Food Research International 50 (2013) 224231

Contents lists available at SciVerse ScienceDirect

Food Research International

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

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

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 s 1 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.

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

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

225

(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.

2.3. 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 mL 1 of each microorganism in the inocula. The counts of probiotic bacteria increase during the fermentation of milk and are maintained above 9 Log CFU mL 1 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. 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).

2.2. Milk preparation Skim milk powder (Molico, Nestl, Araatuba, SP, Brazil) was reconstituted to 12 g100 mL 1 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 mL 1 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 Control Control Control Control Passion Passion Passion Passion 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 Fiber + + + +

fruit fruit fruit fruit

ber ber ber ber

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

226

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): 0 n K 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 Table 2 Spontaneous whey separation in yoghurts during 2 weeks of cold storage and their porosity in freeze-dried samples. Probiotic Treatment Spontaneous whey separation (mL 100 mL 1 of yoghurt) 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 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
e

227

Area of pores (area of porosity100 1 of area of yoghurt gel)

Day 7 5.1 4.6 4.2 4.6 0.8 0.0 0.0 0.4 (0.2) (0.15)d (0.15)c (0.15)d (0.1)b (0.0)a (0.0)a (0.1)b
e

Day 14 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

fruit fruit fruit fruit

ber ber ber ber

*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.

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%).

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

Table 3 Flow behavior of control and passion fruit ber yoghurts predicted by the Power Law model during two weeks of cold storage. Probiotic Treatment Day 1 K1 (Pa s 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 12.15lm 8.10hi 11.49k 9.21j 15.80p 13.83mno 19.02tw 13.16mn n1 0.15bcd 0.30e 0.13bcd 0.20cde 0.08ab 0.17cd 0.09abc 0.08ab 2 (Pa s 1) 5.01cd 3.52a 4.20ab 3.88a 4.48bc 4.11ab 4.70bc 3.78a n2 0.43fgh 0.56j 0.49ghij 0.51hij 0.50hij 0.55j 0.49ghij 0.52ij Day 7 K1 (Pa s 1) 12.40lm 10.75k 11.51klm 11.31kl 18.98rst 18.11rs 20.64st 16.47pq n1 2 (Pa s 1) n2 0.39efg 0.46ghi 0.38defg 0.41fg 0.43fgh 0.48ghi 0.44gh 0.51hij Day 14 K1 (Pa s 1) 14.02no 11.21kl 12.92lmn 12.91lmn 23.39x 18.33rs 21.15t 17.87r n1 2 (Pa s 1) n2 0.37ef 0.43fgh 0.38defg 0.36ef 0.43fgh 0.46ghi 0.39efg 0.45ghi

0.10abcd 5.25cdef 0.17cd 4.85bc 0.24de 5.34e 0.15bcd 0.09abc 0.08ab 0.07a 0.09abc 5.21cde 5.83fg 4.86bc 5.58efg 4.96bcd

0.09abc 5.68efg 0.14bcd 4.87bc 0.11abcd 5.90cd 0.08ab 0.06a 0.09abc 0.07a 0.08ab 5.52ef 5.57ef 5.30cde 6.15fg 5.03cde

fruit fruit fruit fruit

ber ber ber ber

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

30

pq mno no mn mn no

opq nop nop mno

pq opq

30

Thixotropy in the second cycle (%)

Thixotropy in the first cycle (%)

25

25

lm jkl
20

hij gh

hij g

hi

hi

ij

ij ghi

ijk

20

15

15

10

10

cde
5

def cd cde ab cde cd ab cd ab a

NCFM L10 Bl04 B94 NCFM L10 Bl04 B94

cde bc bc ab bc ab ab ab

de bc ab

ab
0 NCFM L10 Bl04 B94 NCFM L10 Bl04 B94 NCFM L10 Bl04 B94 0

ab

NCFM L10 Bl04 B94

d1

d7

d14

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 s 1 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 (P b 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
a

