Food Research International 50 (2013) 224231

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Food Research International

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Rheology, spontaneous whey separation, microstructure and sensorial characteristics

of probiotic yoghurts enriched with passion fruit ber
A.P. Esprito-Santo a, b, A. Lagazzo a, A.L.O.P. Sousa b, P. Perego a, A. Converti a, Maric N. Oliveira b,
a
b

Department of Chemical and Process Engineering, University of Genoa, Via Opera Pia, 15, 16145 Genoa, Italy
Department of Biochemical and Pharmaceutical Technology, So Paulo University, Av. Prof. Lineu Prestes, 580, Bl 16, 05508-900, So Paulo, Brazil

a r t i c l e

i n f o

Article history:
Received 18 July 2012
Accepted 8 September 2012
Keywords:
Yoghurt
Passion fruit ber
Probiotics
Rheology
Microstructure
Sensory

a b s t r a c t
Beyond demonstrated benecial health attributes, passion fruit rinds are a by-product of the fruit pulp industry, rich in total dietary ber, particularly pectin. The aim of this study was to evaluate the inuence of the
addition of passion fruit ber on the whey formation, rheological parameters, microstructure and sensorial
characteristics of probiotic yoghurts. Skim milk bases enriched with 1% of passion fruit ber or not were
heat treated and inoculated with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus,
and divided into four groups according to the probiotic strain added Lactobacillus acidophilus strains L10
and NCFM and Bidobacterium animalis subsp. lactis strains Bl04 and B94. Fermentations were performed
until the pH reached 4.5. Rheological characteristics of yoghurts were determined by a rotational rheometer
in two cycles of shear rate ranging from 0 to 15 s1 in both upward and downward curves. Sensorial analysis
of passion fruit ber yoghurts, either without any probiotic or co-fermented by L. acidophilus L10 or
B. animalis subsp. lactis Bl04, was evaluated against a control yoghurt without ber. Photomicrographs of
freeze-dried yoghurts were made by eld-emission scanning electron microscope (SEM). Thixotropy of
enriched yoghurts was higher than that of their respective controls in the two cycles of shear rate. Apparent
viscosity was signicantly higher in ber yoghurts co-fermented by the lactobacilli than in their controls at
the end of cold storage. Photomicrographs demonstrated that in passion fruit ber yoghurts the casein gel
was more compact and overlaid the ber, while laments of exopolysaccharides were more frequent in
control yoghurts. Appearance, odor and color of the passion fruit ber yoghurts received scores as good,
and the intensity of the passion fruit avor was considered weak by the sensory assessors. Results indicate
that the passion fruit ber is an almost neutral ingredient for the design of new high value-added yoghurt.
2012 Published by Elsevier Ltd.

1. Introduction
During milk fermentation in yoghurt manufacture, the pH decreases
as the lactic acid is produced by the starter culture Streptococcus
thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Casein
begins to aggregate at pH 4.7, isoelectric point, forming a fragile gel
net. At the end of fermentation, the gel of the set-type yoghurt is usually
broken to produce stirred yoghurt and the subsequent operations of
mixing, pumping and packaging impact in its structure, decreasing the
apparent viscosity. However, during thawing to approximately 20 C
and cold storage at 45 C, the stirred yoghurt recovers partially its
structure and viscosity, thus behaving as a pseudoplastic material
(Damin, Minowa, Alcantara, & Oliveira, 2008; Marafon, Sumi,
Alcantara, Tamime, & de Oliveira, 2011; Marafon et al., 2011; Sodini,
Remeuf, Haddad, & Corrieu, 2004; Tamime & Robinson, 2007).
Rheological and organoleptic properties, texture characteristics
and microstructure of yoghurt depend on many factors such as milk
Corresponding author. Tel.: +55 1130913690; fax: +55 1138156386.
E-mail address: monolive@usp.br (M.N. Oliveira).
0963-9969/$ see front matter 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.foodres.2012.09.012

base formulation, bacterial culture selection, production process,

packaging and storage (Tamime & Robinson, 2007). For the development of new fermented milks, the inuence of modications in the
milk base on texture, rheology and sensorial properties of products
has been studied, concerning mainly the lipid content of milk (De
Lorenzi, Pricl, & Torriano, 1995; Esprito-Santo, Perego, Converti, &
Oliveira, 2012; Folkenberg & Martens, 2003), the addition of proteins
to increase total solids (Gastaldi, Lagaude, Marchesseau, & Fuente,
1997; Marafon, Sumi, Granato, et al., 2011; Penna, Converti, &
Oliveira, 2006; Sodini, Lucas, Tissier, & Corrieu, 2005), the total dietary ber (DF) contents (Esprito-Santo et al., 2012; Garca-Prez et
al., 2005; McCann, Fabre, & Day, 2011; Staffolo, Bertola, Martino, &
Bevilacqua, 2004), and the addition of prebiotics (Guggisberg,
Cuthbert-Steven, Piccinah, Butikofer, & Eberhard, 2009; Kipa, Meyerb,
& Jellema, 2006) or calcium (Ozcan-Yilsay, Lee, Horne, & Lucey, 2007;
Singh & Muthukumarappan, 2008).
Specic strains as well as composition of the bacterial cultures
used in fermentation, especially those releasing exopolysaccharides
or combinations of the starter culture with one or more probiotics,
also play an important role in the development of yoghurt structure

