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The role of exopolysaccharide produced by Lactococcus lactis subsp. cremoris in structure formation and recovery of acid milk gels
International Dairy Journal 21 (2011) 656e662
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The role of exopolysaccharide produced by Lactococcus lactis subsp. cremoris in structure formation and recovery of acid milk gels Eleana Kristo*, Zhutuan Miao, Milena Corredig Food Science Department, University of Guelph, Guelph, Ontario N1G 2W1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 June 2010 Received in revised form 2 February 2011 Accepted 9 February 2011
The effect of exopolysaccharide (EPS) produced by Lactococcus lactis subsp. cremoris JFR1 on milk fermentation and recovery of gel structure after stirring was studied using rheology and diffusing wave spectroscopy. Skim milk fermented with an EPS-non producing (EPS) strain of L. lactis served as control. The production of EPS was not sufficient to affect the stages preceding aggregation. No differences were found in the changes in diffusion coefficient, radius and turbidity parameter of casein particles up to the point of gelation between EPSþ and EPS samples. However, the presence of EPS significantly increased the storage modulus and viscosity of milk gel, and reduced the ability of the gel to recover after shearing. This study indicates that although the present EPS does not affect the preliminary stages of gelation, it is produced in sufficient quantities after gelation to significantly alter the structure of milk gels in their set or stirred form. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Many mesophilic and thermophilic lactic acid bacteria (LAB) strains, commonly used in dairy processing, excrete heteropolysaccharides, containing mainly D-glucose, L-rhamnose and D-galactose (Ruas-Madiedo, Hugenholtz, & Zoon, 2002). Exopolysaccharides (EPS) produced by LAB can freely be released into the medium or be attached to the cell. When present free in the medium, they may impart a ropy appearance to the milk gels. LAB that produce EPS are often combined in commercial starters, as the EPS may function as natural stabilisers and thickeners in fermented milk products. In situ production of EPS from LAB may replace the additives and stabilisers that are usually employed to ensure a smooth and creamy texture of fermented milk products. Studies have shown that the effect of EPS in the rheological properties of final fermented milk is more pronounced and with a better outcome when EPS is produced in situ than when added as an ingredient (Doleyres, Schaub, & Lacroix, 2005). The amount of EPS produced by LAB during milk fermentation varies with data reported in literature ranging from 50 to 600 mg L1 (Cerning, 1995). Most often very little is recovered from the fermented milk, suggesting that EPS may influence the texture and stability of fermented products even when it is present in low amounts. Therefore, the desirable structuring properties of EPS
* Corresponding author. Tel.: þ1 519 824 4120; fax: þ1 519 824 6631. E-mail address: [email protected] (E. Kristo). 0958-6946/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2011.02.002
have been attributed to factors other than concentration, such as type of linkage, presence of side groups, branched structure, molecular weight (Ruas-Madiedo et al., 2002; Tuinier et al., 2001), and/or interactions with milk proteins (Ayala-Hernández, Hassan, Goff, Mira de Orduna, & Corredig, 2008; Tuinier, ten Grotenhuis, Holt, Timmins, & de Kruif, 1999). In addition to the dynamic changes occurring to casein micelles, colloidal calcium phosphate, whey proteins and other milk components (Alexander & Dalgleish, 2004; Lucey, Teo, Munro, & Singh, 1997), resulting ultimately in the formation of a protein network, the process of fermentation of milk with LAB producing EPS includes also the dynamics of production and incorporation of EPS. That is to say that EPS is produced gradually by LAB during acidification, and, as the environmental conditions change, the effect of the polysaccharide on the proteins will continue to evolve. It can be hypothesised that the balance of repulsive/attractive interactions of EPS with milk proteins will continuously change during fermentation, and a better understanding of the dynamics will allow a better control of the final characteristics of the milk gel (Girard & Schaffer-Lequart, 2008; Sanchez, Zuniga-Lopez, Schmitt, Despond, & Hardy, 2000). In addition, during processing of stirred type yoghurts and other fermented milk products, the gels are disrupted due to shearing and pumping. Despite the practical importance of the structure breakdown and recovery of these types of gels and the role played by EPS in the gel when shearing is applied, this aspect has not been fully explored (Arshad, Paulsson, & Dejmek, 1993; Girard & Schaffer-Lequart, 2007b).
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Transmission diffusing wave spectroscopy (DWS) is a nondisruptive method that is based on multiple scattering of light from particles in a sample, which allows the study of concentrated food systems (Weitz & Pine, 1993). DWS has been employed to derive information on the changes in the early stages preceding aggregation of casein micelles during acidification or rennet coagulation of milk (Alexander & Dalgleish, 2004). A multitechnique approach, combining DWS, rheology and microscopic observations would successfully describe the dynamics occurring during fermentation, from the beginning stages of casein destabilisation to the formation of the gel network. The objective of this study was to investigate the role of EPS produced in situ from the ropy strain of Lactococcus lactis subsp. cremoris JFR1 on various stages of milk fermentation process, on the formation of gel structure as well as on the recovery of structure after shearing. 2. Materials and methods 2.1. Preparation of strains L. lactis subsp. cremoris (JFR1), a highly ropy strain (Hassan, Frank, & Elsoda, 2003a) was obtained from Dr. Hassan’s collection at South Dakota State University. A L. lactis strain that did not produce EPS was obtained from Chr. Hansen (Hørsholm, Denmark) and was used as a control. The strains were subcultured through two transfers in M17 broth (Difco, Becton Dickinson Co., Sparks, MD, USA) at 30 C for 18 h; 0.2 mL from the prepared microbial suspension of the last transfer was inoculated in tubes with 7 mL of 10% reconstituted skim milk and kept in a freezer (20 C) without incubation until use. To prepare the inoculum for milk fermentation, the frozen strains were incubated for one night at 30 C. Nine decimal dilutions in sterile peptone water (0.1%, v/v) were prepared from the coagulated milk, and 0.2 mL from each dilution (including undiluted sample) was inoculated in tubes with 9 mL of sterilised reconstituted skim milk powder (10%, w/v). The inoculated tubes were mixed in a vortex and incubated at 30 C for 24 h. From these series of tubes, the last dilution showing milk coagulation was used as inoculum (Kristo, Biliaderis, & Tzanetakis, 2003).