Color 6.95 6.68 6.67 6.68 (1.59) (1.39)a (1.41)a (1.35)a

a

Texture 7.11 5.52 5.72 5.66 (1.98) (1.91)a (1.94)a (1.74)a

b

Odor 2.27 5.35 5.67 5.62 (1.04) (1.46)b (1.65)b (1.56)b

a

Intensity of the passion fruit avor 1.25 2.41 3.17 3.29 (0.82) a (1.83)b (2.02)c (2.39)c

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

230

A.P. Esprito-Santo et al. / Food Research International 50 (2013) 224231 Esprito Santo, A. P., Cartolano, N. S., Silva, T. F., Soares, F. A. S. M., Gioielli, L. A., Perego, P., et al. (2012a). Fibers from fruit by-products enhance probiotic viability and fatty acids prole and increase CLA content in yogurts. International Journal of Food Microbiology, 154, 135144. Esprito-Santo, A. P., Perego, P., Converti, A., & Oliveira, M. N. (2012b). Inuence of milk type and addition of passion fruit peel powder on fermentation kinetics, texture prole and bacterial viability in probiotic yogurts. LWT Food Science and Technology, 47(2), 393399. Everett, D. W., & McLeod, R. E. (2005). Interactions of polysaccharide stabilisers with casein aggregates in stirred skim-milk yogurt. International Dairy Journal, 15(11), 11751183. Fernndez-Garca, E., & McGregor, J. U. (1997). Fortication of sweetened plain yogurt with insoluble dietary ber. Z Lebensm Unters Forsch A, 204, 433437. Fischer, P., Pollard, M., Erni, P., Marti, I., & Padar, S. (2009). Rheological approaches to food systems. Comptes Rendus Physique, 10, 740750. Folkenberg, D. M., & Martens, M. (2003). Sensory properties of low fat yogurts. Part A: Effect of fat content, fermentation culture and addition of non-fat dry milk on the sensory properties of plain yogurts. MilchwissenschaftMilk Science International, 58(12), 4851. Garca-Prez, F. J., Lario, Y., Fernandez-Lopez, J., Sayas, E., Prez-Alvarez, J. A., & Sendra, E. (2005). Effect of orange ber addition on yogurt color during fermentation and cold storage. Color Research and Application, 30(6), 457463. Gastaldi, E., Lagaude, A., Marchesseau, S., & Fuente, B. T. (1997). Acid milk gel formation as affected by total solids content. Journal of Food Science, 62, 671687. Guggisberg, D., Cuthbert-Steven, J., Piccinah, P., Butikofer, U., & Eberhard, P. (2009). Rheological, microstructural and sensory characterization of low-fat and whole milk set yogurt as inuenced by inulin addition. International Dairy Journal, 19(2), 107115. Heldman, D. R., & Singh, R. P. (1981). Food process engineering (2nd edn). Westport, CT, USA: AVI Publishing. Janebro, D. I., Queiroz, M. S. R., Ramos, A., Sabaa-Srur, A. U. O., Cunha, M. A. L., & Diniz, M. F. (2008). Effect of the our of the yellow passion fruit peel (Passiora edulis f. avicarpa Deg.) in the glycemic and lipid levels of type 2 diabetes patients. Brazilian Journal of Pharmacognosy, 18, 724732. Kipa, P., Meyerb, D., & Jellema, R. H. (2006). Inulins improve sensoric and textural properties of low-fat yogurts. International Dairy Journal, 16, 10981103. Lamsal, B. P., & Faubion, J. M. (2009). The benecial use of cereal and cereal components in probiotic foods. Food Reviews International, 25, 103114. Laws, A. P., & Marshall, V. M. (2001). The relevance of exopolysaccharides to the rheological properties