A.P. Esprito-Santo et al. / Food Research International 50 (2013) 224231

(Esprito-Santo et al., 2012; Laws & Marshall, 2001; Prasanna,

Grandison, & Charalampopoulos, 2012; Rawson & Marshall, 1997;
Reid et al., 2003; Staffolo et al., 2004; Tamime & Robinson, 2007).
Formulation of new food products with ingredients from fruit
by-products rich in total DF has increased in recent years, being convenient to their association with probiotic bacteria for the promotion
of the intestinal health (Lamsal & Faubion, 2009; Sendra et al., 2008).
Dietary ber can be fractioned into two major groups of components,
the water-insoluble and the water-soluble fraction. While the insoluble fraction stimulates the intestinal peristalsis, the soluble one promotes the selective growth of the indigenous microbiota, acting as a
prebiotic (Sembries et al., 2003). Nawirska and Kwasniewska
(2005) reported the importance of DF intake on a daily basis to prevent obesity, atherosclerosis, heart diseases, gut cancer and diabetes.
Therefore it is healthier to consume the total dietetic ber, instead of
just its prebiotic fraction. One of the promising fruit by-products is
the passion fruit peel, which, in addition to its functional properties
such as the reduction of cholesterol and glucose in blood serum
(Barbalho et al., 2011; Janebro et al., 2008; Medeiros et al., 2009;
Parkar, Stevenson, & Skinner, 2008), it was shown, in the recent
study of our group, the improvement in fatty acid prole and increase
in the conjugated linoleic acid content of probiotic yoghurts added
with passion fruit peel (Esprito Santo et al., 2012a). Moreover,
passion fruit peel ber enhanced the texture parameters of skim
yoghurts during cold storage (Esprito-Santo et al., 2012b). Based on
this background, the present study aimed to evaluate some other
important aspects of the rheology, spontaneous whey separation,
microstructure and sensorial characteristics of probiotic yoghurts
enriched with passion fruit ber.

2. Materials and methods

2.1. Preparation of passion fruit ber and determination of water and oil
holding capacity
Passion fruit by-product was obtained from a fruit pulp manufacturer, located in the city of Jundiai, So Paulo State, Brazil, and
maintained in freezer at 26 C until processing.
The peels of the passion fruit were dried in an oven (Quimis
Q314M-293) at 60 C under airow at 12 air changesmin 1 until
constant weight. The dry peels were reduced to ne powder in a
Bimby processor, TM 31 (Vorwerk, Wuppertal, Germany). Fiber particle size was standardized to less than 17.7 m, measured through
sieves (Granutest, So Paulo, Brazil), and ber powder was stored in
glass pots maintained under refrigeration at 4 C until use.
The water and oil holding capacities of the passion fruit ber (PFF)
were determined by centrifugation (multispeed PK 131, ALC Porta,
Italy) at 25 C, according to Chau and Huang (2004).