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for each separate experiment, to be able to plot the parameters measured by rheology or DWS as a function of pH. 2.3. Diffusing wave spectroscopy Aliquots (1 mL) of milk were poured into a rectangular flat-faced optical glass cuvette (Hellma Canada Limited, Concord, ON, Canada) with a path length 5 mm, which was placed in a water tank maintained at 30 C. The source of light was a solid state diode pumped Nd:YAG (Yttrium Aluminium Garnet) laser (VERDI) 532 nm, maximum output power 2 W (Coherent, Santa Clara, CA, USA). Two glass windows of the water tank allowed incident and scattered light beam to pass. A single fibre optic, that collects the transmitted scattered light, was bifurcated and fed to two matched photomultipliers (HC120-03, Hamamatsu, Loveland, OH, USA) and a correlator (FLEX2K-12 2, Correlator.com, Bridgewater, NJ, USA). The intensity of laser was calibrated daily by means of standard latex spheres with a diameter of 260 nm (Portland Duke Scientific, Palo Alto, CA, USA) dispersed in MilliQ water. Correlation functions and intensity of the transmitted scattered light were measured at intervals of 3 min until the end of fermentation. Data were analysed using specialised software (DWS-Fit, Mediavention Inc., Guelph, ON, Canada). Each experiment was repeated at least 3 times, starting from different batches of fresh skim milk. Transmission DWS detects the intensity of the light scattered multiple times from the dispersed particles and collected after it has travelled the length (L) of the scattering cell. The fluctuation of the scattered light that happens due to the Brownian motion of particles can be characterised by means of the correlation function (Weitz & Pine, 1993). The value of l*, the photon transport mean free path, can be calculated from the total intensity of the light scattered by the sample and the laser intensity measured using 260 nm latex spheres dispersed in water. The diffusion coefficient D (m2 s1) is calculated from the correlation function using a value of 1.34 for n, the refractive index of milk serum. From the diffusion coefficient D, the radius of the particle can be calculated using the StokeseEinstein equation (Weitz & Pine, 1993). The pH of aggregation was determined as the pH value denoting the rapid growth of particle size compared to the original particle size. It was extrapolated from the curves of particle radii as a function of pH using the tangent graphical method at a radius scale of 2 mm.
2.2. Production of fermented milk
2.4. Rheological measurements
Aliquots of 97 mL of pasteurised skim milk (Crown Dairy Ltd., Guelph, ON, Canada) were distributed in sterilised 100 mL glass bottles. Milk was heated at 90 C for 20 min (not including come up time, w10 min) in a water bath to ensure a nearly complete heat denaturation of the whey proteins. The bottles were cooled by placing them into ice water. Three millilitres from the tube containing the inoculum were added into the bottle under aseptic conditions to prevent contamination. Twenty millilitres of inoculated milk was added in the cup of the rheometer for rheological measurements. Also, a few millilitres were added into a tube and the DWS cuvette for pH and DWS measurements, respectively. The cup and bob of the rheometer, the cuvette of DWS, the tube used in pH measurement and the glass electrode were cleaned with ethanol before use. Milk bottles were then incubated at 30 C until pH 4.6 was reached, when the bottles were cooled fast by immersing them into ice water and then stored in the refrigerator at 4 C. The pH of samples was monitored as a function of time with a pH meter AR15 (Fisher Scientific, Mississauga, ON, Canada). The changes of pH as a function of time were analysed by curve fitting using SigmaPlot version 10 (Systat Software, Inc., San Jose, CA, USA)
A Physica MCR 301 rheometer (Anton Paar Germany GmbH, Ostfildern, Germany) with a measuring cell C-PTD200 equipped with a Peltier temperature controller and a concentric cylinder geometry (diameter of cup and bob: 28.92 and 26.66 mm, respectively) was used for rheological measurements. A Julabo circulator (Julabo West, Inc., CA, USA) was used as counter cooling system for the Peltier element. Twenty millilitres of inoculated sample were added into the cup and a solvent trap was used to cover the cup and prevent sample evaporation. Oscillatory measurements were performed at 0.1% strain and frequency of 1 Hz at 30 C. Storage modulus (G0 ), loss modulus (G00 ), and loss tangent (tan d) were tracked for as long as required for the sample to reach pH 4.6. Gelation point was arbitrarily defined as the pH or time when G0 ¼ G00 . Once the samples reached pH 4.6, the temperature in the rheometer cell was decreased to 4 C in 30 min and an oscillatory time sweep measurement was performed for 10 min at 4 C, at 0.1% strain and frequency of 1 Hz, followed by a steady state viscosity measurement over the range of shear rates of 0.1e500 s1. After reaching 500 s1, this shear rate was maintained for 20 min to disrupt the gel, as previously reported (Girard & Schaffer-Lequart, 2007b) and the recovery of structure was then followed by
