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Food Hydrocolloids 39 (2014) 204e214

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Food Hydrocolloids

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Structure development and acidification kinetics in fermented milkcontaining oat b-glucan, a yogurt culture and a probiotic strain

Athina Lazaridou*, Amalia Serafeimidou, Costas G. Biliaderis, Thomas Moschakis,Nikolaos TzanetakisDepartment of Food Science and Technology, School of Agriculture, Aristotle University of Thessaloniki, P.O. Box 235, Thessaloniki GR-541 24, Greece

a r t i c l e i n f o

Article history:Received 29 June 2013Accepted 16 January 2014

Keywords:Oat b-glucanProbioticsYogurt starter cultureMilk gelsRheologyFermentation

* Corresponding author. Tel.: þ30 2310 991671; faxE-mail address: athlazar@agro.auth.gr (A. Lazarido

0268-005X/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodhyd.2014.01.015

a b s t r a c t

The impact of oat b-glucan fortification (1.4%) on the acidification and gelation kinetics of fermentedskimmed milk (12% total solids) as well as the effects of inclusion of a probiotic strain (Lactobacillusparacasei subsp. paracasei B117) into the yogurt starter culture (Lactobacillus delbrueckii subsp. bulgaricusY 6.15 and Streptococcus thermophilus Y 4.10) and fermentation temperature (36, 38 and 40 �C) have beenexplored by dynamic rheometry, pH measurements and microbial enumeration. The viability/growth ofthe mixed culture bacteria was also evaluated upon storage. Incorporation of b-glucan to milk resulted inphase separation between proteins and the added polysaccharide, altering the entire gelation process asmanifested by the development of a transient gel structure at the early stages, followed by significant(P < 0.05) retardation of both protein aggregation and acidification kinetics, and finally the formation ofsignificantly weaker gels compared to control formulations. Furthermore, fortification of milk with b-glucan led to a liquid-like structure (tan d > 1) at the end of fermentation (pH ¼ 4.6) when performed at36 �C. A higher fermentation temperature accelerated the acidification kinetics, yielding a shortergelation time, but the storage modulus values of milk gels decreased. Inclusion of the probiotic strain intothe yogurt starter culture seemed to increase the gelation rate without affecting the gelation time and gelstrength. Viability of all three culture bacteria was enhanced by decreasing fermentation temperatureand during storage. Moreover, the L. paracasei showed good compatibility with the yogurt starter cultureand the addition of b-glucan enhanced the viability of the probiotic strain in the fermented productsthroughout cold storage (4 �C).

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1. Introduction

The (1 / 3), (1 / 4) b-D-glucans (b-glucans) are cell wallpolysaccharides of cereal grains, mostly found in oat and barley andto a lesser extent in rye and wheat. Cereal b-glucans are linearhomopolysaccharides of consecutively linked (1 / 4)-b-D-glucosylresidues in oligomeric segments that are separated by single(1 / 3)-linkages along the polymer chain backbone; i.e., theirstructure consists mostly of cellotriosyl and cellotetraosyl oligo-mers interlinked via b-(1 / 3) glycosidic linkages (Lazaridou,Biliaderis, Micha-Screttas, & Steele, 2004). The potential use ofthis neutral polysaccharide as a food hydrocolloid has been pro-posed based on its rheological characteristics; i.e., viscosityenhancement and gelling properties. Furthermore, b-glucans,mostly extracted from oat and barley, exhibit all the health benefitsof soluble dietary fibers including the reduction of blood serum

: þ30 2310 991632.u).

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cholesterol and the regulation of post-prandial blood glucose levels(Lazaridou et al., 2004; Wood, 2002; Wolever et al., 2010). Besidesconcentration, the molecular/structural features of cereal b-glu-cans, such as molecular weight and ratio of tri- to tetra- cellulosicoligomers determine their rheological behavior, which consecu-tively regulates the technological and nutritional functionality ofthese indigestible polysaccharides (Lazaridou & Biliaderis, 2007).

In 1997, the US Food and Drug Administration (US FDA, 1997)approved a health claim for the reduction of heart disease riskwith adaily consumption of 3 g of b-glucan (a minimum of 0.75 g perserving) originating from oat-formulated products that include oatbran, rolled oats and whole oat flour. FDA has subsequently‘approved’ an additional eligible source of oat b-glucan soluble fiber,the oatrim, which is a soluble fraction of a-amylase-hydrolyzed oatbran or whole oat flour with up to 10% b-glucan content (US FDA,2002). Recently, the European Food Safety Authority (EFSA) hasalso authorized health claims, according to which oat b-glucaningestion leads to the reduction of blood plasma cholesterol con-centrations, which is a major risk factor for the development of

A. Lazaridou et al. / Food Hydrocolloids 39 (2014) 204e214 205

coronary heart disease (EFSA, 2010); the recommended daily intakewas 3 g of oat b-glucan and the claim can be used for food products(portion) containing at least 1 g. Another claimwas also authorizedby EFSA concerning the reduction in post-prandial glycemic re-sponses, at doses of about 4 g per 30 g of available carbohydrates inbread and pasta products. Further to the hypocholesterolemic andhypoglycemic properties, some evidence exists for a prebiotic actionof cereal b-glucans on some Lactobacillus species (Dongowski, Huth,Gebhardt, & Flamme, 2002; Snart et al., 2006). Fermented dairyproducts could become effective delivery systems for cereal b-glu-cans. Previous studies suggest that incorporationof oat andbarleyb-glucans at low levels, up to 0.5%, into fermentedmilk products haveno adverse effects on growth of yogurt starter culture and evenimproved the probiotic viability of some bifidobacteria strainsduring cold storage (Gee, Vasanthan, & Temelli, 2007; Rosburg,Boylston, & White, 2010; Vasiljevic, Kealy, & Mishra, 2007).

On the other hand, incorporation of purified cereal b-glucansinto milk or milk protein derived gels, via acidification with glu-cono-d-lactone (GDL) or by bacteria fermentation, showed thattheir physical and textural attributes can be largely modified due tophase separation between these polysaccharides and milk proteins(Brennan & Tudorica, 2008; Kontogiorgos, Ritzoulis, Biliaderis, &Kasapis, 2006; Lazaridou, Vaikousi, & Biliaderis, 2008; Vasiljevicet al., 2007). These structural modifications, caused bybiopolymer demixing, seem to be governed by the concentrationand the molecular/structural features of the added polysaccharide(Corredig, Sharafbafi, & Kristo, 2011; Kontogiorgos, Tosh, & Wood,2009; Kontogiorgos et al., 2006; Lazaridou & Biliaderis, 2009;Lazaridou et al., 2008; Repin, Scanlon, & Fulcher, 2012).