225

2.3. Microbial cultures

The freeze-dried starter yoghurt culture (CY340, DSM, Moorebank,
NSW, Australia) composed of Streptococcus thermophilus (St) and Lactobacillus delbrueckii subsp. bulgaricus (Lb) and two Lactobacillus acidophilus strains (LAFTI L10, DSM, and NCFM, HOWARU Dophilus,
Danisco, Madison, WI, USA) and two Bidobacterium animalis subsp.
lactis strains (Bl04, Danisco and LAFTI B94, DSM) were used in this
study. The lyophilized cultures were diluted in 50 mL of sterilized
milk inoculums, according to the recommendations of the manufacturer. The amount (g) of the lyophilized cultures was previously determined to reach counts around 6 Log CFU mL1 of each microorganism
in the inocula. The counts of probiotic bacteria increase during the fermentation of milk and are maintained above 9 Log CFU mL1 during
2 weeks of cold storage of the yoghurts, as previously reported by
Esprito Santo et al. (2012a) and Esprito-Santo et al. (2012b).
2.4. Experimental procedure
Four types of probiotic yoghurts were prepared according to the
experimental design presented in Table 1. For the rheology, syneresis
and microstructure analysis, each yoghurt type was prepared in duplicate in two independent batch fermentations (N = 4). The asks
containing 500 mL of the heat treated milk base were inoculated
with 1 mL of yoghurt starter cultures and 1 mL of probiotic culture.
Thereby, each milk ask had, before fermentation, an average count
of 6.4 Log CFU mL 1 of St and 5.9 Log CFU mL 1 of Lb, and the probiotic counts were ~ 6.9 Log CFU mL 1. Afterwards, the asks with
the samples were transferred to a water bath at 42 C and connected
to a CINAC (Cynetique d'acidication, Ysebaert, Frpillon, France)
system (Spinnler & Corrieu, 1989) which recorded the pH decrease.
At pH 4.5, fermentation was stopped and the asks were capped and
cooled to 20 C in an ice bath. Then, the asks were transferred to a laminar ow cabinet and the coagulum was broken with regular upward and
downward movements for 2 min with a sterile perforated disk on a
stainless steel rod. The coagulum of the yoghurts in the 15 L containers
was broken under agitation at 100 rpm by a sterile steel propeller
connected to the agitator already described in Section 2.2. The stirred yoghurts were distributed into 50 mL polypropylene cups, thermally
sealed and stored in refrigerator at 4 C.
2.5. Spontaneous whey separation
After 24 h of fermentation, four cups of each yoghurt type were
carefully homogenized (N = 32) and an aliquot (8 mL) of yoghurt
was collected throughout a 10 mL (1:10) sterile plastic pipette
(Sterilin, Barloworld Scientic, Stafforshire, UK), placed in a shelf pipette holder and maintained in a perpendicular position at 4 C. The
whey formed in the upper phase of the yoghurt was read in the pipette scale. This procedure was repeated for yoghurts stored at 7

2.2. Milk preparation

Skim milk powder (Molico, Nestl, Araatuba, SP, Brazil) was
reconstituted to 12 g100 mL1 in potable water ltered by a water
purier, FR600 (IBBL, So Paulo, Brazil) and divided into two portions:
(i) enriched with PFF powder at 1.0 g100 mL1 of milk and (ii) not
enriched and designed as control. The milk bases were heat treated at
85 C for 15 min in a bath thermostat (A100, Lauda, Knigshofen,
Germany) under agitation at 300 rpm provided by steel two-blade
propeller connected to an agitator (Q250M1 (Quimis, Diadema, Brazil)).
Afterwards, the milk bases for the analysis of rheology, syneresis and
microstructure were divided into sterile glass asks (500 mL) and
those for the sensorial analysis were conditioned in sanitized white
polypropylene containers (15 L), cooled in an ice bath and stored at
4 C for 24 h until inoculation.

Table 1
Experimental design to study the rheology, spontaneous whey separation, microstructure and sensorial characteristics of probiotic yoghurts enriched with passion fruit
ber.
Yoghurt

Probiotic

Fiber

Control
Control
Control
Control
Passion
Passion
Passion
Passion

Lactobacillus acidophilus NCFM

L. acidophilus L10
Bidobacterium animalis subsp. lactis Bl04
B. animalis subsp. lactis B94
L. acidophilus NCFM
L. acidophilus L10
B. animalis subsp. lactis Bl04
B. animalis subsp. lactis B94

+
+
+
+

fruit
fruit
fruit
fruit

ber
ber
ber
ber

: without passion fruit ber; +: with passion fruit ber.

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A.P. Esprito-Santo et al. / Food Research International 50 (2013) 224231

and 14 days after fermentation. The results were expressed in % of

whey formed.
2.6. Rheological measurements
Rheological measurements were carried out in quintuplicate for
each sample, at 5 C, using a rotational Rheometer, R20 (HaakeRotovisco, Karlsruhe, Germany), with a cone and plate geometry
(50 mm diameter and 2 of inclination angle). About 1 mL of the
sample was placed in the center of the stationary plate. The gap
between the plates was 100 m. The controlled-temperature was
assured by a circulating water bath through the jacket surrounding
the rotor and cup assembly. Two cycles of shear rate () ranging
from 0 to 15 s 1 of upward and downward curves were performed,
and the corresponding shear stress () data computed by a RheoWin
software 2.97 (Haake Laboratories, Karlsruhe, Germany). This range
of shear rate was chosen to support, through the apparent viscosity
(app), the weak forces present in the inner structure of the yoghurt,
which were not possible by applying shear rate higher than 15 s 1.
Apparent viscosity (app) was calculated at = 2 s 1 in the downward ow curves of the rst and second cycles. The data obtained
were tted by the Power Law model (Heldman & Singh, 1981):
K