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measurement of G0 , G00 and tan d for 10 h at 4 C using 0.1% strain and frequency of 1 Hz. Flow curves were fitted with the rheological models of HerscheleBulkley and Carreau (Lee & Lucey, 2006). 2.5. Confocal laser scanning microscopy The microstructure of set and stirred fermented milk was observed using a confocal laser scanning inverted microscope (Leica TCS SP2, model Leica DM IRE2, Leica Microsystems CMS GmbH, Mannheim, Germany) with an Ar/Kr visible light laser and 40 (oil) objective. The fluorescent dye rhodamine B (excitation and emission wavelengths 543 and 625 nm, respectively) was used to stain the protein network. Resolution of the acquired digital images was 1024 1024 pixels. Twenty microlitres of 0.2% (w/v) aqueous solution of rhodamine B were mixed with 5 mL inoculated milk; a few drops of the mixture were transferred to a slide with cavity and covered with a cover slip. The slide was placed in an incubator at 30 C until the pH of the sample dropped to pH 4.6 (as indicated from the measurements in pH meter). The slide was subsequently stored at 4 C overnight and used next day to take images of the set fermented milk. Preparation of stirred sample for confocal laser scanning microscopy (CLSM) testing was done by adding a few drops of 0.2% (w/v) of rhodamine B solution on a small portion of fermented milk that was stirred in the rheometer at 4 C, 500 s1 for 20 min, and transferred in a glass bottom dish. The sample was kept at 4 C for 1 h to allow diffusion of the fluorescent dye before observation in the microscope. 2.6. Statistical analysis All experiments were repeated at least three times. The results reported in the tables are means standard deviations. Statistical analysis was carried out using SigmaPlot version 10 (Systat Software, Inc., San Jose, CA, USA). Statistical significance at P < 0.05 was determined using Student’s t-test. Confocal images are representative of at least three observations of each separate replicate. 3. Results and discussion 3.1. Gelation properties The aggregation of casein micelles during fermentation was followed using DWS, and the development of apparent particle radius (diffusion coefficient in inset Fig. 1) and turbidity parameter 1/l* are shown as a function of pH in Fig. 1. The turbidity parameter 1/l* of the control sample increased continuously throughout fermentation. Such behaviour of 1/l* has been previously reported for acidification of heated milk with glucono-d-lactone (GDL) (Alexander & Dalgleish, 2004; Azim, Corredig, Koxholt, & Alexander, 2010) or fermentation of heated milk with a commercial culture (Azim et al., 2010). The increase in the turbidity parameter 1/l* of milk fermented with the EPSþ strain was comparable with that of EPS-free fermented milk, implying that similar changes occurred in the casein micelles during fermentation. The value of 1/l* depends on the physical properties of the micelles as well as particleeparticle interactions, and the results shown in Fig. 1 suggest that the presence of EPSþ culture did not affect physical parameters such as the refractive index contrast or the structural organisation of the micelles. It is important to note that previous work has demonstrated that in the presence of a phase separating polysaccharide, the value of 1/l* decreases, because of structuring of casein micelles in microphase separated clusters (Acero-Lopez, Alexander, & Corredig, 2010). In the present work, no differences were noted in the values of 1/l* between EPSþ and EPS fermented
Fig. 1. Light scattering parameters 1/l* (B, 7), apparent particle radius (C, ;) and (insert) diffusion coefficient (C, ;) measured by diffusing wave spectroscopy as a function of pH during fermentation of milk with the exopolysaccharide- (EPS-) producing (C, B) and EPS-non producing strain (;, 7).
milk samples, suggesting the lack of depletion flocculation in the early stages of acidification. In addition, it is clear from Fig. 1 that up to pH 5.5 no changes occurred in the apparent radius of the casein micelles. When comparing the development of apparent radius (Fig. 1) and diffusion coefficient (Fig. 1 inset) of casein micelles during fermentation of milk with the EPSþ and EPS culture, no significant differences were found between the two treatments. The aggregation of casein micelles was marked by a sharp increase of the apparent particle radius that allowed for determination of a pH of aggregation. This value was not significantly different for EPSþ and EPS fermented milk (5.21 0.07 and 5.23 0.11, respectively). These findings indicate that the production of EPS from L. lactis subsp. cremoris JFR1 does not have a significant effect on the casein micelles in the stages preceding aggregation. An average radius of gyration of 68 and 86 nm has been previously reported (Ayala-Hernández et al., 2008) for the EPS produced by L. lactis subsp. cremoris JFR1 during fermentation of milk permeate supplemented with 2% whey protein isolate at the constant pH of 6.5 and 5.5, respectively. It can be hypothesised that if EPS was produced and interacted with the casein micelles, a thick layer of EPS of the order of the radius of gyration would form, and this would significantly increase the radius of the casein micelles by at least 60e80 nm. However, no changes were noted in the apparent radius and diffusion coefficient of the caseins. It has also been hypothesised that the EPS produced by L. lactis subsp. cremoris JFR1 is negatively charged (Ayala-Hernández et al., 