The yogurt starter culture strains of Streptococcus thermophilus Y4.10 and Lactobacillus bulgaricus Y 6.15 and the probiotic Lactoba-cillus paracasei subsp. paracasei B117 have been previouslyemployed in our laboratory to evaluate the effect of fermentationtemperature, milk total solids content and inoculum level on thegelationeacidification kinetics, and microbial growth in fermentedmilk products by mixed cultures (Kristo, Biliaderis, & Tzanetakis,2003a, 2003b). The same strains were also used in the presentwork, for producing fermented set yogurt-like milk products withthe aim to explore the individual and interaction effects amongthree factors, oat b-glucan (at 1.4%), fermentation temperature andthe probiotic strain (L. paracasei B117), on the rheological behavior,acidification kinetics, and microbial culture viability. To ourknowledge there are no studies on the impact of cereal b-glucanson acidification, gelation and microbiological properties of milkproducts fermented by yogurt starter culture bacteria and/or pro-biotic strains, particularly when these soluble fibers are added atrelatively high concentration, sufficient to fulfill the health claimrequirements for hypocholesterolemic action.

2. Materials and methods

2.1. Microbial cultures

Yogurt starter culture bacteria of S. thermophilus (S. thermophilus)Y 4.10 and Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) Y6.15 as well as the probiotic strain L. paracasei subsp. paracasei(L. paracasei) B117 were obtained from the microbial collection ofthe FoodMicrobiology and Hygiene Laboratory, Aristotle Universityof Thessaloniki, Greece. Allmicrobial strains havebeen isolated fromtraditional Greek yogurts and goat cheese. Skim milk powder(Regilait UCA, Sr. Martin, Belle-Roche, France), an instant productpurchased from a local retail market, was found to contain 34.7%(w/w) protein, 50.5% (w/w) lactose and 0.8% (w/w) fat.

The cultures were kept at �80 �C in a mixture of MRS broth(Oxoid Ltd, UK) and glycerol (Riedel-de-Haen GMbH, Germany)

(70:30) for lactobacilli, and in a mixture of M17 broth (Oxoid) andglycerol (70:30) for S. thermophilus. The conditions of propagationand maintenance of the cultures, as well as the preparation pro-tocol before their use in the fermented milks are described in detailby Kristo et al. (2003a, 2003b).

2.2. Isolation and molecular characterization of oat b-glucan

A b-glucan isolate was obtained by further purification of anoatmeal b-glucan concentrate (OATLY, Lund, Sweden). The purifi-cation protocol involved aqueous extraction of the polysaccharide(45 �C � 1 h) using a slurry of the oatmeal concentrate (1:30 sol-ids:liquid), followed by enzyme digestion (90 �C� 3 h, pH 4.5) witha preheated (90 �C � 30 min to eliminate any b-glucanase sideactivity) thermostable a-amylase (Termamyl 120L, Novozymes A/S,Bagsvaerd, Denmark) added to the slurry at 1% (v/v) to hydrolyzethe co-solubilized starch. The slurry was stored at 25 �C for 18 h (pH4.5) and then centrifuged (2800� g for 20min) for removal of someproteins. Following neutralization and concentration (under vac-uum, 80 �C) of the supernatant, the b-glucan was precipitated withtwo volumes of ethanol (4 �C � 18 h). Finally, the polysaccharidewas re-suspended in 2-propanol (25 �C � 18 h), filtered and dried(35 �C � 18 h) to obtain the b-glucan isolate; this material due tothe solvent exchange process exhibited high water solubility. Thepurity of this preparation (70% b-glucan d.b.) as well as the b-glucancontent of the initial oatmeal concentrate (19% d.b.) were assessedby the mixed-linkage (1 / 3), (1 / 4) b-D-glucan assay kit, ob-tained from Megazyme International Ltd (Bray, Ireland).

The apparent peak molecular weight (Mwp) of the purifiedb-glucan was found 117 � 103 Da, using a high performance sizeexclusion chromatography (HPSEC) system combined with arefractive index (RI) detector; details of the chromatographicmethod are given elsewhere (Lazaridou et al., 2004). The b-glucanmolecular weight markers employed for calibration of the systemwere isolated in our laboratory (Lazaridou et al., 2004) and theirmolecular size was determined with a light scattering technique(Skendi, Biliaderis, Lazaridou, & Izydorczyk, 2003).

2.3. Preparation of fermented milk products

Fermented milk products were prepared with reconstitutedskim milk alone or fortified with the oat b-glucan isolate using thestandard yogurt starter culture (ST), comprising of L. bulgaricus andS. thermophilus, with or without inclusion of the L. paracasei pro-biotic strain (PR). Milk products were fortified with 2% of the oat b-glucan isolate; i.e., the b-glucan content was 1.4% in the finalproduct. This b-glucan level aimed at meeting the health claimrequirements for reduction of serum cholesterol that could beaccomplished by a daily ingestion of at least 220 g of fermentedproduct which provides more than 3 g of b-glucan. The milkproducts were prepared according to the process of set-yogurtproduction; fermentation was carried out at three different tem-peratures, 36, 38 and 40 �C.

For milk products made without b-glucan, the skim milk pow-der (12% w/v) was dissolved in distilled water. For the b-glucanfortified products, the proper amount of b-glucan isolate was firstlydissolved in distilled water at 80 �C under stirring, followed bycooling to 35e40 �C and addition of the skimmilk powder. Portionsof 100 ml from all milk dispersions were placed in previouslysterilized (121 �C � 15 min) glass bottles with screw caps, and thedispersions were heat-treated at 90 �C for 15 min, cooled andstored at 4e5 �C; inoculation with the culture was carried outunder aseptic conditions at 25 �C. In formulations where only theyogurt starter culture was used, S. thermophilus and L. bulgaricuswere added in a ratio of 1:1 (v/v), whereas for products containing

Table 1Analysis of variance and significance (P values) of the general linear model effects for the gelation, acidification and microbiological parameters of fermented milk products.

