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Rheology, spontaneous whey separation, microstructure and sensorial characteristicsof probiotic yoghurts enriched with passion fruit berA.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 Department of Chemical and Process Engineering, University of Genoa, Via Opera Pia, 15, 16145 Genoa, Italyb Department of Biochemical and Pharmaceutical Technology, So Paulo University, Av. Prof. Lineu Prestes, 580, Bl 16, 05508-900, So Paulo, Brazil

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

Article history:Received 18 July 2012Accepted 8 September 2012

Keywords:YoghurtPassion fruit berProbioticsRheologyMicrostructureSensory

Beyond demonstrated bene cial health attributes, passion fruit rinds are a by-product of the fruit pulp indus-try, rich in total dietary ber, particularly pectin. The aim of this study was to evaluate the in uence of theaddition of passion fruit ber on the whey formation, rheological parameters, microstructure and sensorialcharacteristics of probiotic yoghurts. Skim milk bases enriched with 1% of passion fruit ber or not wereheat 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 L10and NCFM and Bi dobacterium animalis subsp. lactis strains Bl04 and B94. Fermentations were performeduntil the pH reached 4.5. Rheological characteristics of yoghurts were determined by a rotational rheometerin two cycles of shear rate ranging from 0 to 15 s 1 in both upward and downward curves. Sensorial analysisof passion fruit ber yoghurts, either without any probiotic or co-fermented by L. acidophilus L10 orB. 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. Apparentviscosity was signi cantly higher in ber yoghurts co-fermented by the lactobacilli than in their controls atthe end of cold storage. Photomicrographs demonstrated that in passion fruit ber yoghurts the casein gelwas 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 indicatethat 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

Duringmilk fermentation in yoghurt manufacture, thepH decreasesas the lactic acid is produced by the starter culture Streptococcusthermophilus and Lactobacillus delbrueckii subsp. bulgaricus . Caseinbegins to aggregate at pH 4.7, isoelectric point, forming a fragile gelnet. At the endof fermentation,the gelof the set-typeyoghurt is usuallybroken to produce stirred yoghurt and the subsequent operations of mixing, pumping and packaging impact in its structure, decreasing theapparent viscosity. However, during thawing to approximately 20 Cand cold storage at 4 5 C, the stirred yoghurt recovers partially itsstructure 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 characteristicsand microstructure of yoghurt depend on many factors such as milk

base formulation, bacterial culture selection, production process,packaging and storage ( Tamime & Robinson, 2007 ). For the develop-ment of new fermented milks, the in uence of modi cations in themilk base on texture, rheology and sensorial properties of productshas been studied, concerning mainly the lipid content of milk ( DeLorenzi, Pricl, & Torriano, 1995; Esprito-Santo, Perego, Converti, &Oliveira, 2012; Folkenberg & Martens, 2003 ), the addition of proteinsto 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 die-tary ber (DF) contents ( Esprito-Santo et al., 2012; Garca-Prez etal., 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 ).

Speci c strains as well as composition of the bacterial culturesused in fermentation, especially those releasing exopolysaccharidesor combinations of the starter culture with one or more probiotics,also play an important role in the development of yoghurt structure

Food Research International 50 (2013) 224 231

Corresponding author. Tel.: +55 1130913690; fax: +55 1138156386.E-mail address: [email protected] (M.N. Oliveira).

0963-9969/$ see front matter 2012 Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.foodres.2012.09.012

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http://dx.doi.org/10.1016/j.foodres.2012.09.012http://dx.doi.org/10.1016/j.foodres.2012.09.012http://dx.doi.org/10.1016/j.foodres.2012.09.012mailto:[email protected]://dx.doi.org/10.1016/j.foodres.2012.09.012http://www.sciencedirect.com/science/journal/09639969http://www.sciencedirect.com/science/journal/09639969http://dx.doi.org/10.1016/j.foodres.2012.09.012mailto:[email protected]://dx.doi.org/10.1016/j.foodres.2012.09.012