2008), and at pH > 5.6 this should cause microphase separation of the casein micelles by means of a depletion flocculation mechanism (Tuinier et al., 1999); again, the present work showed no differences in the turbidity parameter and the diffusion coefficient between the control and the milk fermented with EPSþ culture. It is also important to point out that an increase of the viscosity of the continuous phase due to the presence of EPS should also result in a lower diffusivity of the casein micelles compared to the control. Previous authors (Ruas-Madiedo & Zoon, 2003) reported a range of viscosity values from 2 to 3.6 mPa s for serums obtained from milk fermented with four strains of EPSþ L. lactis subsp. cremoris. It can be assumed that the EPS produced in the stages before gelation caused an increase in the viscosity of the serum. For example, if the viscosity of the serum due to the presence of EPS were 3 mPa s, i.e., three times greater than the background viscosity value used in calculations of particle radii from DWS, the diffusion coefficient
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would be significantly lower (about three times) than the one measured in the present study. Again, as no such changes in diffusion coefficient were observed in the EPSþ fermented milk, it can be concluded that if any EPS was produced by L. lactis subsp. cremoris JFR1 during fermentation, it was at levels below detection by DWS, which, on the other hand, is very sensitive to changes in background viscosity, refractive index contrast, particleeparticle interactions and mobility of the casein micelles. The fermentation of skim milk inoculated with either EPSþ or EPS strain was performed in situ in the rheometer and the development of gel structure was followed using small amplitude shear strain oscillatory testing. Table 1 summarises the rheological parameters measured during acidification of milk fermented with EPSþ and EPS strains. Milk fermented with EPSþ strain required more time to reach gelation point (Tgel) as well as pH 4.6 (TpH 4.6) than milk fermented with the EPS-free culture. However, the rate of pH drop below pH w6.2 was similar for both samples (data not shown). The production of EPS did not have a significant effect (P > 0.05) on the pH of gelation (pHgel) (Table 1), which is in agreement with previously published results (Van Marle & Zoon, 1995). It is important to note that, as mentioned above, the values of diffusion coefficient of the micelles did not vary between EPSþ and EPS samples; therefore, a similar behaviour between the two systems at the pH of gelation was expected. The values of pH of gelation determined from rheological data (5.30 and 5.29 for EPSþ and EPS samples, respectively) were in agreement with the pH of aggregation measured by DWS (5.21 and 5.23 for milk gels fermented with EPSþ and EPS strains, respectively). Fig. 2 shows representative profiles of G0 , G00 , and tan d of milk samples fermented with EPSþ or EPS strains as a function of pH. Although EPS did not show any impact on the fermentation process before and during aggregation of casein micelles, differences were noted at the later stages of the gelation process. The presence of EPS contributed to a stiffer gel network with significantly higher (P < 0.05) G0 values at the end of fermentation (Table 1 and Fig. 2). Previous authors (Girard & Schaffer-Lequart, 2007b) correlated the higher firmness of some of the EPS containing milk gels to stronger caseinecasein interactions due to depletion flocculation caused by a combination of negative charge, sulphate groups and high molecular weight of these EPS. However, stiffer gels could also derive from bridging of the polysaccharide molecules with whey proteins and casein particles within the network (Acero-Lopez et al., 2010). Other studies have reported lower G0 values (Doleyres et al., 2005; Hassan, Corredig, & Frank, 2001; Hassan, Ipsen, Janzen, & Qvist, 2003b; Hess, Roberts, & Ziegler, 1997) or no difference (Girard & Schaffer-Lequart, 2007a; Van Marle & Zoon, 1995) on the G0 values when milk was fermented with an EPSproducing culture compared with a control. Table 1 Effect of exopolysaccharide (EPS) production on the rheological parameters measured during fermentation of milk with EPS-producing (EPSþ) and EPS-non producing (EPS) cultures. Parametera
EPSþ
EPS
Tgel (min) TpH 4.6 (min) pHgel G0 pH 4.6 (Pa) Tan dmax Tan dpH 4.6 pHtand max
370 8a 544 20a 5.30 0.07a 324 48a 0.409 0.002a 0.319 0.012a 4.82 0.09a
279 17b 402 35b 5.29 0.05a 249 39b 0.388 0.003b 0.297 0.011b 4.88 0.07a
a Definitions are: Tgel, time (min) at which G0 ¼ G00 ; TpH 4.6, time (min) from inoculation to pH 4.6; pHgel, pH at which G0 ¼ G00 ; G0 pH 4.6, storage modulus at pH 4.6; Tan dmax, maximum in tan d; Tan dpH 4.6, tan d at pH 4.6; pHtand max, pH at which the maximum in tan d is observed. Different superscript letters in the same row indicate significant differences determined by Student’s t-test (P < 0.05); standard deviation is also shown.
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Fig. 2. Rheological parameters G0 (C, ;), G00 ( , ) and tan d (B, 7) measured during fermentation of milk containing exopolysaccharide- (EPS-) producing (C, , B) and EPS-non producing (;, , 7) strain at 30 C as a function of pH. Curves are representative runs (see Table 1 for statistical analysis).