0
n

where is the shear stress, K the consistency index and n the Power
Law index that describes the ow behavior of the uid as shearthinning/pseudoplastic (n b 1) or shear-thickening/dilatant (n > 1)
(Fischer, Pollard, Erni, Marti, & Padar, 2009). A recent study of our
group demonstrated that major alterations in the texture prole of
PFF yoghurts occur during the rst two weeks of cold storage. Thus,
the rheological characterization of the yoghurts was focused at days
1, 7 and 14.
2.7. Microstructural analyses
Three cups of each yoghurt type were freeze-dried in a lyophilizer
(L4KR 118, BOC Edwards, So Paulo, Brazil) after 1 day of storage at
4 C, as described by Damin, Alcntara, Nunes, and Oliveira (2009).
Afterwards, the samples were stuck on stubs with double-face tape
and coated with 15 nm of a goldpalladium layer applied by a
cathodic coater, E 5100 (Polaron, Hertfordshire, West Sussex, UK).
Six elds of each sample were observed in a eld-emission scanning
electron microscope (SEM) (JSM-7401-F, JEOL, Akishima, Japan), operating at a voltage of 5.0 kV, and photomicrographs were registered
under magnications from 1000 to 10,000. Images were analyzed
using the software Image Pro Plus v.4.5.1 (Media Cybernetics, Silver
Spring, MD, USA) as JPG les, and to three images at 10,000 of magnication of each sample were applied the highest contrast to highlight the pores in the casein network, while the areas of the pores
were marked and measured through a CINAG system of planimetry
(Centro de Informtica na Agricultura, Faculdade de Cincias
Agronmicas, UNESP, Botucatu, Brazil) according to Brito and
Deschamps (2001), and expressed as area of porosity100 1 of area
of yoghurt's gel.
2.8. Sensorial analysis
For the sensorial analysis, were chosen passion fruit ber yoghurts
co-fermented by L. acidophilus L10 and B. animalis subsp. lactis Bl04,
which showed the highest apparent viscosities (app) compared to
their respective controls. Natural control yoghurt and PFF yoghurt
without probiotic were used to evaluate the inuence of PFF and probiotic strain, respectively, on the sensorial perception. In each session,
four samples packed in white cups and identied with random
three-digit codes were served to the untrained assessors (170) in a

randomized order. The assessors were students and members of the

staff of the University of So Paulo, So Paulo, Brazil, and frequent
consumers of avored yoghurts and passion fruit. Between one sample and the next they were instructed to cleanse their palates with
crackers and tap water. The attributes of appearance, color and odor
were scored out according to a hedonic scale (1 = dislike extremely,
3 = dislike moderately, 5 = neither like nor dislike, 7 = like moderately, and 9 = like extremely). The intensity of the passion fruit avor
was evaluated in a 9-point structured scale from very weak (on the
left) to very strong passion fruit avor (on the right). Analyses were
performed on day 4 in four sessions being two in the morning and
two in the afternoon. Microbiological safety of yoghurt samples was
examined before sensory evaluation regarding yeasts and molds
using Yeast and Mold Petrilm (3 M Company, St. Paul, MN, USA)
incubated at 21 C, as well as Escherichia coli and total coliforms
using Coliform Petrilm (3 M Company) incubated at 37 C. Sensory
evaluation tests were approved by the Ethics Committee in Research
of the Faculty of Pharmaceutical Sciences, So Paulo University
(process number CEP 474, June 30, 2008).
2.9. Statistical analyses
Analysis of variance (ANOVA) was applied to the experimental
data, and the means among different groups were compared by the
Tukey test at P b 0.05 using the software Statistica 8.0 (Statsoft,
Tulsa, OK, USA).
3. Results and discussion
3.1. Spontaneous whey separation
Quantitative evaluation of whey spontaneously separated from
the protein gel is a useful approach to represent, particularly from
the consumer point of view, the aspect of yoghurt during cold storage,
and the instability of the casein network in yoghurts can be evaluated
through the measurement of the spontaneous syneresis (Lucey,
2002).
As shown in Table 2, composition of co-cultures in yoghurts
enriched with passion fruit ber had no signicant effect (P > 0.05)
on whey separation, which was likely due to the fact that all probiotic
strains tested produce exopolysaccharides with recognized waterholding capacity (Barrangou et al., 2009; Laws & Marshall, 2001;
Prasanna et al., 2012; Ruas-Madiedo, Gueimonde, Margolles,
Losreyes-Gaviln, & Seppo, 2006). During cold storage, control
yoghurts produced more whey (P b 0.05), varying from 4.2% to 5.1%,
than passion fruit ber or PFF yoghurts (0.00.8%), especially those
co-fermented by L. acidophilus NCFM (P b 0.05). Water and oil holding
capacities of PFF were 3.8 mLg 1 and 4.9 gg 1, respectively, which
are in accordance to the ndings of Yapo and Kof (2008). These
results suggest that the water-holding capacity of pectin present in
PFF reduced signicantly the amount of whey formed in yoghurts
containing it. According to Everett and McLeod (2005), pectin is, in
fact, constituted by anionic hydrocolloids that are classied as
adsorbing polysaccharides, which interact with the casein network
increasing its ability to entrap the whey phase.
The post-acidication is one of the factors that can increase the
production of whey in yoghurts (Lucey, 2002). As reported in a previous study of our group (Esprito-Santo et al., 2012b), in this study the
addition of passion fruit ber decreased the pH (measured in pH
meter model Q-400M1, Quimis, So Paulo, Brazil) of the yoghurts.
In PFF yoghurts the pH varied from 4.5 to 4.2 and in control yoghurts
it varied from 4.5 to 4.4 during the storage period (data not shown).
However, during the two weeks of storage, the whey formed in PFF
yoghurts was signicantly lower than in their controls, which
reinforces the importance of the pectins in the PFF to preserve the
yoghurt structure.