in milk fermented with ropy strains of lactic acid bacteria. International Dairy Journal, 11, 709721. Lucey, J. A. (2002). Formation and physical properties of milk protein gels. Journal of Dairy Science, 85(2), 281294. Marafon, A., Sumi, A., Alcantara, M., Tamime, A., & de Oliveira, M. (2011). Optimization of the rheological properties of probiotic yoghurts supplemented with milk proteins. Lwt Food Science and Technology, 44(2), 511519. Marafon, A. P., Sumi, A., Granato, D., Alcntara, M. R., Tamime, A. Y., & Oliveira, M. N. (2011). Effects of partially replacing skimmed milk powder with dairy ingredients on rheology, sensory proling, and microstructure of probiotic stirred-type yogurt during cold storage. Journal of Dairy Science, 94(11), 53305340. McCann, T. H., Fabre, F., & Day, L. (2011). Microstructure, rheology and storage stability of low-fat yogurt structured by carrot cell wall particles. Food Research International, 44(4), 884892. Medeiros, J., Diniz, M., Srur, A., Pessoa, M., Cardoso, M., & de Carvalho, D. (2009). Tests of clinical toxicology of the yellow passion fruit peel (Passiora edulis f. avicarpa), as food with health property. Revista Brasileira de Farmacognosia-Brazilian Journal of Pharmacognosy, 394399. Nawirska, A., & Kwasniewska, M. (2005). Dietary bre fractions from fruit and vegetable processing waste. Food Chemistry, 91, 221225. Ozcan-Yilsay, T., Lee, W. -J., Horne, D., & Lucey, J. A. (2007). Effect of trisodium citrate on rheological and physical properties and microstructure of yogurt. Journal of Dairy Science, 90, 16441652. Parkar, S., Stevenson, D., & Skinner, M. (2008). The potential inuence of fruit polyphenols on colonic microora and human gut health. International Journal of Food Microbiology, 295298. Penna, A. L. B., Converti, A., & Oliveira, M. N. (2006). Simultaneous effects of total solids content, milk base, heat treatment temperature and sample temperature on the rheological properties of plain stirred yogurt. Food Technology and Biotechnology, 44(4), 515518. Prasanna, P. H. P., Grandison, A. S., & Charalampopoulos, D. (2012). Effect of dairy-based protein sources and temperature on growth, acidication and exopolysaccharide production of Bidobacterium strains in skim milk. Food Research International, 47, 612. Rawson, H. L., & Marshall, V. M. (1997). Effect of ropy strains of Lactobacillus delbrueckii ssp.bulgaricus and Streptococcus thermophilus on rheology of stirred yogurt. International Journal of Food Science and Technology, 32, 213220. Reid, G., Sanders, M. E., Gaskins, R., Gibson, G. R., Mercenier, A., Rastall, R., et al. (2003). New scientic paradigms for probiotics and prebiotics. Journal of Clinical Gastroenterology, 37(2), 105118. Ribeiro, M. M., Minim, V. P. R., Minim, L. A., Arruda, A. C., Ceresino, E. B., Carneiro, H. C. F., et al. (2010). Yogurt market research in the city of Belo Horizonte, Brazil. Revista Ceres, 57(2), 151156. Ruas-Madiedo, P., Gueimonde, M., Margolles, A., Losreyes-Gaviln, C. G., & Seppo, S. (2006). Exopolysaccharides produced by probiotic strains modify the adhesion of probiotics and enteropathogens to human intestinal mucus. Journal of Food Protection, 69(8), 20112015.