A maximum in tan d was observed in both samples at pH 4.8 (Fig. 2 and Table 1). The maximum in tan d can be attributed to a partial loosening of the caseinecasein interactions due to solubilisation of colloidal calcium phosphate, which causes an increase in charge repulsion between micelles, before the enhancement of mutual casein interactions as the pH decreases towards the isoelectric point of caseins and the repulsion between casein particles is reduced (Lucey et al., 1997). Other authors (Van Marle & Zoon, 1995) reported also no significant difference in the pH of tan dmax during gel formation of milk acidified with different EPS-producing cultures and GDL. As was shown from DWS measurements, no effect of EPS on the caseins in the stages preceding gelation was observed. However, the values of tan dmax and tan d plateau at pH 4.6 were significantly higher for the samples containing EPS compared with the control samples (Table 1), implying a stronger contribution of the viscous component to the overall viscoelastic character of the protein network of gels made with the EPSþ strain. A higher tan d of casein gels indicates that a higher number of proteineprotein bonds relax, allowing more rearrangements of the network structure (van Vliet, van Dijk, Zoon, & Walstra, 1991). Furthermore, the time at which EPS production takes place may have a significant contribution on the formation and rearrangements of casein network (Ruas-Madiedo & Zoon, 2003). The EPS that may be synthesised after the aggregation of the casein micelles commences, will be limited mainly in pockets around bacteria. Such separate domains of protein aggregates and pockets with EPS will be under continuous dynamic changes as the pH drops towards the isoelectric point of caseins (pH 4.6) and more EPS is produced in the pores. This dynamic environment would allow for thicker strands (higher G0 ) and fast and intense rearrangements to occur in the range of pH 5.3e4.6, which could count for the higher tan dmax and tan d at pH 4.6 values for EPSþ gels. The viscosity of fermented milk as a function of shear rate measured in steady state after cooling of samples at 4 C is depicted in Fig. 3. As the shear rate increased the happ of both samples decreased, a relationship that shows shear thinning characteristics of these gels. The presence of EPS brought about significantly higher apparent viscosity of fermented milk compared with the sample without EPS (Table 2), which is in agreement with previous published work (Doleyres et al., 2005; Haque, Richardson, & Morris, 2001; Hassan et al., 2003b). Furthermore, the gels showed different viscosity profiles; when the gel containing EPS was sheared, the
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Fig. 3. Apparent viscosity as a function of shear rate measured at 4 C for gels fermented with exopolysaccharide- (EPS-) producing (C) and EPS-non producing (;) strains. The data are average of three replicate experiments. Error bars indicate standard deviations. The Carreau fit is indicated by a solid line.
Fig. 4. Storage modulus (G0 ) of milk gels fermented with the exopolysaccharide- (EPS-) producing (C) or EPS-non producing (;) before structural breakdown (interval I) and recovery of G0 after shearing (interval III). Interval II represents shearing of sample at 500 s1 for 20 min, 4 C.
viscosity at low shear rates showed a tendency to level off (Fig. 3); this was not the case for EPS samples, where the viscosity decreased continuously with shear rate from the beginning of the shear rate range studied. The rheological model of HerscheleBulkley, which includes a yield stress term, did not fit to stress/strain functions of fermented milk containing EPS, while it fitted well to the data of control samples (R2 ¼ 0.98) (data not shown). On the other hand, the Carreau model, which includes a zero shear and infinite shear viscosity term fitted well (R2 ¼ 0.98) the EPSþ flow data, but did not fit the experimental curves of milk fermented with the EPS-free culture (Fig. 3), once again confirming the difference in their flow behaviour.
contained EPS compared with the ones made with EPS strains. The ratios of G0 after 60 min or 600 min of recovery over G0 before shearing, expressed in percentage, were calculated and used as indicators of the capability of the structure of milk gels to recover after shearing. Milk gels containing EPS showed much lower structure recovery compared to the control gels. Thus, EPSþ gels, 1 h after shearing stopped, recovered 0.3 0.07% of their original G0 compared with 9.5 5.2% for EPS fermented milk (Fig. 4). After 10 h of recovery these figures were 0.7 0.07 and 13.2 6.4%, respectively. This is in agreement with the reports of other authors (Folkenberg et al., 2006; Girard & Schaffer-Lequart, 2007b), who also showed that yoghurts containing EPS have a reduced ability to recover the structure after shearing compared with EPS-free gels. In contrast, it has been previously suggested that EPS could, at least partially, protect yogurt against the breakdown of the protein network or impart some resistance of yoghurt structure to stirring (Folkenberg, Dejmek, Skriver, & Ipsen, 2005).
3.2. Structural breakdown and recovery of the gels at 4 C The recovery of the gel structure after shearing at 500 s1 for 20 min was followed for 10 h at 4 C using dynamic oscillatory measurements at 1 Hz and 0.1% strain. The development of storage modulus G0 with time before shearing (interval I) and during recovery after shearing (interval III) is shown in Fig. 4. There was a fast increase in G0 over the first few minutes after ceasing of shearing and a much slower, but steady increase of G0 thereafter for both samples (Fig. 4, interval III). Similar recovery profiles of stirred yogurts have been reported by others (Arshad et al., 1993; Haque et al., 2001). Stirring caused lower G0 values for samples containing EPS compared with EPS gels (Fig. 4), and this was in contrast with the results of the undisturbed gel, where G0 vas higher for EPSþ. Similarly, Doleyres et al. (2005) and Hassan et al. (2003b) reported lower storage modulus values of stirred yogurts that Table 2 Apparent viscosity at selected shear rates for gels fermented with exopolysaccharide- (EPS-) producing (EPSþ) and EPS-non producing (EPS) strains.a Shear rate (s1)
Apparent viscosity (Pa s) EPSþ
11 47 121
EPS a
1.75 0.48 0.46 0.04a 0.27 0.06a
0.45 0.05b 0.20 0.02b 0.13 0.01b
a The data are average of three replicate experiments and values are means standard deviation. Measurements were performed at 4 C; values within the same row, followed by different superscript letters are significantly different (P < 0.05) as determined by Student’s t-test.