Factors P Values

Gelation parametersf Acidification parametersf Microbiological parameters

G0max tan dmin IE Gt pH5h Vm Tm Te S. thermophilus L. bulgaricus L. paracasei

Culture (CU)a 0.377 0.388 0.002 0.877 0.001 0.554 0.018 0.513 <0.001 <0.001 e

b-Glucan (BG)b <0.001 <0.001 <0.001 <0.001 <0.001 0.003 0.001 <0.001 <0.001 0.466 <0.001Fermentation

temperature (FT)c<0.001 0.026 0.195 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Storage time (ST)d ee e e e e e e e <0.001 <0.001 <0.001CU � BG 0.325 0.381 0.869 0.013 0.003 0.009 0.380 0.019 0.007 <0.001 e

CU � FT 0.001 0.184 <0.001 0.064 0.239 0.161 0.079 0.010 0.115 0.027 e

BG � FT <0.001 0.023 0.455 0.009 0.266 0.002 0.115 0.001 0.001 0.424 0.033CU � BG � FT 0.001 0.185 <0.001 0.003 0.002 0.958 0.387 0.120 0.211 0.010 e

CU � ST e e e e e e e e <0.001 0.383 e

BG � ST e e e e e e e e 0.031 0.023 0.001CU � BG � ST e e e e e e e e 0.004 0.051 e

FT x ST e e e e e e e e 0.066 0.031 0.177CU � FT � ST e e e e e e e e 0.203 0.251 e

BG � FT � ST e e e e e e e e <0.001 0.095 0.106CU � BG � FT � ST e e e e e e e e 0.001 0.231

a Factor was examined at two levels: (1) milk fermented by yogurt starter culture without the probiotic strain and (2) milk fermented by yogurt starter culture and theprobiotic strain.

b Factor was examined at two levels: (1) milk without b-glucan supplementation and (2) milk with b-glucan supplementation.c Factor was examined at three levels: (1) milk fermented at 36 �C, (2) milk fermented at 38 �C and (3) milk fermented at 40 �C.d Factor was examined at three levels: (1) fermented milk product at the end of fermentation (pH ¼ 4.6), (2) fermented milk product stored at 4 �C for 7 days and (3)

fermented milk product stored at 4 �C for 14 days.e Factor or interactions were not examined.f G0

max: Maximum of G0 value; tan dmin :Minimum tan d value; IE: [(d log G0)/dt]max; Gt: Gelation time (G0 ¼ G00); Vm:Maximum acidification rate; Tm: Time to reachmaximumacidification rate; Te : Time to reach a pH 4.6; pH5h : pH value after 5 h fermentation.

A. Lazaridou et al. / Food Hydrocolloids 39 (2014) 204e214206

the probiotic strain, S. thermophilus, L. bulgaricus and L. paracaseiwere utilized at a ratio of 1:1:1 (v/v), at a total inoculum concen-tration of 3% (v/v) in both cases. Three bottles of milk (100 ml each)were prepared and used as follows: one for continuous monitoringof pH during fermentation, one for transferring its content into therheometer to follow the rheological responses and one for probingthe changes in viable counts of the culture. All treatments wereconducted at least in triplicates.

2.4. Rheological measurements

The gelation of milk formulations was monitored in a concentriccylinder geometry (cup and bob diameters, 28.92 and 26.66 mm,respectively) of a Physica MCR 300 Rheometer (Physica Mes-stechnic GmbH, Stuttgart, Germany) by applying dynamic oscilla-tory testing. The measuring fixture in the rheometer was set at thedesired temperature (36 or 38 or 40 �C). Milk inoculated with theculture was immediately poured into the cup of the rheometer anda thin layer of paraffin oil was added at the top of the dispersion toprevent evaporation. Storage and loss moduli (G0, G00), and losstangent (tan d ¼ G00/G0), were monitored at a fixed frequency (1 Hz)and strain (1%) throughout the time course of gel formation (atconstant temperature), until plateau values for G0 and tan d werereached.

2.5. Acidification activity measurements

Changes in pH during fermentation were monitored continu-ously by means of a glass electrode pH meter (Hanna Instruments,Padova, Italy) according to the method of Spinnler and Corrieu(1989). The electrode was disinfected with 70% (v/v) ethanol so-lution, rinsed with sterile distilled water and placed in the milkbottles under aseptic conditions after inoculation of milk with theculture. Fermentation was carried out in a circulating water bath(�0.2 �C) preset at the desired temperature and the pH was auto-matically recorded in 15-min intervals.

2.6. Microbiological analysis

Following milk inoculation with the predetermined culture, thebottleswere incubatedat the required temperature until a pH4.6wasreached, fast cooled by immersing them into ice water and subse-quently stored at 4 �C. Enumeration of bacteria, using the pour platetechnique, was carried out at the end of fermentation (when a pH 4.6was reached) and after 7 and 14 days storage of the products at 4 �C.Plates containing30e300 colonieswere enumerated and recorded ascolony forming units per milliliter of culture (cfu mL�1). The specificsubstrates and conditions of incubation used for enumeration of thethree different bacteria are described elsewhere (Kristo et al., 2003b).

2.7. Statistical analysis

A factorial experimental design was employed in a completelyrandomized way. For the gelation and acidification parameters athree factor (2 cultures � 2 b-glucan � 3 fermentation tempera-tures) experiment was designed, whereas for the bacterial countsthe effects of four (2 cultures � 2 b-glucan � 3 fermentationtemperatures � 3 storage times) factors were explored. The levelsof each factor and the interactions among factors are shown inTable 1. The data were analyzed by ANOVA according to the generallinear model using the SPSS statistical software (version 16.0 SPSSInc., Chicago, IL), and differences betweenmeans were identified bythe least square difference (LSD) test (a ¼ 0.05 significance level).The significance (P values) of the factor effects and their in-teractions for the gelation, acidification and microbiological pa-rameters were calculated by the general linear model.

3. Results and discussion

3.1. Effect of oat b-glucan on gel structure formation of fermentedmilk products

The evolution of G0, tan d and pH of a representative fermentedmilk preparation supplemented with oat b-glucan and its

A. Lazaridou et al. / Food Hydrocolloids 39 (2014) 204e214 207

respective control formulation (product without b-glucan) areshown in Fig. 1a. All control products gave the characteristic gela-tion kinetic profile of microbially acidified heat-treated milk(Biliaderis, Khan, & Blank,1992; Haque, Richardson, &Morris, 2001;Kristo, Miao, & Corredig, 2011; Lucey, Tamehana, Singh, & Munro,1998). At the beginning of the process, the G0 value was low(w0.01 Pa) and tan d > 10, indicative of liquid-like behavior, fol-lowed by an abrupt increase of the G0 and decrease of tan d to avalue lower than 1, implying the establishment of a three-dimensional gel structure (Fig. 1a). The turning point of G0 curvewas evidenced at pH around 5.4 (Fig. 1a) and concurred with asharp peak in the d log G0/dt vs. time curve, named elasticityincrement (IE) (Fig. 1b); this parameter is a measure of the gelationrate (i.e., a high IE value indicates rapid gelation; Böhm & Kulicke,1999). For the non-fortified with b-glucan products the gelationtime (Gt), i.e., the time at which tan d¼ 1, was reached at a pH valuearound 5.3 which is in agreement with previous reports on gelationand acidification kinetics of fermented heat-treated milk gels(Azim, Corredig, Koxholt, & Alexander, 2010; Hassan, Corredig, &Frank, 2002; Kristo et al., 2011; Lee & Lucey, 2006). It has beenpreviously reported that preheating of milk shifts the pH of gelationfrom 4.9e5.1 to 5.3e5.5 due to aggregation of denaturated wheyproteins and their association with the casein micelles that rendersproteins more susceptible to aggregation as their net repulsivecharge is decreased (Corredig et al., 2011; Guyomarc’h, Queguiner,Law, Horne, & Dalgleish, 2003; Lucey, 2002; Lucey & Singh, 1998).