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(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 fruitby-products rich in total DF has increased in recent years, being con-venient to their association with probiotic bacteria for the promotionof 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 insolu-ble fraction stimulates the intestinal peristalsis, the soluble one pro-motes the selective growth of the indigenous microbiota, acting as aprebiotic ( Sembries et al., 2003 ). Nawirska and Kwasniewska(2005) reported the importance of DF intake on a daily basis to pre-vent 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 isthe passion fruit peel, which, in addition to its functional propertiessuch 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 recentstudy of our group, the improvement in fatty acid pro le and increasein the conjugated linoleic acid content of probiotic yoghurts addedwith passion fruit peel ( Esprito Santo et al., 2012a ). Moreover,passion fruit peel ber enhanced the texture parameters of skimyoghurts during cold storage ( Esprito-Santo et al., 2012b ). Based onthis background, the present study aimed to evaluate some otherimportant aspects of the rheology, spontaneous whey separation,microstructure and sensorial characteristics of probiotic yoghurtsenriched with passion fruit ber.

2. Materials and methods

2.1. Preparation of passion fruit ber and determination of water and oilholding capacity

Passion fruit by-product was obtained from a fruit pulp manufac-turer, located in the city of Jundiai, So Paulo State, Brazil, andmaintained in freezer at 26 C until processing.

The peels of the passion fruit were dried in an oven (QuimisQ314M-293) at 60 C under air ow at 12 air changesmin 1 untilconstant weight. The dry peels were reduced to ne powder in aBimby processor, TM 31 (Vorwerk, Wuppertal, Germany). Fiber parti-cle size was standardized to less than 17.7 m, measured throughsieves (Granutest, So Paulo, Brazil), and ber powder was stored inglass 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) wasreconstituted to 12 g100 mL 1 in potable water ltered by a waterpuri er, 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) notenriched and designed as control. The milk bases were heat treated at85 C for 15 min in a bath thermostat (A100, Lauda, Knigshofen,Germany) under agitation at 300 rpm provided by steel two-bladepropeller connectedto an agitator (Q250M1(Quimis, Diadema, Brazil)).Afterwards, the milk bases for the analysis of rheology, syneresis andmicrostructure were divided into sterile glass asks (500 mL) andthose for the sensorial analysis were conditioned in sanitized whitepolypropylene containers (15 L), cooled in an ice bath and stored at

4 C for 24 h until inoculation.

2.3. Microbial cultures

The freeze-dried starter yoghurt culture (CY340, DSM, Moorebank,NSW, Australia) composed of Streptococcus thermophilus (St) and Lac-tobacillus delbrueckii subsp. bulgaricus (Lb) and two Lactobacillus aci-dophilus strains (LAFTI L10, DSM, and NCFM, HOWARU Dophilus,Danisco, Madison, WI, USA) and two Bi dobacterium animalis subsp.lactis strains (Bl04, Danisco and LAFTI B94, DSM) were used in this

study. The lyophilized cultures were diluted in 50 mL of sterilizedmilk inoculums, according to the recommendations of the manufac-turer. The amount (g) of the lyophilized cultures was previously deter-mined to reach counts around 6 Log CFU mL 1 of each microorganismin the inocula. The counts of probiotic bacteria increase during the fer-mentation of milk and are maintained above 9 Log CFU mL 1 during2 weeks of cold storage of the yoghurts, as previously reported byEsprito Santo et al. (2012a) and Esprito-Santo et al. (2012b) .

2.4. Experimental procedure

Four types of probiotic yoghurts were prepared according to theexperimental design presented in Table 1. For the rheology, syneresisand microstructure analysis, each yoghurt type was prepared in du-

plicate in two independent batch fermentations (N=4). The askscontaining 500 mL of the heat treated milk base were inoculatedwith 1 mL of yoghurt starter cultures and 1 mL of probiotic culture.Thereby, each milk ask had, before fermentation, an average countof 6.4 Log CFU mL 1 of St and 5.9 Log CFU mL 1 of Lb, and the pro-biotic counts were ~6.9 Log CFU mL 1 . Afterwards, the asks withthe samples were transferred to a water bath at 42 C and connectedto a CINAC (Cynetique d'acidi cation , Ysebaert, Frpillon, France)system ( Spinnler & Corrieu, 1989 ) which recorded the pH decrease.