3.3. Microstructure of intact and stirred fermented milk Micrographs of set and stirred fermented milk prepared with EPSþ and EPS strains are shown in Fig. 5. The protein network was stained with rhodamine B and appears red in the images. Unstirred gels made with the EPSþ strain showed a network of thick, continuous protein aggregates, with large void spaces (Fig. 5a). On the other hand, control gels, fermented with EPS culture showed a network of fine protein strands and small pores (Fig. 5c). For the set-type gels (Fig. 5a and c) the micrographs are similar to those published by others (Hassan, Frank, & Qvist, 2002; Hassan et al., 2003b), who reported the formation of thick, dense protein strands in the presence of EPS with EPS being located in the channels of serum between protein aggregates. These results also would confirm the results shown in rheological testing (Fig. 2). Therefore, it can be concluded that the EPS was present in the serum pores and caused the formation of thicker strands. As discussed above, if a considerable amount of EPS was produced after the onset of aggregation of casein micelles, it would be entrapped in pockets between casein clusters and constrained around the bacteria cells. Such spatial constrain will reduce the probability of possible interactions of EPS with casein micelles and induce an intensification of the mutual interactions of casein particles in the
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Fig. 5. Confocal laser scanning micrographs of unstirred (a, c) and stirred (b, d) milk samples fermented with EPSþ (exopolysaccharide producing) strain (a, b) and EPS (EPS-non producing) strain (c, d). Bar ¼ 50 mm. (For interpretation of reference to color in this figure, please refer to the web version of this article.)
presence of highly concentrated regions of EPS. Other authors (Sanchez et al., 2000) have previously reported a very compact casein network with large pores in GDL acidified skim milk in the presence of 0.1% xanthan gum, which they attributed mainly to an incompatibility between casein particles and xanthan. The microstructure of stirred gels is shown in Fig. 5b and d. Shearing changed the microstructure of EPSþ fermented milk causing the formation of a larger number of pores and channels and less compact protein aggregates. It has been previously shown (Hassan et al., 2002, 2003b) that stirring results in formation of channels that contain EPS, concentrated in larger strands than those observed in the undisturbed gels. This was attributed to intense mutual interactions and aggregation of EPS molecules in the continuous serum phase due to incompatibility with casein micelles. The microstructure of EPSþ fermented milk after shearing (Fig. 5b) shows much less connectivity between protein aggregates, most likely because of pockets enriched with EPS that surround the casein aggregates. This type of microstructure would explain the lower ability to recover after structural breakdown due to shearing that was observed in Fig. 4. In EPS gels, stirring resulted in a more aggregated protein network with more connectivity between protein strands and smaller pores compared to the stirred gels containing EPS (Fig. 5d), in agreement with previous reports (Hassan et al., 2003b). It is important to note that the microstructure of the stirred control gels does not seem very different from that of the intact gel with the greatest noticeable difference being a more dense and aggregated
structure after stirring. This type of more spanning structure with greater connectivity is more likely to show better recovery after shearing compared with the structure of fermented milk gels containing EPS. 4. Conclusions The results obtained from DWS measurements of fermentation with EPSþ and EPS cultures suggested that EPS produced by L. lactis subsp. cremoris JFR1 during milk fermentation at 30 C did not affect the preceding stages of gelation as it did not change the properties of the casein micelles or their aggregation behaviour. While the presence of EPS did not change the pH of gelation, it affected the formation and rheological properties of the three dimensional network of casein micelles. The presence of EPS resulted in a stiffer gel, with a higher viscous component (higher tan dmax and tan dpH 4.6), which is translated into greater ability for rearrangements of the casein micelles during gel formation. Substantial effect of EPS was seen after cooling of the gel at 4 C, when a microstructure of large, dense protein clusters and large void pores was observed. After shearing, the microstructure of gels containing EPS changed and the gels showed a lower ability to recover than gels not containing EPS. It is hypothesised that there was little production of EPS up to the point of gelation, and the EPS produced after casein micelle aggregation concentrated in pockets around protein clusters, playing an important role in the formation and properties the EPSþ gel.
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Acknowledgements The authors would like to thank Dr. A. Hassan for donating the JFR1 culture. This work was funded by the Natural Sciences and Engineering Council of Canada and the Ontario Dairy Council through the Industrial Research Chair program.
References Acero-Lopez, A., Alexander, M., & Corredig, M. (2010). Diffusing wave spectroscopy and rheological studies of rennet-induced gelation of skim milk in the presence of pectin and k-carrageenan. International Dairy Journal, 20, 328e335. Alexander, M., & Dalgleish, D. G. (2004). Application of transmission diffusing wave spectroscopy to the study of gelation of milk by acidification and rennet. Colloids and Surfaces B: Biointerfaces, 38, 83e90. Arshad, M., Paulsson, M., & Dejmek, P. (1993). Rheology of buildup, breakdown, and rebodying of acid casein gels. Journal of Dairy Science, 76, 3310e3316. Ayala-Hernández, I., Hassan, A., Goff, H. D., Mira de Orduna, R., & Corredig, M. (2008). Production, isolation and characterization of exopolysaccharides produced by Lactococcus lactis subsp. cremoris JFR1 and their interaction with milk proteins: effect of pH and media composition. International Dairy Journal, 18, 1109e1118. Azim, Z., Corredig, M., Koxholt, M., & Alexander, M. (2010). Sol gel transitions during acid gelation of milk containing modified waxy maize starch. Differences between chemical and bacterial acidification measured using rheological and spectroscopic techniques. International Dairy Journal, 20, 785e791. Cerning, J. (1995). Production of exopolysaccharides by lactic acid bacteria and dairy propionibacteria. Lait, 75, 463e472. Doleyres, Y., Schaub, L., & Lacroix, C. (2005). Comparison of the functionality of exopolysaccharides produced in situ or added as bioingredients on yogurt properties. Journal of Dairy Science, 88, 4146e4156. Folkenberg, D. M., Dejmek, P., Skriver, A., & Ipsen, R. (2005). Relation between sensory texture properties and exopolysaccharide distribution in set and in stirred yoghurts produced with different starter cultures. Journal of Texture Studies, 36, 174e189. Folkenberg, D. M., Dejmek, P., Skriver, A., Skov Guldager, H., & Ipsen, R. (2006). Sensory and rheological screening of exopolysaccharide producing strains of bacterial yoghurt cultures. International Dairy Journal, 16, 111e118. Girard, M., & Schaffer-Lequart, C. (2007a). Gelation and resistance to shearing of fermented milk: role of exopolysaccharides. International Dairy Journal, 17, 666e673. Girard, M., & Schaffer-Lequart, C. (2007b). Gelation of skim milk containing anionic exopolysaccharides and recovery of texture after shearing. Food Hydrocolloids, 21, 1031e1040. Girard, M., & Schaffer-Lequart, C. (2008). Attractive interactions between selected anionic exopolysaccharides and milk proteins. Food Hydrocolloids, 22, 1425e1434. Haque, A., Richardson, R. K., & Morris, E. R. (2001). Effect of fermentation temperature on the rheology of set and stirred yogurt. Food Hydrocolloids, 15, 593e602.