A.P. Esprito-Santo et al. / Food Research International 50 (2013) 224231

227

Table 2
Spontaneous whey separation in yoghurts during 2 weeks of cold storage and their porosity in freeze-dried samples.
Probiotic

Spontaneous whey separation (mL 100 mL1 of

yoghurt)

Treatment

Day 1
Lactobacillus acidophilus NCFM
L. acidophilus L10
Bidobacterium animalis subsp. lactis Bl04
B. animalis subsp. lactis B94
L. acidophilus NCFM
L. acidophilus L10
B. animalis subsp. lactis Bl04
B. animalis subsp. lactis B94

Control
Control
Control
Control
Passion
Passion
Passion
Passion

Day 7
e

fruit
fruit
fruit
fruit

ber
ber
ber
ber

5.1
4.6
4.2
4.6
0.8
0.0
0.0
0.4

5.0 (0.0)
4.2 (0.15)c
4.2 (0.15)c
4.6 (0.15)d
0.4 (0.1)b
0.0 (0.0)a
0.0 (0.0)a
0.4 (0.1)b

Area of pores (area of porosity1001

of area of yoghurt gel)

Day 14
e

(0.2)
(0.15)d
(0.15)c
(0.15)d
(0.1)b
(0.0)a
(0.0)a
(0.1)b

5.1
4.6
4.2
4.6
0.8
0.0
0.0
0.4

(0.1)f
(0.15)e
(0.15)d
(0.15)e
(0.1)c
(0.1)a
(0.1)a
(0.1)b

30.5
33.6
30.9
31.2
24.7
22.3
22.1
23.6

(2.8)d
(3.1)ef
(2.7)d
(3.0)de
(1.5)bc
(1.2)a
(1.2)a
(1.4)ab

*Means (N = 32) (standard deviation) with different superscript letters in the same column differ signicantly (P b 0.05).
**Determined on day 1; N = 24.

In the second cycle, the structure recovery ranged from 3.4 to 7.7%
and from 0.5 to 3.3% in PFF and control yoghurts, respectively, being
generally higher in yoghurts supplemented with PFF (Fig. 1).
In the rst cycle of shear rate, the apparent viscosity (app) varied
from 41.5 to 62.7 Pas in control yoghurts and from 44.3 to 91.6 Pas
in PFF yoghurts during the storage period (Fig. 2). All PFF yoghurts increased signicantly their app; however, in general this pattern was
not followed by the control yoghurts (Fig. 2), indicating that the
structure of PFF yoghurts was possibly stabilized by positive interactions between pectins from the fruit and exopolysaccharides produced by the bacteria. On the other hand, in the second cycle of
shear rate, PFF and control yoghurts showed practically the same
mean values of app, which varied from 8.1 to 16.3 Pas in control yoghurts and from 10.2 to 18.4 Pas in PFF yoghurts, respectively
(Fig. 2). The second cycle of shear rate reduced signicantly app in
all yoghurts. However, since PFF yoghurts presented the highest
app in the rst cycle, it is evident, especially after 14 days of cold
storage, that they suffered greater damage in their structure than
the controls, probably because the bers acted as knives, breaking
the proteinprotein interactions during tests, which also reduced
the thixotropy in the second cycle (Figs. 1, 2 and 3).
The decrease in K values (Table 3), combined to the partial structure recovery between the two cycles of shear rate (Fig. 1), indicates
that both control and PFF yoghurts presented shear rate-thinning and
thixotropic properties, which are typical of yoghurts (McCann et al.,
2011; Rawson & Marshall, 1997; Staffolo et al., 2004). Moreover, different rheological behaviors of control and PFF yoghurts may have
been the result of positive interaction of pectin present in PFF with
both protein and exopolysaccharides, hence helping the yoghurts to
partially recover their structure (thixotropy) during the decrease of
shear rate. According to Lucey (2002) and Everett and McLeod
(2005), pectins have demonstrated to interact with the positive
charges on the surface of casein micelles, promoting strengthening