At pH 4.5, fermentationwasstopped andthe asks were capped andcooled to 20 C inan ice bath. Then, the asks were transferred to a lam-inar owcabinet andthe coagulumwasbroken with regular upwardanddownward movements for 2 min with a sterile perforated disk on astainless steel rod. The coagulum of the yoghurts in the 15 L containers

was broken under agitation at 100 rpm by a sterile steel propellerconnectedto theagitator already describedin Section 2.2 . The stirred yo-ghurts were distributed into 50 mL polypropylene cups, thermallysealed and stored in refrigerator at 4 C.

2.5. Spontaneous whey separation

After 24 h of fermentation, four cups of each yoghurt type werecarefully homogenized (N=32) and an aliquot (8 mL) of yoghurtwas collected throughout a 10 mL (1:10) sterile plastic pipette(Sterilin,BarloworldScienti c, Stafforshire, UK), placed in a shelf pi-pette holder and maintained in a perpendicular position at 4 C. Thewhey formed in the upper phase of the yoghurt was read in the pi-pette scale. This procedure was repeated for yoghurts stored at 7

Table 1Experimental design to study the rheology, spontaneous whey separation, microstruc-ture and sensorial characteristics of probiotic yoghurts enriched with passion fruit

ber.

Yoghurt Probiotic Fiber

Control Lactobacillus acidophilus NCFM Control L. acidophilus L10 Control Bi dobacterium animalis subsp. lactis Bl04 Control B. animalis subsp. lactis B94 Passion fruit ber L. acidophilus NCFM +Passion fruit ber L. acidophilus L10 +Passion fruit ber B. animalis subsp. lactis Bl04 +Passion fruit ber B. animalis subsp. lactis B94 +

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

225 A.P. Esprito-Santo et al. / Food Research International 50 (2013) 224 231


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

2.6. Rheological measurements

Rheological measurements were carried out in quintuplicate foreach sample, at 5 C, using a rotational Rheometer, R20 (Haake-Rotovisco, Karlsruhe, Germany), with a cone and plate geometry

(50 mm diameter and 2 of inclination angle). About 1 mL of thesample was placed in the center of the stationary plate. The gapbetween the plates was 100 m. The controlled-temperature wasassured by a circulating water bath through the jacket surroundingthe rotor and cup assembly. Two cycles of shear rate ( ) rangingfrom 0 to 15 s 1 of upward and downward curves were performed,and the corresponding shear stress ( ) data computed by a RheoWinsoftware 2.97 (Haake Laboratories, Karlsruhe, Germany). This rangeof 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 down-ward ow curves of the rst and second cycles. The data obtainedwere tted by the Power Law model ( Heldman & Singh, 1981 ):

K 0 n

where is the shear stress, K the consistency index and n the PowerLaw index that describes the ow behavior of the uid as shear-thinning/pseudoplastic ( n b 1) or shear-thickening/dilatant ( n >1)(Fischer, Pollard, Erni, Marti, & Padar, 2009 ). A recent study of ourgroup demonstrated that major alterations in the texture pro le of PFF yoghurts occur during the rst two weeks of cold storage. Thus,the rheological characterization of the yoghurts was focused at days1, 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 at4 C, as described by Damin, Alcntara, Nunes, and Oliveira (2009) .Afterwards, the samples were stuck on stubs with double-face tapeand coated with 15 nm of a gold palladium layer applied by acathodic coater, E 5100 (Polaron, Hertfordshire, West Sussex, UK).Six elds of each sample were observed in a eld-emission scanningelectron microscope (SEM) (JSM-7401-F, JEOL, Akishima, Japan), op-erating at a voltage of 5.0 kV, and photomicrographs were registeredunder magni cations from 1000 to 10,000. Images were analyzedusing the software Image Pro Plus v.4.5.1 (Media Cybernetics, SilverSpring, MD, USA) as JPG les, and to three images at 10,000 of mag-ni cation of each sample were applied the highest contrast to high-light the pores in the casein network, while the areas of the poreswere marked and measured through a CINAG system of planimetry

(Centro de Informtica na Agricultura, Faculdade de CinciasAgronmicas, UNESP, Botucatu, Brazil) according to Brito andDeschamps (2001) , and expressed as area of porosity100 1 of areaof yoghurt's gel.