In the early stages of the gelation process of control milkpreparations, a ‘local maximum tan d’ (tan dpeak) was observed atpH 4.8e4.9 before tan d decreases again to a minimum (tan dmin),where the ultimate gel structure is established (Fig.1a). This peak intan d has been attributed to a loosening of the gel network structurethrough solubilization of colloidal calcium phosphate, resulting in apartial micellar disintegration, and/or to transition of the milk gelfrom a network dominated by denaturated whey proteinecaseinassociations at a high pH to a network structure dominated bycaseinecasein interactions at a lower pH (Azim et al., 2010;

Fig. 1. Time and pH dependence of G0 and tan d (a), and time dependence of d log G0/dt (b) f(ST-40C) or with b-glucan (ST-40C-BGL); inset shows the time dependence of G0 and tan d fora frequency of 1 Hz, strain 1% and at 40 �C.

Biliaderis et al., 1992; Hassan et al., 2002; Kristo et al., 2003a,2003b; 2011; Lucey, 2002; Lucey & Singh, 1998; Lucey et al.,1998). Upon acidification of the control milk preparations, theonset of the ‘local’ increase of tan d coincided with a change in theslope of G0 curve between the pH values of 5.1 and 4.7, reflecting theformation of casein network (Fig. 1a). Guyomarc’h et al. (2003)found that in heat-treated acidified milk, soluble aggregates ofwhey proteins precipitate at their overall pI (w5.3), thus initiatingan early gelation by interacting with the casein micelles. On theother hand, the micelle-bound whey protein aggregates modify themicelle surface, resulting in micelle destabilization at pH 5.1instead of 4.7. As the isoelectric point of caseins is approached andthus, the repulsions between casein particles decrease and thehydrophobic interactions are enhanced, rapid aggregation causesextensive rearrangements in the protein network and/or incorpo-ration of additional protein clusters into the gel network (Lucey,2002; Lucey & Singh, 1998; Lucey et al., 1998); as a result, there isa continuous increase of G0 before the attainment of a maximumvalue ðG0

maxÞ at pH 4.2 (Fig. 1a). Beyond this pH value, a slightdecrease of G0 was observed and this could be attributed to increasein net positive charge between caseins, enhancing their electro-static repulsions (Lucey et al., 1998; Moschakis, Murray, &Dickinson, 2010).

For the b-glucan containing milk preparations, even at thebeginning of the acidification process, the system exhibited arheological behavior of a slightly structured networkwith relativelyhigh values of G0 (w1 Pa) and tan d close to 1 (Fig. 1a). The higher G0

values of b-glucan fortified milk, with two orders of magnitudehigher than those of the unfortified samples, could be explained bythe increase in total solids content and the phase separation be-tween the milk proteins and the added polysaccharide. This hy-pothesis was verified by examining the behavior of aqueousdispersions of the b-glucan isolate alone at 2% w/v, a concentrationequal to that employed in the fortified milk gels and at the sametemperatures that fermentation took place. Clearly, the poly-saccharide dispersions alone showed much lower G0 (10�7 Pa) and

or milks fermented by yogurt starter culture (S. thermophilus and L. bulgaricus) withoutaqueous dispersion of b-glucan isolate at 2% w/v (BGL). All samples were monitored at

Fig. 2. Gelation parameters of fermented milk products (monitored at a frequency of1 Hz and strain 1%). ST: milk fermented by yogurt starter culture (S. thermophilus and L.bulgaricus); (ST þ PR): milk fermented by yogurt starter culture and the probioticstrain (L. paracasei); 40C, 38C and 36C: products fermented at 40 �C, 38 �C and 36 �C,respectively. BGL: milk supplemented with b-glucan. Differences between means(n ¼ 3) were compared by the LSD test at a ¼ 0.05.

A. Lazaridou et al. / Food Hydrocolloids 39 (2014) 204e214208

higher tan d (106) values compared to fermented milks fortifiedwith b-glucan (Fig. 1a, inset). Thus, mixing of the neutral, non-adsorbing, b-glucans with proteins most likely leads to depletionflocculation events, resulting in stronger proteineprotein in-teractions and thereby enhancement of elasticity of the compositesystem. Consistent with these views are the findings of anotherstudy on bulk phase separation between sodium caseinate and oatb-glucan aqueous in mixed dispersions at neutral pH; such dem-ixing phenomena were found to depend largely on the molecularweight and concentration of the polysaccharide (Lazaridou &Biliaderis, 2009). In the latter study, a 3.3% sodium caseinatee2%b-glucan mixture was examined, having similar contents of caseinand b-glucan and the same polysaccharide molecular weight(110 � 103) with the respective system of the present work, andseemed to be on the boundary zone of the diagram for a two-phaseseparated biopolymer system. Even at lower polysaccharide con-centrations, phase separation was noted in mixtures of concen-trated milk or whey protein isolate with b-glucans having highmolecular weight (>106) (Corredig et al., 2011; Kontogiorgos et al.,2009). Recently, Repin et al. (2012) studying aqueous mixed dis-persions of skimmed milk with a barley b-glucan concentrate,Glucagel, claimed that the driving force for phase separation wasdepletion flocculation of casein micelles in the presence of thepolysaccharide; above a certain volume fraction of casein micellesand Glucagel concentration the system phase separated as tran-sient gel. Bourriot, Garnier, and Doublier (1999) also found thatwhen non-absorbing polysaccharides, such as guar gum or locustbean gum, were added to micellar caseins at pH 7.0, the rheologicalbehavior resembled that of a network-like structure involvingaggregated casein micelles due to localized phase separation phe-nomena, as evidenced by microscopic observations. Tanaka (2000)has described this phenomenon as ‘viscoelastic phase separation’,underlying the formation of a transient gel in the initial stage ofphase separation of asymmetric mixtures, such as colloidal sus-pensions and non-adsorbing polymers; in such systems, theappearance of an ‘interaction network’ occurs due to polymer-induced depletion attraction forces between the colloidal particles.