3.2. Rheological measurements

Yoghurt samples were subjected to two cycles of low shear rate
(0 15 s 1) to minimize the impact of the presence of total dietary ber from passion fruit on the shear analysis performed using a
rotational rheometer as well as to shed light on the weak forces acting
in the yoghurt structure, such as polysaccharideprotein complexes
(Fernndez-Garca & McGregor, 1997), that cannot be properly
evidenced at high shear rate.
The two cycles of shear rate in upwarddownward ow curves
tted by Power Law model resulted in adequate correlation coefcients R 2 0.968, indicating that this model is more adequate to represent the experimental data (Table 3). The mean values of n were b 1,
thus pointing out a pseudoplastic behavior of yoghurts, as already
reported (Fischer et al., 2009; Tamime & Robinson, 2007).
In most of the cases, the consistency index of the rst cycle (K1)
increased signicantly during storage (P b 0.05), varying from 8.10
to 14.02 Pas 1 in control yoghurts and being signicantly higher
in all passion fruit ber yoghurts (13.16 K1 23.39 Pas 1)
(Table 3). Strengthening of proteinprotein complexes forming yoghurt structure may have been responsible for the increase in K1 during the two weeks of storage (Damin et al., 2008; Rawson & Marshall,
1997). However, in the second cycle, the mean values of K2 decreased,
whereas n2 increased, but no signicant differences were noticed between PFF yoghurts and their respective controls, except in those
co-fermented by B. lactis Bl04, where K2 was signicantly higher in
the presence of ber.
Thixotropy was measured separately in the two cycles as the area
between the upward and downward slopes of shear rate, which
represents the ability of samples to recover their structure during
the decrease of (Fig. 1). Considering the whole period of storage,
thixotropy in the rst cycle was signicantly higher in all PFF yoghurts (24.030.1%) than in their respective controls (14.022.0%).

Table 3
Flow behavior of control and passion fruit ber yoghurts predicted by the Power Law model during two weeks of cold storage.
Probiotic

Lactobacillus acidophilus NCFM

L. acidophilus L10
Bidobacterium animalis subsp.
lactis Bl04
B. animalis subsp. lactis B94
L. acidophilus NCFM
L. acidophilus L10
B. animalis subsp. lactis Bl04
B. animalis subsp. lactis B94

Treatment

Day 1

Control
Control
Control
Control
Passion
Passion
Passion
Passion

fruit
fruit
fruit
fruit

ber
ber
ber
ber

Day 7

Day 14

K1
(Pa s1)

n1

2
(Pa s1)

n2

K1
(Pa s1)

n1

12.15lm
8.10hi
11.49k

0.15bcd
0.30e
0.13bcd

5.01cd
3.52a
4.20ab

0.43fgh
0.56j
0.49ghij

12.40lm
10.75k
11.51klm

9.21j
15.80p
13.83mno
19.02tw
13.16mn

0.20cde
0.08ab
0.17cd
0.09abc
0.08ab

3.88a
4.48bc
4.11ab
4.70bc
3.78a

0.51hij
0.50hij
0.55j
0.49ghij
0.52ij

11.31kl
18.98rst
18.11rs
20.64st
16.47pq

2
(Pa s1)

2
(Pa s1)

n2

K1
(Pa s1)

n1

0.10abcd 5.25cdef
0.17cd
4.85bc
0.24de
5.34e

0.39efg
0.46ghi
0.38defg

14.02no
11.21kl
12.92lmn

0.09abc 5.68efg
0.14bcd 4.87bc
0.11abcd 5.90cd

0.37ef
0.43fgh
0.38defg

0.15bcd
0.09abc
0.08ab
0.07a
0.09abc

0.41fg
0.43fgh
0.48ghi
0.44gh
0.51hij

12.91lmn
23.39x
18.33rs
21.15t
17.87r

0.08ab
0.06a
0.09abc
0.07a
0.08ab

0.36ef
0.43fgh
0.46ghi
0.39efg
0.45ghi

5.21cde
5.83fg
4.86bc
5.58efg
4.96bcd

5.52ef
5.57ef
5.30cde
6.15fg
5.03cde

n2

Means (N = 40) with different superscript letters in the same column differ signicantly (P b 0.05). Abbreviations: K1 and K2 are the consistency index of upward curves of the rst
and the second cycles, respectively; n1 and n2 are the Power Law index that indicates the ow behavior in upward curves of the rst and the second cycle, respectively.