2.8. Sensorial analysis

For the sensorial analysis, were chosen passion fruit ber yoghurtsco-fermented by L. acidophilus L10 and B. animalis subsp. lactis Bl04,which showed the highest apparent viscosities ( app ) compared totheir respective controls. Natural control yoghurt and PFF yoghurtwithout probiotic were used to evaluate the in uence of PFF and pro-biotic strain, respectively, on the sensorial perception. In each session,four samples packed in white cups and identi ed with random

three-digit codes were served to the untrained assessors (170) in a

randomized order. The assessors were students and members of thestaff of the University of So Paulo, So Paulo, Brazil, and frequentconsumers of avored yoghurts and passion fruit. Between one sam-ple and the next they were instructed to cleanse their palates withcrackers and tap water. The attributes of appearance, color and odorwere scored out according to a hedonic scale (1 = dislike extremely,3 = dislike moderately, 5 = neither like nor dislike, 7 = like moder-ately, and 9= like extremely). The intensity of the passion fruit avor

was evaluated in a 9-point structured scale from very weak (on theleft) to very strong passion fruit avor (on the right). Analyses wereperformed on day 4 in four sessions being two in the morning andtwo in the afternoon. Microbiological safety of yoghurt samples wasexamined before sensory evaluation regarding yeasts and moldsusing Yeast and Mold Petri lm (3 M Company, St. Paul, MN, USA)incubated at 21 C, as well as Escherichia coli and total coliformsusing Coliform Petri lm (3 M Company) incubated at 37 C. Sensoryevaluation tests were approved by the Ethics Committee in Researchof 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 experimentaldata, and the means among different groups were compared by theTukey 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 fromthe protein gel is a useful approach to represent, particularly fromthe consumer point of view, the aspect of yoghurt duringcold storage,and the instability of the casein network in yoghurts can be evaluatedthrough the measurement of the spontaneous syneresis ( Lucey,2002 ).

As shown in Table 2, composition of co-cultures in yoghurtsenriched with passion fruit ber had no signi cant effect ( P >0.05)on whey separation, which was likely due to the fact that all probioticstrains tested produce exopolysaccharides with recognized water-holding capacity ( Barrangou et al., 2009; Laws & Marshall, 2001;Prasanna et al., 2012; Ruas-Madiedo, Gueimonde, Margolles,Losreyes-Gaviln, & Seppo, 2006 ). During cold storage, controlyoghurts produced more whey ( P b 0.05), varying from 4.2% to 5.1%,than passion fruit ber or PFF yoghurts (0.0 0.8%), especially thoseco-fermented by L. acidophilus NCFM (P b 0.05). Water and oil holdingcapacities of PFF were 3.8 mLg 1 and 4.9 gg 1 , respectively, whichare in accordance to the ndings of Yapo and Kof (2008) . Theseresults suggest that the water-holding capacity of pectin present inPFF reduced signi cantly the amount of whey formed in yoghurts

containing it. According to Everett and McLeod (2005) , pectin is, infact, constituted by anionic hydrocolloids that are classi ed asadsorbing polysaccharides, which interact with the casein networkincreasing its ability to entrap the whey phase.

The post-acidi cation is one of the factors that can increase theproduction of whey in yoghurts ( Lucey, 2002 ). As reported in a previ-ous study of our group ( Esprito-Santo et al., 2012b ), in this study theaddition of passion fruit ber decreased the pH (measured in pHmeter 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 yoghurtsit 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 PFFyoghurts was signi cantly lower than in their controls, whichreinforces the importance of the pectins in the PFF to preserve the

yoghurt structure.



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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 die-tary ber from passion fruit on the shear analysis performed using arotational rheometer as well as to shed light on the weak forces actingin the yoghurt structure, such as polysaccharide protein complexes(Fernndez-Garca & McGregor, 1997 ), that cannot be properlyevidenced at high shear rate.