Furthermore, two characteristic peaks, a small peak in G0 curveðG0

peakÞ at pH of 5.7e5.8 followed by a broad peak in tan d curve(tan dpeak) at pH 5.5e5.6, were noted during acidification of the b-glucan supplemented milks and before a three-dimensional gelnetwork with tan d value lower than 1 occurred (Fig. 1a); thesepeaks could be also ascribed to phase separation between milkproteins and b-glucans. With dropping of pH toward the milkproteins’ isoelectric point during fermentation, the polysaccharide-induced attraction of proteins is altered, possibly leading to inter-molecular associations which can enhance the G0 value. However,with further decrease of pH, the thermodynamic incompatibilitybetween milk proteins and b-glucans is enhanced, leading to phaseseparation and thus, result in reduced G0 values; i.e., the systembecomes more liquid-like (appearance of a tan dpeak and a localized‘minimum’ in the G0 function, Fig. 1a). The subsequent gradualdecrease of tan d starting at pH of 5.4, concurringwith initiation of aslow increase of the G0, can be ascribed to the aggregation of wheyprotein-bound caseins. The latter phenomenon also coincided withthe turning point of the G0 curve of the control preparations,showing that in fermented milk products, the pH where acid-induced casein aggregation commences is not affected by theadded polysaccharide. The aforementioned changes in the G0 ki-netic profiles of the b-glucan containing samples during acidifica-tion are also manifested in the d log G0/dt vs. time curve by thepresence of positive and negative alternating peaks (Fig. 1b).Similarly to our findings, when purified exopolysaccharide (EPS)isolates from S. thermophilus were added to milk gels, resulted inappearance of a shoulder in the d log G0/dt curve; this response was

attributed to phase separation phenomena (Mende, Peter, Bartels,Rohm, & Jaros, 2013). Depletion-induced phase separation hasbeen also reported when an EPS from lactic bacteria was added todenaturedwhey protein particles (Tuinier, Dhont, & de Kruif, 2000).In another study, in which acidification of aggregated whey

Table

2Effect

ofcu

lture, b

-glucanan

dferm

entation

tempe

rature

onthege

lation

andacidification

param

etersof

ferm

entedmilk

products.

Factors

Leve

lsGelationparam

etersb

Acidification

param

etersb

G0 max

(Pa)

tand m

inI E

(h�1

)Gt(m

in)

pH5h

Vm(m

UpH/m

in)

T m(m

in)

T e(m

in)

Culture

1Yog

urt

starter

culture

a15

1.90

a (n¼

18)

0.57

a (n¼

18)

15.01a(n

¼15

)33

2.94

a (n¼

15)

5.12

a (n¼

18)

8.68

a (n¼

18)

240.00

a (n¼

18)

501.16

a (n¼

18)

2Yog

urt

startercu

lture

andprobiotic

strain

a14

8.28

a (n¼

18)

0.70

a (n¼

18)

25.18

b(n

¼15

)32

5.43

a (n¼

15)

5.38

b(n

¼18

)8.94

a (n¼

18)

285.00

b(n

¼18

)51

5.49

a (n¼

18)

b-G

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A. Lazaridou et al. / Food Hydrocolloids 39 (2014) 204e214 209

proteins fortified with various polysaccharides, using glucono-d-lactone (GDL), was monitored microscopically, it was found thatthe first signs of phase separation were visible at pH 5.6 (de Jong,Jan Klok, & van de Velde, 2009); the mixed gels of whey proteinswith a non-adsorbing polysaccharide (locust been gum) revealeda protein rich phase and a serum polysaccharide rich phase. ThispH coincided with the tan dpeak range observed in the kineticrheological profiles of the present work when b-glucan wasincluded in the acidified milk products (Fig. 1a). The magnitude oftan dpeak was more pronounced in the kinetic profiles of b-glucancontaining milk products fermented at 36 and 38 �C (data notshown).

Overall, the addition of oat b-glucan to the fermented milkproduct largely affected the gelation kinetic profiles, as man-ifested by the longer gelation time (Gt), and occurring at lower pHof 4.7e4.9, compared to control milk gels (Fig. 1a). The peak in thed log G0/dt (IE value) vs. time curve for the b-glucan supplementedgels was considerably lower, compared to control milk prepara-tions, indicating a slower gelation rate in the presence of thepolysaccharide (Fig. 1b). Moreover, relatively weak gel networkswere obtained, showing higher tan dmin values (w0.9) andconsiderably lower G0

max than milk gels without b-glucan (Figs. 1aand 2). Similar gelation profiles to those of milk gels fermented at40 �C (Fig. 1a) were also observed for b-glucan fortified milk at38 �C (data not shown), but with lower G0 and higher tan dmin

values as well as a longer gelation time (Fig. 2). At even lowerfermentation temperature (36 �C) protein aggregation was slowerin the presence of b-glucan (data not shown). For the latterproducts, the tan dmin values were higher than 1 (Fig. 2), implyinga liquid-like rheological response and an incomplete gelationprocess, based on the adopted gelation criterion of tan d ¼ 1.

3.2. Effect of b-glucan, probiotic strain and fermentationtemperature on rheological and acidification properties offermented milk products: a factorial analysis approach

The effects of inclusion of the probiotic strain (L. paracasei) intothe yogurt starter culture, b-glucan addition and fermentationtemperature on the rheological, acidification and microbiologicalparameters of fermented milks are summarized in Table 1. Thesignificant effects of these factors and their interactions arespecified by the P values using the general linear model; i.e., fac-tors or their interactions having P values lower than 0.05, indicatea significant effect of the examined parameters. Moreover, themean values of the examined parameters for each factor, pre-sented in Table 2, point to the trends for these parameters as afunction of b-glucan addition, probiotic strain inclusion andfermentation temperature as well.

3.2.1. Rheological propertiesAnalysis of variance confirmed and supported our observations

that all rheological parameters were significantly affected by b-glucan inclusion in the milk formulations (Table 1). The rheolog-ical parameters, with the exception of IE, were also affectedsignificantly by the fermentation temperature, as shown by therespective P values for G0

max, tan dmin and Gt. In contrast, inclusionof L. paracasei into the conventional yogurt starter culture did nothave a significant impact on the rheological parameters withexception of the gelation rate which increased (Table 1); i.e., ahigher mean value of IE for milk products containing the probioticstrain was noted compared to products fermented merely withthe yogurt starter culture (Table 2).

Confirming the results discussed in the previous section, forproducts containing b-glucans the mean value of Gt increased andthat of IE decreased compared to control formulations (Table 2).

Fig. 3. Effect of b-glucan on acidification kinetic profiles of fermented milk (monitoredat frequency 1 Hz, strain 1% and 38 �C). Milks fermented by yogurt starter culture(S. thermophilus and L. bulgaricus) and the probiotic strain (L. paracasei) at 38 �C.(ST þ PR)-38C: control milk, and (ST þ PR)-38C-BGL: milk supplemented withb-glucan.