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. References
Barbalho, S. M., Damasceno, D. C., Spada, A. P., Lima, I. E., Arajo, A. C., Guiguer, E. L., et al. (2011). Effects of Passiora edulis on the metabolic prole of diabetic Wistar rat offspring. J Med Food, 14(12), 14901495. Barrangou, R., Briczinski, E. P., Traeger, L. L., Loquasto, J. R., Richards, M., Horvath, P., et al. (2009). Comparison of the complete genome sequences of Bidobacterium animalis subsp. lactis DSM 10140 and Bl-04. Journal of Bacteriology, 191(13), 41444151. Brito, C. J. F. A., & Deschamps, F. C. (2001). Tissue anatomy in different fractions of elephant grass cultivars (Pennisetum purpureum Schumach.). Revista Brasileira de Zootecnia, 30(5), 14091417. Chau, C. F., & Huang, Y. L. (2004). Characterization of passion fruit seed bres A potential bre source. Food Chemistry, 85, 189194. Damin, M. R., Alcntara, M. R., Nunes, A. P., & Oliveira, M. N. (2009). Effects of milk supplementation with skim milk powder, whey protein concentrate and sodium caseinate on acidication kinetics, rheological properties and structure of nonfat stirred yogurt. LWT Food Science and Technology, 42, 17441750. Damin, M. R., Minowa, E., Alcantara, M. R., & Oliveira, M. N. (2008). Effect of cold storage on culture viability and some rheological properties of fermented Milk prepared with yogurt and probiotic bacteria. Journal of Texture Studies, 39, 4055. De Lorenzi, L., Pricl, S., & Torriano, G. (1995). Rheological behavior of low-fat and full-fat stirred yogurt. International Dairy Journal, 5, 661671. Dhawan, K., Dhawan, S., & Sharma, A. (2004). Passiora: a review update. Journal of Ethnopharmacology, 94, 123.

A.P. Esprito-Santo et al. / Food Research International 50 (2013) 224231 Sembries, S., Dongowski, G., Jacobasch, G., Mehrlander, K., Will, F., & Dietrich, H. (2003). Effects of dietary bre-rich juice colloids from apple pomace extraction juices on intestinal fermentation products and microbiota in rats. British Journal of Nutrition, 90, 607615. Sendra, E., Fayos, P., Lario, Y., Fernandez-Lopez, J., Sayas-Barbera, E., & Perez-Alvarez, J. A. (2008). Incorporation of citrus bers in fermented milk containing probiotic bacteria. Food Microbiology, 25, 1321. Sendra, E., Kuri, V., Fernandez-Lopez, Sayas-Barbera, J. E., Navarro, C., & Prez-Alvarez, J. A. (2010). Viscoelastic properties of orange ber enriched yogurt as a function of ber dose, size and thermal treatment. LWT Food Science and Technology, 43, 708714. Singh, G., & Muthukumarappan, K. (2008). Inuence of calcium fortication on sensory, physical and rheological characteristics of fruit yogurt. LWT Food Science and Technology, 41, 11451152. Sodini, I., Lucas, A., Tissier, J. P., & Corrieu, G. (2005). Physical properties and microstructure of yogurts supplemented with milk protein hydrolysates. International Dairy Journal, 15, 2935.

231

Sodini, I., Remeuf, F., Haddad, S., & Corrieu, G. (2004). The relative effect of milk base, starter, and process on yogurt texture: A review. Critical Reviews in Food Sciences and Nutrition, 44, 113137. Spinnler, H. E., & Corrieu, G. (1989). Automatic method to quantify starter activity based on pH measurement. Journal of Dairy Research, 56, 755764. Staffolo, M. D., Bertola, N., Martino, M., & Bevilacqua, A. (2004). Inuence of dietary ber addition on sensory and rheological properties of yogurt. International Dairy Journal, 14, 263268. Tamime, Y. A., & Robinson, R. K. (2007). Yoghurt: Science and technology (3rd ed.). Boca Raton, FL, USA: CRC. Tuorila, H., & Cardello, A. V. (2002). Consumer responses to an off-avor in juice in the presence of specic health claims. Food Quality and Preference, 13, 561569. Yapo, B. M., & Kof, K. L. (2008). Dietary ber components in yellow passion fruit rinds A potential ber source. Journal of Agricultural and Food Chemistry, 56, 58805883.