The two cycles of shear rate in upward downward ow curvestted by Power Law model resulted in adequate correlation coef -

cients R 2 0.968, indicating that this model is more adequate to rep-resent the experimental data ( Table 3 ). The mean values of n were b 1,thus pointing out a pseudoplastic behavior of yoghurts, as alreadyreported ( Fischer et al., 2009; Tamime & Robinson, 2007 ).

In most of the cases, the consistency index of the rst cycle ( K 1 )increased signi cantly during storage ( P b 0.05), varying from 8.10to 14.02 Pas 1 in control yoghurts and being signi cantly higherin all passion fruit ber yoghurts (13.16 K 1 23.39 Pas 1)(Table 3). Strengthening of protein protein complexes forming yo-ghurt structure may have been responsible for the increase in K 1 dur-ing the two weeks of storage ( Damin et al., 2008; Rawson & Marshall,1997 ). However, in the second cycle, the mean values of K 2 decreased,whereas n 2 increased, but no signi cant differences were noticed be-tween PFF yoghurts and their respective controls, except in thoseco-fermented by B. lactis Bl04, where K 2 was signi cantly higher inthe presence of ber.

Thixotropy was measured separately in the two cycles as the areabetween the upward and downward slopes of shear rate, whichrepresents the ability of samples to recover their structure duringthe decrease of (Fig. 1). Considering the whole period of storage,thixotropy in the rst cycle was signi cantly higher in all PFF yo-ghurts (24.0 30.1%) than in their respective controls (14.0 22.0%).

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, beinggenerally higher in yoghurts supplemented with PFF ( Fig. 1).

In the rst cycle of shear rate, the apparent viscosity ( app ) variedfrom 41.5 to 62.7 Pas in control yoghurts and from 44.3 to 91.6 Pasin PFF yoghurts during the storage period ( Fig. 2). All PFF yoghurts in-creased signi cantly their app ; however, in general this pattern wasnot followed by the control yoghurts ( Fig. 2), indicating that thestructure of PFF yoghurts was possibly stabilized by positive interac-tions between pectins from the fruit and exopolysaccharides pro-duced by the bacteria. On the other hand, in the second cycle of shear rate, PFF and control yoghurts showed practically the samemean values of app , which varied from 8.1 to 16.3 Pas in control yo-ghurts and from 10.2 to 18.4 Pas in PFF yoghurts, respectively(Fig. 2). The second cycle of shear rate reduced signi cantly app inall yoghurts. However, since PFF yoghurts presented the highest app in the rst cycle, it is evident, especially after 14 days of coldstorage, that they suffered greater damage in their structure thanthe controls, probably because the bers acted as knives, breakingthe protein protein interactions during tests, which also reducedthe thixotropy in the second cycle ( Figs. 1, 2 and 3).

The decrease in K values ( Table 3), combined to the partial struc-ture recovery between the two cycles of shear rate ( Fig. 1), indicatesthat both control and PFF yoghurts presented shear rate-thinning andthixotropic properties, which are typical of yoghurts ( McCann et al.,2011; Rawson & Marshall, 1997; Staffolo et al., 2004 ). Moreover, dif-ferent rheological behaviors of control and PFF yoghurts may havebeen the result of positive interaction of pectin present in PFF withboth protein and exopolysaccharides, hence helping the yoghurts topartially 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 positivecharges on the surface of casein micelles, promoting strengthening

Table 2Spontaneous 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)

Area of pores (area of porosity100 1

of area of yoghurt gel)

Day 1 Day 7 Day 14

Lactobacillus acidophilus NCFM Control 5.0 (0.0)e 5.1 (0.2) e 5.1 (0.1) f 30.5 (2.8) d

L. acidophilus L10 Control 4.2 (0.15) c 4.6 (0.15) d 4.6 (0.15) e 33.6 (3.1) ef

Bi dobacterium animalis subsp. lactis Bl04 Control 4.2 (0.15) c 4.2 (0.15) c 4.2 (0.15) d 30.9 (2.7) d