228

A.P. Esprito-Santo et al. / Food Research International 50 (2013) 224231

35

35

opq

pq

Thixotropy in the first cycle (%)

mno
25

no
mn

no

nop
mno

mn
lm
jkl

20

hij

hij

hi

hi

ij

ijk

ij
ghi

15

gh

30

opq
nop

10

Thixotropy in the second cycle (%)

pq

30

25

20

15

10

NCFM L10 Bl04 B94

NCFM L10 Bl04 B94

NCFM L10 Bl04 B94

d1

d7

d14

cde

cd

ab
0

def

cde

ab

ab

cde

cde
cd
ab

cd
bc

ab

bc
ab

de
bc

bc
ab

ab

ab

ab

NCFM L10 Bl04 B94

NCFM L10 Bl04 B94

NCFM L10 Bl04 B94

d1

d7

d14

Fig. 1. Thixotropy of control and passion fruit ber yoghurts. Means (N = 40) with different letters are signicantly different (P b 0.05). Abbreviations: NCFM and L10: yoghurts
co-fermented by Lactobacillus acidophilus NCFM and L10, respectively; Bl04 and B94: yoghurts co-fermented by Bidobacterium animalis subsp. lactis Bl04 and B94, respectively.
d1, d7 and d14: samples taken after 1, 7 and 14 days of cold storage after fermentation. Control Passion fruit ber.

of casein network and stabilization of yoghurt structure. Even though,

pectinprotein interactions are weaker than the proteinprotein ones
in yoghurt structure (Fernndez-Garca & McGregor, 1997), these
linkages may have been unable to hold up the second cycle of shear
rate (Table 3 and Fig. 1). Rheological data are consistent with the texture attributes of a previous work (Esprito-Santo et al., 2012b), that
reported higher rmness, consistency and cohesiveness of PFF skim
yoghurts than their respective controls. Sendra et al. (2010) observed
that the addition of orange ber at doses lower than 0.6 g100 mL 1
weakened the yoghurt's structure, but at higher amounts, water absorption counterbalanced this negative effect and strengthened the

casein network. Similar behavior may have occurred in this study

when PFF was added to yoghurts.
3.3. Microstructure
SEM micrographs of control and enriched yoghurts co-fermented by
B. lactis subsp. animalis Bl04 are presented in Fig. 3 showing signicant
differences. A normal structure of non-fat yoghurt with a casein network surrounded by a high number pores of whey could be seen as described by many authors (Damin et al., 2009; Marafon, Sumi, Granato, et
al., 2011; Ozcan-Yilsay et al., 2007; Sodini et al., 2005). Measurements of

Fig. 2. Apparent viscosity (app) at = 2 s1 of control and passion fruit ber yoghurts in the two cycles of upwarddownward curves of shear rate. Means (N = 40) with different
letters are signicantly different (P b 0.05). Abbreviations: NCFM and L10: yoghurts co-fermented by Lactobacillus acidophilus NCFM and L10, respectively; Bl04 and B94: yoghurts
co-fermented by Bidobacterium animalis subsp. lactis Bl04 and B94, respectively. d1, d7 and d14: samples taken after 1, 7 and 14 days of cold storage after fermentation. Control
Passion fruit ber.

A.P. Esprito-Santo et al. / Food Research International 50 (2013) 224231

229

the area of pores (Table 2) point out that PFF yoghurts had higher density
of the casein network than their respective controls (Pb 0.05), consistent
with a lower number of pores and a higher number of cross-links between strands (Fig. 3a compared to Fig. 3b). In control yoghurts, but
not in PFF yoghurts, exopolysaccharides formed several laments and
covered some areas of the protein gel (Fig. 3b). Also this observation
can be explained by the presence of pectin in PFF, which may have
interacted with proteins and bacterial exopolysaccharides forming a
more compact structure with greater apparent viscosity and ability to entrap the whey phase (Fig. 2, Table 2). The passion fruit ber presented a
needle-shaped blade and was clearly observed in the micrographs
nesting the casein gel and, sometimes, the yoghurt bacteria (Fig. 3c).
3.4. Sensorial characteristics
Results of hedonic tests summarized in Table 4 emphasize that the
four types of yoghurt assayed had no signicant differences among
them (P > 0.05) in the scores of appearance and color, which varied
from 6.6 to 7.0 (like moderately), in spite of the evident light yellowish color of PFF yoghurts.
However, natural yoghurts had higher scores (P b 0.05) for texture
than the PFF ones, which were scored as having a sandy or graininess
mouth feeling by some of the assessors, even though the particle size
of bers was less than 17.7 m. This result can be ascribed not only to
the amount or size of PFF in yoghurts but also to the shape of bers,
which had edges like stones (Fig. 3c) capable of sensitizing the
mouth more than if they had spherical and smooth shape. PFF yoghurts with or without probiotic bacteria received higher mean
scores for odor (neither like nor dislike) than the natural ones (dislike
moderately) (Table 4). Nonetheless, the passion fruit avor was more
intense (P b 0.05) in PFF yoghurts co-fermented by L. acidophilus L10
(3.2) and B. animalis subsp. lactis Bl04 (mean score = 3.3) than in
PFF yoghurt without probiotics (mean score = 2.1). In a previous
study of our group, the pH and total titratable acidity of PFF yoghurts
were not signicantly different from their respective controls
(Esprito-Santo et al., 2012b). Thus, the enhancement of the passion
fruit avor observed in PFF yoghurts co-fermented by L. acidophilus
and B. animalis subsp. lactis is unlikely due to the post-acidication
during storage. Therefore, these probiotic bacteria may have produced or transformed compounds that reinforce the avor of the
Passiora edulis peels which is due, mainly, to esters, 3-methylthiohexan-1-ol, 2-methyl-4-propyl-1, 3-oxathione enantiomers and
edulans I and II (Dhawan, Dhawan, & Sharma, 2004). However, considering only the probiotic yoghurts, the strain of probiotic bacteria
had no signicant effect on odor and passion fruit avor perceived
by the panelists. Similarly, Sendra et al. (2008) observed that probiotic bacteria used in yoghurts supplemented with citrus ber had no inuence on those parameters.
Participants of the sensory panel were familiarized in eating
fruit-avored yoghurt (97.6%), but not to consume natural yoghurt.
These data are in agreement with Ribeiro et al. (2010), who reported
that the Brazilian yoghurt market is dominated by fruit-avored yoghurt (about 95% of the market), and the colorful and sweet yoghurts
are those preferred by the consumers. Therefore, some sensory properties of the natural yoghurt, such as appearance, color, smell and