Hassan, A. N., Corredig, M., & Frank, J. F. (2001). Viscoelastic properties of yogurt made with ropy and non-ropy exopolysaccharides producing cultures. Milchwissenschaft e Milk Science International, 56, 684e687. Hassan, A. N., Frank, J. F., & Elsoda, M. (2003a). Observation of bacterial exopolysaccharide in dairy products using cryo-scanning electron microscopy. International Dairy Journal, 13, 755e762. Hassan, A. N., Frank, J. F., & Qvist, K. B. (2002). Direct observation of bacterial exopolysaccharides in dairy products using confocal scanning laser microscopy. Journal of Dairy Science, 85, 1705e1708. Hassan, A. N., Ipsen, R., Janzen, T., & Qvist, K. B. (2003b). Microstructure and rheology of yogurt made with cultures differing only in their ability to produce exopolysaccharides. Journal of Dairy Science, 86, 1632e1638. Hess, S. J., Roberts, R. F., & Ziegler, G. R. (1997). Rheological properties of nonfat yogurt stabilized using Lactobacillus delbrueckii ssp. bulgaricus producing exopolysaccharide or using commercial stabilizer systems. Journal of Dairy Science, 80, 252e263. Kristo, E., Biliaderis, C. G., & Tzanetakis, N. (2003). Modelling of rheological, microbiological and acidification properties of a fermented milk product containing a probiotic strain of Lactobacillus paracasei. International Dairy Journal, 13, 517e528. Lee, W. J., & Lucey, J. A. (2006). Impact of gelation conditions and structural breakdown on the physical and sensory properties of stirred yogurts. Journal of Dairy Science, 89, 2374e2385. Lucey, J. A., Teo, C. T., Munro, P. A., & Singh, H. (1997). Rheological properties at small (dynamic) and large (yield) deformations of acid gels made from heated milk. Journal of Dairy Research, 64, 591e600. Ruas-Madiedo, P., Hugenholtz, J., & Zoon, P. (2002). An overview of the functionality of exopolysaccharides produced by lactic acid bacteria. International Dairy Journal, 12, 163e171. Ruas-Madiedo, P., & Zoon, P. (2003). Effect of exopolysaccharide-producing Lactococcus lactis strains and temperature on the permeability of skim milk gels. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 213, 245e253. Sanchez, C., Zuniga-Lopez, R., Schmitt, C., Despond, S., & Hardy, J. (2000). Microstructure of acid-induced skim milkelocust bean gumexanthan gels. International Dairy Journal, 10, 199e212. Tuinier, R., ten Grotenhuis, E., Holt, C., Timmins, P. A., & de Kruif, C. G. (1999). Depletion interaction of casein micelles and an exocellular polysaccharide. Physical Review E, 60, 848e856. Tuinier, R., van Casteren, W. H. M., Looijesteijn, P. J., Schols, H. A., Voragen, A. G. J., & Zoon, P. (2001). Effects of structural modifications on some physical characteristics of exopolysaccharides from Lactococcus lactis. Biopolymers, 59, 160e166. Van Marle, M. E., & Zoon, P. (1995). Permeability and rheological properties of microbially and chemically acidified skim-milk gels. Netherlands Milk Dairy Journal, 49, 47e65. van Vliet, T., van Dijk, H. J. M., Zoon, P., & Walstra, P. (1991). Relation between syneresis and rheological properties of particle gels. Colloid and Polymer Science, 269, 620e627. Weitz, D. A., & Pine, D. J. (1993). Diffusing wave spectroscopy. In W. Brown (Ed.), Dynamic light scattering, the methods and some applications (pp. 652e720). Oxford, UK: Oxford University Press.