B. animalis subsp. lactis B94 Control 4.6 (0.15) d 4.6 (0.15) d 4.6 (0.15) e 31.2 (3.0) de

L. acidophilus NCFM Passion fruit ber 0.4 (0.1) b 0.8 (0.1) b 0.8 (0.1) c 24.7 (1.5) bc

L. acidophilus L10 Passion fruit ber 0.0 (0.0) a 0.0 (0.0) a 0.0 (0.1) a 22.3 (1.2) a

B. animalis subsp. lactis Bl04 Passion fruit ber 0.0 (0.0) a 0.0 (0.0) a 0.0 (0.1) a 22.1 (1.2) a

B. animalis subsp. lactis B94 Passion fruit ber 0.4 (0.1) b 0.4 (0.1) b 0.4 (0.1) b 23.6 (1.4) ab

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

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

Probiotic Treatment Day 1 Day 7 Day 14

K1(Pa s 1)

n1 2(Pa s 1)

n2 K1(Pa s 1)

n1 2(Pa s 1)

n2 K1(Pa s 1)

n1 2(Pa s 1)

n2

Lactobacillus acidophilus NCFM Control 12.15lm 0.15bcd 5.01cd 0.43fgh 12.40 lm 0.10abcd 5.25cdef 0.39efg 14.02 no 0.09abc 5.68efg 0.37ef

L. acidophilus L10 Control 8.10hi 0.30e 3.52a 0.56 j 10.75 k 0.17cd 4.85bc 0.46ghi 11.21 kl 0.14bcd 4.87bc 0.43fgh

Bi dobacterium animalis subsp.lactis Bl04

Control 11.49 k 0.13bcd 4.20ab 0.49ghij 11.51 klm 0.24de 5.34e 0.38defg 12.92 lmn 0.11 abcd 5.90cd 0.38defg

B. animalis subsp. lactis B94 Control 9.21 j 0.20cde 3.88a 0.51hij 11.31 kl 0.15bcd 5.21cde 0.41fg 12.91 lmn 0.08ab 5.52ef 0.36ef

L. acidophilus NCFM Passion fruit ber 15.80 p 0.08ab 4.48bc 0.50hij 18.98 rst 0.09abc 5.83 fg 0.43fgh 23.39 x 0.06a 5.57ef 0.43fgh

L. acidophilus L10 Passion fruit ber 13.83 mno 0.17cd 4.11 ab 0.55 j 18.11 rs 0.08ab 4.86bc 0.48ghi 18.33 rs 0.09abc 5.30cde 0.46ghi

B. animalis subsp. lactis Bl04 Passion fruit ber 19.02 tw 0.09abc 4.70bc 0.49ghij 20.64st 0.07 a 5.58efg 0.44gh 21.15 t 0.07a 6.15 fg 0.39efg

B. animalis subsp. lactis B94 Passion fruit ber 13.16 mn 0.08ab 3.78a 0.52 ij 16.47 pq 0.09abc 4.96bcd 0.51hij 17.87 r 0.08ab 5.03cde 0.45ghi

Means (N=40) with different superscript letters in the same column differ signi cantly ( P b 0.05). Abbreviations: K 1 and K 2 are the consistency index of upward curves of the rst

and the second cycles, respectively; n1 and n 2 are the Power Law index that indicates the ow behavior in upward curves of the rst and the second cycle, respectively.



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of casein network and stabilization of yoghurt structure. Even though,pectin protein interactions are weaker than the protein protein onesin yoghurt structure ( Fernndez-Garca & McGregor, 1997 ), theselinkages may have been unable to hold up the second cycle of shearrate ( Table 3 and Fig. 1). Rheological data are consistent with the tex-ture attributes of a previous work ( Esprito-Santo et al., 2012b ), thatreported higher rmness, consistency and cohesiveness of PFF skimyoghurts than their respective controls. Sendra et al. (2010) observedthat the addition of orange ber at doses lower than 0.6 g100 mL 1

weakened the yoghurt's structure, but at higher amounts, water ab-sorption counterbalanced this negative effect and strengthened the

casein network. Similar behavior may have occurred in this studywhen PFF was added to yoghurts.