eps

fb

Fig. 3. Microstructure of yoghurts co-fermented by Bidobacterium lactis subsp. animalis

Bl04 at 10,000 magnication of (a1) passion fruit ber yoghurt, (a2) passion fruit ber
yoghurt shown with the highest contrast, (b) control yoghurt, and (c) passion fruit ber
yoghurt. Bar=1 m. Abbreviations: eps = laments of exopolysaccharides ; fb =
passion fruit ber.

Table 4
Scores of the sensory evaluation of passion fruit ber yoghurts.
Yoghurt type
Control natural skim yoghurt
PFF yoghurt
PFF yoghurt co-fermented by Lactobacillus acidophilus L10
PFF yoghurt co-fermented by Bidobacterium animalis subsp. lactis Bl04

Appearance
7.03
6.83
6.84
6.74

(1.51)
(1.44)a
(1.45)a
(1.33)a

Color
6.95
6.68
6.67
6.68

Texture
a

(1.59)
(1.39)a
(1.41)a
(1.35)a

7.11
5.52
5.72
5.66

Intensity of the passion fruit avor

Odor
b

(1.98)
(1.91)a
(1.94)a
(1.74)a

2.27
5.35
5.67
5.62

(1.04)
(1.46)b
(1.65)b
(1.56)b

Mean (N = 170) (standard deviation). Values with different superscript letters in the same column are signicantly different (P b 0.05).

1.25
2.41
3.17
3.29

(0.82) a
(1.83)b
(2.02)c
(2.39)c

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A.P. Esprito-Santo et al. / Food Research International 50 (2013) 224231

even the intensity of passion fruit avor may have been underestimated or confused by the assessors, not accustomed to its
characteristics.
Fernndez-Garca and McGregor (1997) reported that the supplementation of yoghurt with oat ber reduced the score for texture, but
the authors refereed to the healthy benets of the new product to
justify its use. Tuorila and Cardello (2002) observed that consumers
are, indeed, more prone to accept off-avors and some unpleasant
characteristics depending on the functional claim of the food product.
Notes attributed to avor, for all enriched yoghurts, revealed a weak
or very weak passion fruit avor, and a minimum, but signicant, inuence on texture, which are important features for the development
of new functional foods, as well as broader possibilities in the formulation of avors.
4. Conclusions
Enriching probiotic yoghurt with passion fruit ber or PFF offered
a scaffold that strengthened the yoghurt's structure, and increases apparent viscosity during storage. In contrast, these bers also act as
cutting blades during shearing, breaking the casein cross-links and
dropping down the apparent viscosity of yoghurts.
The two cycles at low shear rate were effective to discriminate the
rheological behavior of control and enriched PFF yoghurts, minimizing the destructive effect of the analysis during rheological tests in
a rotational rheometer.
SEM micrographs showed that in PFF yoghurts the casein gel overlaid the ber and sometimes was nestled in it, but laments of
exopolysaccharides were more evident in control yoghurts.
The attributes of appearance, odor and color of PFF yoghurts were
considered neutral or good, and the intensity of the passion fruit avor was deemed weak by the sensory assessors.
Results indicate that PFF, when added at a dose of 1 g100 mL 1,
is an almost neutral ingredient and a promising avenue in designing
of new high value-added probiotic yoghurts.
Acknowledgments
The authors wish to thank DuPont Brasil Ltda (Cotia, So Paulo,
Brazil) and Globalfood (So Paulo, Brazil) for providing the cultures,
De Marchi for the donation of passion fruit by-product, Dr. C. Bona
from the Botany Department of Federal University of Paran, Brazil,
for the help with the pore's area analysis, and nally for FAPESP,
CNPq and CAPES for the nancial support.
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