A. Lazaridou et al. / Food Hydrocolloids 39 (2014) 204e214210

Similarly, Kristo et al. (2011) found that milks fermented with anEPS-producing strain exhibited longer gelation times compared tothe respective fermentation products of the EPS-free culture.Moreover, the weakening effect of b-glucan on milk protein gelnetwork structure was also evidenced by the significant decrease ofG0max and increase of tan dmin mean values when b-glucan was

included in the acidified milk gels (Table 2). In another study byLazaridou et al. (2008), a mixed 12% skim milk-2% b-glucan system,having similar composition and a comparable polysaccharide mo-lecular weight (140 � 103) with the b-glucan fortified milks of thepresent work, exhibited similar rheological responses; i.e., themixed gels showed a considerable decrease in G0 and increase in thetan d values. In contrast, other studies on cereal b-glucan supple-mented dairy products, such as curds and acidified gels, revealedthat the addition of polysaccharide reinforces the casein network,as manifested by an increase of G0 of the b-glucan-fortified products(Brennan & Tudorica, 2008; Tudorica, Jones, Kuri, & Brennan, 2004).The divergence in the rheological responses of such mixed systemsmay originate from differences in molecular features and concen-tration of the added polysaccharide that can significantly affect thegel strength of the phase separated system. In a relevant study,when an oat b-glucan isolate was added to a set yogurt, hardness(as assessed by large deformation mechanical testing) increased upto an optimal fortification level of 0.1% (Gustaw, 2008); moreover,the viscosity of the product was not affected by polysaccharideincorporation up to 0.2%, whereas with further increase of b-glucanconcentration to 0.3% the viscosity was dropped drastically. Un-fortunately, the molecular weight of the polysaccharide is notspecified in most of the studies on mixed milk-b-glucan systems,which renders the comparison of the results difficult. Overall,thermodynamic incompatibility between milk proteins and poly-saccharides has been proposed to affect the composite networkproperties for several non-adsorbing (e.g. guar gum, locust beangum and xanthan), or adsorbing (pectin, l-carrageenan) poly-saccharides added to stirred yogurt formulations (Everett &McLeod, 2005; Ramirez-Santiago et al., 2010); inclusion of theformer stabilizers into yogurt formulations up to a certain level of1 g L�1 results in lower G0 values than for unfortified products.

Concerning the effect of fermentation temperature, the meanvalue of G0 was similar for products fermented at either 36 �C or38 �C, whereas it decreased with further increase in fermentationtemperature to 40 �C (Table 2). According to previous studies, thehigher G0 values of milk gels acidified either chemically or by bac-terial cultures at lower incubation temperature are ascribed to thehigher voluminosity of the casein micelles due to weaker hydro-phobic interactions, thus weaker particle rearrangements,compared to milk gels formed at higher temperatures (Arshad,Paulsson, & Dejmek, 1993; Kristo et al., 2003b; Lee & Lucey, 2006;Lucey et al., 1998; Walstra, 1998). A higher voluminosity meansthat casein micelles can form more extensive and stronger inter-molecular associations with a large number of protein moleculesinvolved in the junctions, yielding a continuous and firmer gelnetwork. At high temperatures, the hydrophobic interactions areenhanced leading to smaller colloidal particle voluminosity and acoarser-heterogeneous gel network. In contrast, the tan d meanvalue of milk gels prepared at 36 �C was higher than those ofproducts fermented at 38 and 40 �C (Table 2). The lack of a cleartrend in the mean values of G0 and tan d as a function of fermen-tation temperature can be explained by the significant (P < 0.05)interaction effect between the fermentation temperature and b-glucan fortification (BG � FT, Table 1). Thus, the strong weakeningeffect of b-glucan addition to the gel network structure of fer-mented products at 36 �C is reflected by the very high tan d values(Fig. 2); instead, for the unfortified milk products, the tan d valueswere not affected by fermentation temperature. Arshad et al. (1993)

also found no dependence of tan d values on gelation temperatureof chemically acidified milk gels. In contrast, other studies (Kristoet al. 2003b; Lee & Lucey, 2006) showed a decrease of tan dmin ofthe cultured milk products with decreasing fermentationtemperature.

The mean values of IE and Gt for gels fermented at 36 �C arederived only from the values obtained for the unfortified products,since the addition of b-glucans hindered gel network formation atthis temperature (Table 2 and Fig. 2). Between milk products fer-mented at 38 and 40 �C, the gelation time decreased at highfermentation temperature; the latter trendwas also obvious amongunfortified milks over the entire range of fermentation tempera-tures, 36e40 �C. The observation of shorter gelation time at higherfermentation temperatures concurs with the findings of otherstudies (Arshad et al., 1993; Kristo et al., 2003a, 2003b; Lee & Lucey,2006; Lucey et al., 1998).

3.2.2. Acidification propertiesAlthough inclusion of b-glucan into milk formulations did not

influence the pH changes throughout gelation of the products fer-mented at 40 �C, differences in the acidification rates wereobserved between the b-glucan fortified and the control productsat lower fermentation temperatures of 38 and 36 �C (Fig. 3). In thelatter cases, the polysaccharide addition retarded acidification, asindicated by the lower maximum acidification rates (Vm), calcu-lated from the respective dpH/dt vs. time curves (Fig. 3, inset). Thisis in accordance with the longer time at which maximum acidifi-cation rate was noted (Tm), the longer time to reach a pH of 4.6 (Te),and the higher pH at a fermentation time interval of 5 h (pH5h),compared to the respective values of control milk gels (Figs. 3 and4). Analysis of variance showed that b-glucan enrichment andfermentation temperature of milk gels had a significant impact(P < 0.05) to all acidification parameters (Table 1) and their meanvalues revealed the same trends between the b-glucan fortified andcontrol products (Table 2). Other reports also revealed that milkfermented with EPS-producing bacteria strains require a longertime to reach a pH of 4.6 than milk products fermented with EPS-free cultures (Haque et al., 2001; Kristo et al. 2011).

Both gelation and acidification kinetics indicate that the extentof phase separation phenomena increased as fermentation tem-perature decreased from 40 to 36 �C; the latter may also reflect the

Fig. 4. Acidification parameters of milk fermented products. ST: milk fermented byyogurt starter culture (S. thermophilus and L. bulgaricus). (ST þ PR): milk fermented byyogurt starter culture and the probiotic strain (L. paracasei); 40C, 38C and 36C: productsfermented at 40 �C, 38 �C and 36 �C, respectively. BGL: milk supplemented with b-glucan. Differences between means (n ¼ 3) were compared by the LSD test at a ¼ 0.05.

A. Lazaridou et al. / Food Hydrocolloids 39 (2014) 204e214 211

deviation from the optimum growth temperature ofS. thermophilus, the bacterium primarily responsible for the abruptpH decline at the early stages of milk fermentation (Walstra,Wouters, & Geurts, 2006, chap. 22). In agreement with our

findings for milk fermented at 40 �C, other relevant studies haveshown that there was no pronounced effect of the b-glucan on theacidification curves of milk fermented at 42 �C by yogurt starterbacteria, when oat fiber or barley b-glucan were included in theformulations at 1.32 and 0.5%, respectively (Fernandez-Garcia,McGregor, & Traylor, 1998; Gee et al., 2007). The acidification ki-netics seemed to be affected by the strain composition of the starterculture, and the fermentation temperature as well as by their in-teractions (Table 1). However, using a starter culture that containsL. paracasei there was no significant effect on Vm and Te i.e., thecommon problem of slower acidification during yogurt manufac-ture when probiotic cultures are used (Sarkar, 2008) was notobserved in the present work (Tables 1 and 2, and Fig. 4).