3.3. Microstructure

SEM micrographs of control andenriched yoghurts co-fermentedbyB. lactis subsp. animalis Bl04 are presented in Fig. 3 showing signi cantdifferences. A normal structure of non-fat yoghurt with a casein net-worksurrounded by a high number pores of whey could be seen as de-scribedbymanyauthors ( Damin et al.,2009;Marafon,Sumi, Granato, etal.,2011;Ozcan-Yilsayet al.,2007;Sodiniet al., 2005 ). Measurementsof

0

5

10

15

20

25

30

35

NCFM L10 Bl04 B94 NCFM L10 Bl04 B94 NCFM L10 Bl04 B940

5

10

15

20

25

30

35

NCFM L10 Bl04 B94 NCFM L10 Bl04 B94NCFM L10 Bl04 B94

T h i x o

t r o p y

i n t h e s e c o n

d c y c l e

( % )

T h i x o

t r o p y

i n t h e f i r s

t c y c l e

( % )

aab baba ab ab

ab ababababbc bc

bccdcd cdcde

cde cde

bc

cde dedef

gghghi

hihihijhij ij ijijk

kllm

mnmnmno mnono

nonopnop

opqpq pqopq

d1 d7 d14 d1 d7 d14

Fig. 1. Thixotropy of control and passion fruit ber yoghurts. Means (N=40) with different letters are signi cantly different ( P b

0.05). Abbreviations: NCFM and L10: yoghurtsco-fermented by Lactobacillus acidophilus NCFM and L10, respectively; Bl04 and B94: yoghurts co-fermented by Bi dobacterium 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.

Fig. 2. Apparent viscosity ( app ) at =2 s 1 of control and passion fruit ber yoghurts in the two cycles of upward downward curves of shear rate. Means (N=40) with different

letters are signi cantly different ( P b 0.05). Abbreviations: NCFM and L10: yoghurts co-fermented by Lactobacillus acidophilus NCFM and L10, respectively; Bl04 and B94: yoghurtsco-fermented by Bi dobacterium animalis subsp. lactis Bl04 and B94, respectively. d1,d7 andd14:samples takenafter 1, 7 and14 daysof cold storage afterfermentation. Control

Passion fruit ber.

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the areaof pores ( Table 2 ) point outthatPFFyoghurts hadhigher densityof the casein network than their respective controls ( P b 0.05), consistentwith a lower number of pores and a higher number of cross-links be-tween strands ( Fig. 3a compared to Fig. 3b). In control yoghurts, butnot in PFF yoghurts, exopolysaccharides formed several laments andcovered some areas of the protein gel ( Fig. 3b). Also this observationcan be explained by the presence of pectin in PFF, which may haveinteracted with proteins and bacterial exopolysaccharides forming a

more compact structurewithgreater apparentviscosityand ability to en-trap the whey phase ( Fig. 2, Table 2). The passion fruit ber presented aneedle-shaped blade and was clearly observed in the micrographsnesting the casein gel and, sometimes, the yoghurt bacteria ( Fig. 3c).

3.4. Sensorial characteristics

Results of hedonic tests summarized in Table 4 emphasize that thefour types of yoghurt assayed had no signi cant differences amongthem ( P >0.05) in the scores of appearance and color, which variedfrom 6.6 to 7.0 (like moderately), in spite of the evident light yellow-ish color of PFF yoghurts.

