.00) and cinnamon-yogurt with
1.6  108 cfu/g (p ¼ 0.00) has significantly higher viable count of
LA5 compared to control-yogurt (3.5  107 cfu/g). The results for
LA5 are comparable to the levels of other commercial probiotics
such as L. delbrueckii ssp. bulgaricus with levels of 105 cfu/ml in
probiotic stirred-type yogurt (Marafon et al., 2011).
5.2.2. Survival of Bb12 in yogurt
Similar to yogurts with LA5, the presence of spices such as
cardamom or nutmeg did not affect the viable count of Bb12 on day
1 compared to control-yogurt. But the presence of spice cinnamon
resulted in a higher Bb12 count on day1 of storage compared to all
the other yogurts (p ¼ 0.05). The viable count of Bb12 increased
from day 1 to day 14 in all yogurts. But from day 14 to day 28 of
storage, all yogurts had a reduced viable count except cardamom
with the fastest rate occurring with the nutmeg-yogurt. On day 28,
the viable count of cinnamon-yogurt (2.7  106 cfu/g) was not
significantly different from control-yogurt. The cardamom-yogurt
(p ¼ 0.00) resulted in significantly higher viable count
(8.6  106 cfu/g) and nutmeg (0.009) had the lowest count
(1.37  106 cfu/g) compared to control yogurt (2.1  106 cfu/g).
Several studies have reported that the survival of probiotic
bifidobacteria is often low in yogurt (Gonzalez et al., 2011; Lourens
& Viljoen, 2001; Shah, 2000). In this study the viable numbers of
both probiotics were higher and more so when spices cardamom
and cinnamon were used, but not with nutmeg. The level of Bb12 in
these yogurts was also consistent with the level of Bifidobacter-
iumanimalis ssp. lactisin probiotic-stirred yogurt (Marafon et al.,
2011).
Several methods have been recommended to increase the sur-
vival of probiotics in yogurt, especially oxygen-sensitive bifido-
bacteria (Ebel, Martin, Le, Gervais, & Cachon, 2011), but these also
affect the sensory characteristics of the yogurt, especially the
texture (Granato et al., 2010; Kailasapathy, 2006) which is not the
case in this study and suggests that the spices used in this study
enhanced the sensory properties without affecting the probiotic
viable count even after four weeks of refrigerated storage.
5.2.3. pH changes in yogurt during storage
The pH values of yogurt products at 4  C storage are presented in
Fig. 2. An overall decline of pH of yogurts occurred during refrig-
erated storage. The pH for all the yogurts reduced (p < 0.05) from
the initial values of 4.5 to between 4.1 by day 28 of storage. But the
addition of spices did not seem to affect the pH of the samples
significantly compared with control. The above results are in line
with the findings of Behrad et al. (2009), who reported that pH for
control-yogurt was approximately the same as pH of spice-yogurts.
260 V.V. Illupapalayam et al. / LWT - Food Science and Technology 55 (2014) 255e262

Fig. 2. pH values of probiotics-yogurts with spice and without spices during refrig-
Fig. 3. DPPH radical inhibition capacity of water extracts from plain LA5 and spice-
erated (4  C) storage. (Standard error was zero). a e pH values of LA5 yogurts with
yogurts at day 28. (The error bars represents standard error of means). a e DPPH
spice and control-yogurt during refrigerated (4  C) storage. b e pH values of Bb12
radical inhibition capacity of water extracts from LA5 yoghurt with spices and control-
yogurts with spice and control-yogurt during refrigerated (4  C) storage.
yogurt during refrigerated (4  C) storage. b e DPPH radical inhibition capacity of water
extracts from Bb1 yogurt with spices and control-yogurt during refrigerated (4  C)
storage.