Reduction in fermentation temperature resulted in sloweracidification kinetics, as reflected by the higher pH5h, Tm, and Te andthe lower Vm values of milk products fermented at 36 and 38 �C,compared to products incubated at 40 �C (Table 2 and Fig. 4). Thehigher acidification rate of milk products fermented at 40 �C couldoriginate from the selective growth of the thermophilic bacteriumS. thermophilus. In agreement with our results, Kristo et al. (2003a,2003b) has modeled the acidification process of milk productsfermented by yogurt starter culture containing or not the probioticstrain of L. paracasei B 117, using response surface methodology,and found an increase in Tm and Te values and a decrease in acidi-fication rate (Vm) with a decline of fermentation temperature from44 to 36 �C. It is worthy here to note, that a medium acidificationrate is preferable during production of stirred yogurt since it resultsin a non-excessive lactic acid production, a more homogeneousstructure of the coagulum and a greater viscosity of the finalproduct (Beal, Skokanova, Latrille, Martin, & Corrieu, 1999). Inthe present work, the conditions of achieving both low Vm andhigh G0

max values were observed when control milk samples werefermented at 38 �C (Table 2, and Figs. 2 and 4).

3.3. Culture viability in fermented milk products

The means for viable count numbers of yogurt starter bacteriaand the probiotic strain are given in Table 3, describing the impactof L. paracasei inclusion, b-glucan fortification, fermentation tem-perature and storage time of the milk products on the viability ofmicroorganisms. All these factors did have a significant effect onthe counts of each culture bacterium, with the exception of b-glucan addition on L. bulgaricus counts (Table 1). Among thedifferent levels of each factor examined, the mean numbers ofyogurt starter culture, S. thermophilus and L. bulgaricus, ranged from106 to 108 cfu/ml, while the values for the probiotic strain,L. paracasei, varied between 107 and 108 cfu/ml (Table 3); the aboveviable counts of the probiotic strain and yogurt starter culturebacteria are satisfactory for providing health benefits from con-sumption of the milk products (Lamsal & Faubion, 2009; Lourens-Hattingh & Viljoen, 2001; Mohammadi & Mortazavian, 2011).

Inclusion of L. paracasei into the culture showed an increase ofcell viability of both conventional yogurt starter bacteria (Table 3).This observation points to the good compatibility of L. paracaseiwith S. thermophilus and L. bulgaricus; similar results were reportedby Kristo et al. (2003b) using the same yogurt culture bacteriastrains. Also, for other subspecies of L. paracasei, such as ssp. tol-erans, no inhibitory activity has been found towards yogurt startercultures grown in association with the probiotic strain(Maragkoudakis et al., 2006). Furthermore, Donkor, Henriksson,Vasiljevic, and Shah (2006) found that the viability ofL. delbrueckii ssp. bulgaricus was enhanced significantly in thepresence of a mixture of probiotic bacteria, Lactobacillus acidophi-lus, Bifidobacterium lactis and L. paracasei in yogurt during coldstorage.

Table 3Effect of culture, b-glucan, fermentation temperature and storage time on the viability of culture bacteria of fermented milk products.

Factors Levels Streptococcus thermophilusY 4.10log (cfu/ml)

Lactobacillus delbrueckiissp. bulgaricus Y 6.15log (cfu/ml)

Lactobacillus paracaseissp. paracasei B 117log (cfu/ml)

Culture 1 Yogurt starter culturea 7.38a(n ¼ 54) 6.79a(n ¼ 54)2 Yogurt starter culture and probiotic straina 7.90b(n ¼ 54) 8.05b(n ¼ 54)

b-Glucan 1 Without b-glucan supplementationa 7.89b(n ¼ 54) 7.39a(n ¼ 54) 7.77a(n ¼ 27)2 With b-glucan supplementationa 7.39a(n ¼ 54) 7.45a(n ¼ 54) 8.27b(n ¼ 27)

Fermentation temperature 1 Fermented at 36 �Ca 7.86b(n ¼ 36) 7.76b(n ¼ 36) 8.43c(n ¼ 18)2 Fermented at 38 �Ca 7.95b(n ¼ 36) 7.55b(n ¼ 36) 7.99b(n ¼ 18)3 Fermented at 40 �Ca 7.11a(n ¼ 36) 6.95a(n ¼ 36) 7.64a(n ¼ 18)

Storage time 1 End of fermentation (pH ¼ 4.6),a 7.25a(n ¼ 36) 6.79a(n ¼ 36) 7.30a(n ¼ 18)2 Stored at 4 �C � 7 daysa 7.74b(n ¼ 36) 7.65b(n ¼ 36) 8.25b(n ¼ 18)3 Stored at 4 �C � 14 daysa 7.93c(n ¼ 36) 7.81b(n ¼ 36) 8.52c(n ¼ 18)

a Values are themeans of n determinations given in parentheses; means of each specified parameter for a certain factor are calculated using the respective values of all levelsof the other factors. Values followed by the same letter in the same column and for the same factor are not significantly different (P > 0.05); means compared using the LSDtest.

Fig. 5. Changes in the number of L. paracasei B117 of milk fermented products at theend of fermentation (N) and after 7 (N7) and 14 (N14) days of storage at 4 �C. (STþ PR):milk fermented by yogurt starter culture (S. thermophilus and L. bulgaricus) and theprobiotic strain (L. paracasei); 40C, 38C and 36C: products fermented at 40 �C, 38 �Cand 36 �C, respectively. BGL: milk supplemented with b-glucan. Differences betweenmeans (n ¼ 3) were compared by the LSD test at a ¼ 0.05.

A. Lazaridou et al. / Food Hydrocolloids 39 (2014) 204e214212

Low fermentation temperatures (36 and 38 �C) seem to favor thegrowth of both yogurt starter bacteria (S. thermophilus andL. bulgaricus) as showed by their higher mean viable cell numberscompared to fermentation at 40 �C (Table 3). Moreover, there was agradual increase in viability of the probiotic strain with decreasingfermentation temperature; the highest number of cells ofL. paracasei was noted at 36 �C that seems reasonable for thismesophilic bacterium.