However, natural yoghurts had higher scores ( P b 0.05) for texturethan the PFF ones, which were scored as having a sandy or graininess

mouth feeling by some of the assessors, even though the particle sizeof bers was less than 17.7 m. This result can be ascribed not only tothe amount or size of PFF in yoghurts but also to the shape of bers,which had edges like stones ( Fig. 3c) capable of sensitizing themouth more than if they had spherical and smooth shape. PFF yo-ghurts with or without probiotic bacteria received higher meanscores for odor (neither like nor dislike) than the natural ones (dislikemoderately) ( Table 4). Nonetheless, the passion fruit avor was moreintense ( 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 inPFF yoghurt without probiotics (mean score= 2.1). In a previousstudy of our group, the pH and total titratable acidity of PFF yoghurtswere not signi cantly different from their respective controls(Esprito-Santo et al., 2012b ). Thus, the enhancement of the passionfruit avor observed in PFF yoghurts co-fermented by L. acidophilusand B. animalis subsp. lactis is unlikely due to the post-acidi cationduring storage. Therefore, these probiotic bacteria may have pro-duced or transformed compounds that reinforce the avor of thePassi ora edulis peels which is due, mainly, to esters, 3-methyl-thiohexan-1-ol, 2-methyl-4-propyl-1, 3-oxathione enantiomers andedulans I and II ( Dhawan, Dhawan, & Sharma, 2004 ). However, con-sidering only the probiotic yoghurts, the strain of probiotic bacteriahad no signi cant effect on odor and passion fruit avor perceivedby the panelists. Similarly, Sendra et al. (2008) observed that probiot-ic bacteria used in yoghurts supplemented with citrus ber had no in-

uence 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 reportedthat the Brazilian yoghurt market is dominated by fruit- avored yo-ghurt (about 95% of the market), and the colorful and sweet yoghurtsare those preferred by the consumers. Therefore, some sensory prop-erties of the natural yoghurt, such as appearance, color, smell and

Table 4Scores of the sensory evaluation of passion fruit ber yoghurts.

Yoghurt type Appearance Color Texture Odor Intensity of the passion fruit avor

Control natural skim yoghurt 7.03 (1.51) a 6.95 (1.59) a 7.11 (1.98) b 2.27 (1.04) a 1.25 (0.82) a

PFF yoghurt 6.83 (1.44) a 6.68 (1.39) a 5.52 (1.91) a 5.35 (1.46) b 2.41 (1.83) b

PFF yoghurt co-fermented by Lactobacillus acidophilus L10 6.84 (1.45) a 6.67 (1.41) a 5.72 (1.94) a 5.67 (1.65) b 3.17 (2.02) c

PFF yoghurt co-fermented by Bi dobacterium animalis subsp. lactis Bl04 6.74 (1.33)a 6.68 (1.35) a 5.66 (1.74) a 5.62 (1.56) b 3.29 (2.39) c

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

0.05).

a

b

eps

fb

c

Fig. 3. Microstructure of yoghurts co-fermented by Bi dobacterium lactis subsp. animalisBl04 at 10,000 magni cation of (a1) passion fruit ber yoghurt, (a2) passion fruit beryoghurt shown with the highest contrast, (b) control yoghurt, and (c) passion fruit beryoghurt. Bar=1 m. Abbreviations: eps = laments of exopolysaccharides ; fb =passion fruit ber.



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even the intensity of passion fruit avor may have been under-estimated or confused by the assessors, not accustomed to itscharacteristics.

Fernndez-Garca and McGregor (1997) reported that the supple-mentation of yoghurt with oat ber reduced the score for texture, butthe authors refereed to the healthy bene ts of the new product to justify its use. Tuorila and Cardello (2002) observed that consumersare, 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 weakor very weak passion fruit avor, and a minimum, but signi cant, in-

uence on texture, which are important features for the developmentof new functional foods, as well as broader possibilities in the formu-lation of avors.

4. Conclusions

Enriching probiotic yoghurt with passion fruit ber or PFF offereda scaffold that strengthened the yoghurt's structure, and increases ap-parent viscosity during storage. In contrast, these bers also act ascutting blades during shearing, breaking the casein cross-links anddropping down the apparent viscosity of yoghurts.

The two cycles at low shear rate were effective to discriminate therheological behavior of control and enriched PFF yoghurts, minimiz-ing the destructive effect of the analysis during rheological tests ina rotational rheometer.

SEM micrographs showed that in PFF yoghurts the casein gel over-laid 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 wereconsidered neutral or good, and the intensity of the passion fruit a-vor 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 designingof 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. Bonafrom 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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passion fruit fiber

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