In contrast to these findings, the study by Amirdivani and Baba

(2011) reported that herbal-yogurts had faster rates of pH reduc-
tion than control-yoghurt. The observed pH range of 4.4 to 4.1 in
the present study is still within the range that was reported by
other researchers (Behrad et al., 2009; Singh et al., 2011).
In both LA5 and Bb12 products, the increase in viable count of
probiotics during the first 7 days coincided with the marked
reduction in pH recorded in all the samples. In a similar way, the
sharp increase in viable count of these probiotics from day 7 to day
14 coincided with the stable pH recorded (no change in pH) during
this period in all this samples. These results indicate that, the
reduction in viable count in all yogurts may be attributed to the acid
accumulation as the results of growth and fermentation as reported
in Shah (2000). Altogether the conversion of lactose to lactic acid,
the composition of bacterial starter culture, and duration of storage
and fermentation temperature could be the reason for the decrease
in pH during storage (Singh et al., 2011).
5.3. DPPH inhibition assay
The addition of spices, considered to have antimicrobial activity,
increased the antioxidant activity of yogurts compared to control-
yogurt at all storage periods (Fig. 3). The highest antioxidant ac-
tivity was recorded on day 1 for cardamom-yogurt (79.5 percent)
followed by nutmeg and cinnamon-yogurt (69 percent) and
control-yogurt (57 percent) in LA5 group of yoghurt products. In
yogurt products containing Bb12, the highest antioxidant activity
was recorded on day 1 for nutmeg (70 percent) followed by
cardamom (57 percent) and cinnamon-yogurts (57 percent).
Cardamom-yogurt showed the highest antioxidant activity at all
storage periods (LA5 or Bb12) compared to other spice and control-
yogurts. In contrast to these findings, Behrad et al. (2009) observed
that highest antioxidant activity of spice added yogurts occurred on
day 7 of storage. Similarly, Amirdivani and Baba (2011) also
observed that highest DPPH inhibition of herbal-yogurts occurred
after 7 days of refrigeration.
At the end of shelf-life (day 28) (Fig. 3) in yogurt product con-
taining LA5, the highest antioxidant activity was recorded for cin-
namon yogurt (35 percent) followed by cardamom yogurt (25.3
percent) and nutmeg (16 percent) which is consistent with the
viability of probiotics. Control-yogurt showed the lowest antioxi-
dant activity significantly (p < 0.05) different from cinnamon
yogurt. Similarly in yogurt products containing Bb12, the highest
antioxidant activity was recorded for cinnamon (41 percent) fol-
lowed by cardamom (32 percent) and nutmeg yogurts (30 percent).
Bb12 control-yogurt showed the lowest antioxidant activity and
was significantly different (p < 0.05) to the other three spice based
probiotic-yogurts.
The DPPH inhibition observed in the present studies may be
attributed to the combined effect of spices and metabolically active
yogurt bacteria (Papadimitriou et al., 2007).
 V.V. Illupapalayam et al. / LWT - Food Science and Technology 55 (2014) 255e262
Bb12 is one of the most adaptable Bifidobacteria to deal with the
human gastrointestinal environment as well as being an aero-
tolerant bifidobacterium species and this makes this bifidobac-
teria very suitable as a functional food component. Independent of
the continued microbial growth during refrigerated storage, which
may have altered some of the phenolic compounds and hence their
antioxidant activities (Blum, 1998), the maintaining of or increased
viable levels of both probiotics, LA5 and Bb12, in the probiotic-
yogurts supplemented with spices may indicate the combined
probiotic-yogurts with spices as a strong antioxidant active func-
tional food that may also protect other oxygen-sensitive bifido-
bacteria against the oxygen toxicity.
6. Conclusions
In conclusion, probiotic-yogurt containing strains of LA5 and
Bb12 and spice oleoresins of cardamom; nutmeg and cinnamon
was successfully manufactured with acceptable sensory properties
with viable probiotic counts up to the acceptable range and
increased antioxidant activity at 28 days. This is the first description
of probiotic-yogurt in combination with spice oleoresins that has
sensory attributes suitable for human consumption and provides
health promoting properties based upon antioxidant potential. The
present studies determined that the selected spices in this study
did not alter the sensory profile of probiotic-yogurt. However, the
sample size of spices and probiotics used in this study was limited
to 3 spices in combination with 2 probiotics. Sensory profiling of
yogurt with other probiotics and spices is needed for manufac-
turers considering incorporating the healthful ingredients into
their products. The study characterized by a significant increase in
survival of both B. animalis ssp. lactis (Bb12) and L. acidophilus (LA5)
during storage without affecting the fermentation kinetics and
providing strong antioxidant activity. However, other oxygen-
sensitive bifidobacteria need to be examined as to the efficacy of
the yoghurt-containing spices in enhancing probiotic viability. Also
further studies would include analysing enzyme activity in flavo-
noid production, the levels of volatile fatty acids, especially buty-
rate, produced in the yogurts for their functionally for gut health.
Acknowledgements
The authors wish to thank the Chr.Hansen Pty., Ltd, Australia,
Victoria University, Victoria for their assistance.
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