The health benefits sought by consumption of fermented milkproducts containing yogurt starter and probiotic bacteria can bedelivered when high viable cell numbers of these bacteria aremaintained in the product throughout their entire shelf-life (Sarkar,2008). The numbers of viable cells of all bacteria species, particu-larly of L. paracasei, exceeded the satisfactory acceptable threshold(>107e108 cfu/ml) at the end of refrigerated storage and showed aslight increase of their counts (culture viability) with storage at 4 �Cup to two weeks; both Lactobacilli increased their population morethan one log cycle (Table 3). Donkor et al. (2006) found thatS. thermophilus, L. bulgaricus and L. paracasei L26 in yogurt main-tained constant viable counts during storage, while Kearney et al.(2011) reported a stable population for L. paracasei NFBC 338 anda decline of the yogurt starter strains during cold storage of fer-mented products for 28 days. In the present work, the L. paracaseissp. paracasei B 117 strain exhibited an enhanced survival (108 cfu/ml), even after 14 days of storage and the highest level of countsamong the three bacteria species after 7 and 14 days storage(Table 3); i.e., the probiotic strain is dominating over both theyogurt starter bacteria, S. thermophilus and L. bulgaricus.

Supplementation of fermented products with b-glucan resultedin a decline of the S. thermophilus viable cell number, while it didnot seem to affect the viability of L. bulgaricus (Table 3). On theother hand, there was a slight, but significant increase in the countsof the probiotic L. paracasei strain with the inclusion of b-glucan,suggesting a stimulating or protective role of the polysaccharide onits growth. In previous studies, the addition of barley and oat b-glucan concentrates in both real and yogurt model systems (Geeet al., 2007; Rosburg et al., 2010; Vasiljevic et al., 2007), as well asthe presence of a microbial b-glucan produced in situ in yogurt froma recombinant probiotic strain, L. paracasei NFBC 338 (Kearneyet al., 2011), did not show an effect on growth and viability ofconventional yogurt starter cultures. On the other hand, Gee et al.(2007) reported that yogurt starter cultures may degrade thecereal b-glucans added to a yogurt model system, as evidenced byviscosity reduction; however, this ability was dependent on thestrain and availability of other nutrients (lactose). Vasiljevic et al.

(2007) have found that oat and barley b-glucan significantlyimproved the growth of the probiotic strain Bifidobacterium ani-malis ssp. lactis Bb-12� during fermentation and cold storage ofyogurt. Other in vitro studies pointed out that selected probioticand yogurt strains possess the enzymes required for b-glucandegradation and thus have the ability to utilize this polysaccharidefor their growth (Jaskari et al., 1998; Lambo, Oste, & Nyman, 2005).However, in the present work, the viable cell counts of L. paracaseiat the end of the fermentation process in the b-glucan-supple-mented product did not differ from those of the respective controlformulations (without b-glucan, Fig. 5). Similarly, Crittenden et al.(2002) found that pure barley b-glucan was not fermentedin vitro by a wide range of probiotic lactobacilli bacteria, including aL. paracasei strain (VTT E-94510).

Factorial analysis clearly indicated that viability of L. paracaseiwas significantly affected by interactions between factors such as b-glucan and fermentation temperature as well as b-glucan and

A. Lazaridou et al. / Food Hydrocolloids 39 (2014) 204e214 213

storage time (P < 0.05) (Table 1). As a result, among the varioustested milk formulations and processing-storage conditions, thehighest number, w109 viable cfu/ml, was observed in b-glucancontaining products fermented at 36 �C after 7 and 14 days ofstorage (Fig. 5). Rosburg et al. (2010) also showed a time-dependentprotective effect of oat b-glucan on the viability of Bifidobacteriumlongum during cold storage of a yogurt product and this wasattributed to a protective effect, rather than a prebiotic action;these researchers hypothesized that due to the viscosity enhance-ment properties of b-glucans oxygen diffusion is reduced in thefinal product, resulting in more favorable growth conditions for theprobiotic strain. The present findings suggest that inclusion of oatb-glucan in combinationwith a selected L. paracasei probiotic strainin fermented milk products (at 36 �C) may provide the healthbenefits from both the probiotic bacterium population and thesoluble fiber. Further to the hypocholesterolemic and hypo-glycaemic properties, b-glucan could also exert a prebiotic functionon the flora of the intestinal tract. Previous animal studies haveshowed that supplementation of diet with cereal b-glucansenhanced lactobacilli growth in the intestinal tract of rats(Dongowski et al., 2002; Snart et al., 2006). In vitro experimentsalso supported a possible strain to strain variation in lactobacillispecies to utilize oligosaccharides (degree of polymerization 3 or 4)derived from b-glucan hydrolyzates (Snart et al., 2006). On theother hand, in vitro fermentation of oat and barley b-glucans byfaecal microbiota, including a large range of lactobacilli and bifi-dobacteria, displayed no apparent prebiotic potential, unless b-glucan hydrolyzates were used as substrates (Hughes, Shewry,Gibson, McCleary, & Rastall, 2008).

4. Conclusions

In the present work, analysis of variance was used for deter-mining the individual and interaction effects that three differentfactors, namely incorporation of oat b-glucan, fermentation tem-perature and inclusion of a probiotic strain (L. paracasei B117),might have on the rheological behavior, acidification kinetics, andculture growth of skimmed milk systems fermented by yogurtcultures. Fortification of milk with 1.4% oat b-glucan could fulfill thehealth claim requirements of EFSA and FDA for reduced risk of heartdisease by lowering the blood serum cholesterol levels. Poly-saccharide inclusion into milk at this concentration reduced theprotein aggregation and acidification processes due to phase sep-aration between the milk proteins and the b-glucan as evidencedby dynamic rheometry even at the early stages of the gelation. Thephase-separated systems yielded weaker milk gels at the end offermentation (pH ¼ 4.6). Phase separation phenomena and retar-dation of gelation in b-glucan fortified milk became more pro-nounced with decreasing fermentation temperature from 40 to36 �C. On the other hand, inclusion of the probiotic strain into theyogurt starter culture increased the gelation rate of the milk pro-teins without altering the gelation time and gel strength of the finalproduct. The bacteria of both yogurt starter culture and the pro-biotic strain not only maintained their viability at levels beneficialfor consumer health, but also slightly increased their counts uponstorage (14 days at 4 �C). Moreover, the examined probiotic strain ofL. paracasei exhibited good compatibility with the yogurt starterculture. A protective role of oat b-glucan on growth of the probioticstrain was also seen since the inclusion of the polysaccharideincreased the counts of its viable cells during storage; w 108e109 viable cfu/ml, was found in the b-glucan containing skimmedmilk products fermented at 36 �C after 7 and 14 days of storage.Although milk fermentation at 36 �C resulted in b-glucan enrichedproducts with a liquid-like structure, like a drinkable yogurt, ratherthan a set yogurt, one may still obtain the health benefits from both

the probiotic action (L. paracasei) and the b-glucan hypocholester-olemic effect, by consumption of this type of fermented dairyproducts.

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