Contents lists available at ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
The role of exopolysaccharide produced by Lactococcus lactis subsp. cremoris in structure formation and recovery of acid milk gels Eleana Kristo*, Zhutuan Miao, Milena Corredig Food Science Department, University of Guelph, Guelph, Ontario N1G 2W1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 June 2010 Received in revised form 2 February 2011 Accepted 9 February 2011
The effect of exopolysaccharide (EPS) produced by Lactococcus lactis subsp. cremoris JFR1 on milk fermentation and recovery of gel structure after stirring was studied using rheology and diffusing wave spectroscopy. Skim milk fermented with an EPS-non producing (EPS) strain of L. lactis served as control. The production of EPS was not sufficient to affect the stages preceding aggregation. No differences were found in the changes in diffusion coefficient, radius and turbidity parameter of casein particles up to the point of gelation between EPSþ and EPS samples. However, the presence of EPS significantly increased the storage modulus and viscosity of milk gel, and reduced the ability of the gel to recover after shearing. This study indicates that although the present EPS does not affect the preliminary stages of gelation, it is produced in sufficient quantities after gelation to significantly alter the structure of milk gels in their set or stirred form. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Many mesophilic and thermophilic lactic acid bacteria (LAB) strains, commonly used in dairy processing, excrete heteropolysaccharides, containing mainly D-glucose, L-rhamnose and D-galactose (Ruas-Madiedo, Hugenholtz, & Zoon, 2002). Exopolysaccharides (EPS) produced by LAB can freely be released into the medium or be attached to the cell. When present free in the medium, they may impart a ropy appearance to the milk gels. LAB that produce EPS are often combined in commercial starters, as the EPS may function as natural stabilisers and thickeners in fermented milk products. In situ production of EPS from LAB may replace the additives and stabilisers that are usually employed to ensure a smooth and creamy texture of fermented milk products. Studies have shown that the effect of EPS in the rheological properties of final fermented milk is more pronounced and with a better outcome when EPS is produced in situ than when added as an ingredient (Doleyres, Schaub, & Lacroix, 2005). The amount of EPS produced by LAB during milk fermentation varies with data reported in literature ranging from 50 to 600 mg L1 (Cerning, 1995). Most often very little is recovered from the fermented milk, suggesting that EPS may influence the texture and stability of fermented products even when it is present in low amounts. Therefore, the desirable structuring properties of EPS
* Corresponding author. Tel.: þ1 519 824 4120; fax: þ1 519 824 6631. E-mail address: [email protected] (E. Kristo). 0958-6946/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2011.02.002
have been attributed to factors other than concentration, such as type of linkage, presence of side groups, branched structure, molecular weight (Ruas-Madiedo et al., 2002; Tuinier et al., 2001), and/or interactions with milk proteins (Ayala-Hernández, Hassan, Goff, Mira de Orduna, & Corredig, 2008; Tuinier, ten Grotenhuis, Holt, Timmins, & de Kruif, 1999). In addition to the dynamic changes occurring to casein micelles, colloidal calcium phosphate, whey proteins and other milk components (Alexander & Dalgleish, 2004; Lucey, Teo, Munro, & Singh, 1997), resulting ultimately in the formation of a protein network, the process of fermentation of milk with LAB producing EPS includes also the dynamics of production and incorporation of EPS. That is to say that EPS is produced gradually by LAB during acidification, and, as the environmental conditions change, the effect of the polysaccharide on the proteins will continue to evolve. It can be hypothesised that the balance of repulsive/attractive interactions of EPS with milk proteins will continuously change during fermentation, and a better understanding of the dynamics will allow a better control of the final characteristics of the milk gel (Girard & Schaffer-Lequart, 2008; Sanchez, Zuniga-Lopez, Schmitt, Despond, & Hardy, 2000). In addition, during processing of stirred type yoghurts and other fermented milk products, the gels are disrupted due to shearing and pumping. Despite the practical importance of the structure breakdown and recovery of these types of gels and the role played by EPS in the gel when shearing is applied, this aspect has not been fully explored (Arshad, Paulsson, & Dejmek, 1993; Girard & Schaffer-Lequart, 2007b).
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Transmission diffusing wave spectroscopy (DWS) is a nondisruptive method that is based on multiple scattering of light from particles in a sample, which allows the study of concentrated food systems (Weitz & Pine, 1993). DWS has been employed to derive information on the changes in the early stages preceding aggregation of casein micelles during acidification or rennet coagulation of milk (Alexander & Dalgleish, 2004). A multitechnique approach, combining DWS, rheology and microscopic observations would successfully describe the dynamics occurring during fermentation, from the beginning stages of casein destabilisation to the formation of the gel network. The objective of this study was to investigate the role of EPS produced in situ from the ropy strain of Lactococcus lactis subsp. cremoris JFR1 on various stages of milk fermentation process, on the formation of gel structure as well as on the recovery of structure after shearing. 2. Materials and methods 2.1. Preparation of strains L. lactis subsp. cremoris (JFR1), a highly ropy strain (Hassan, Frank, & Elsoda, 2003a) was obtained from Dr. Hassan’s collection at South Dakota State University. A L. lactis strain that did not produce EPS was obtained from Chr. Hansen (Hørsholm, Denmark) and was used as a control. The strains were subcultured through two transfers in M17 broth (Difco, Becton Dickinson Co., Sparks, MD, USA) at 30 C for 18 h; 0.2 mL from the prepared microbial suspension of the last transfer was inoculated in tubes with 7 mL of 10% reconstituted skim milk and kept in a freezer (20 C) without incubation until use. To prepare the inoculum for milk fermentation, the frozen strains were incubated for one night at 30 C. Nine decimal dilutions in sterile peptone water (0.1%, v/v) were prepared from the coagulated milk, and 0.2 mL from each dilution (including undiluted sample) was inoculated in tubes with 9 mL of sterilised reconstituted skim milk powder (10%, w/v). The inoculated tubes were mixed in a vortex and incubated at 30 C for 24 h. From these series of tubes, the last dilution showing milk coagulation was used as inoculum (Kristo, Biliaderis, & Tzanetakis, 2003).
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for each separate experiment, to be able to plot the parameters measured by rheology or DWS as a function of pH. 2.3. Diffusing wave spectroscopy Aliquots (1 mL) of milk were poured into a rectangular flat-faced optical glass cuvette (Hellma Canada Limited, Concord, ON, Canada) with a path length 5 mm, which was placed in a water tank maintained at 30 C. The source of light was a solid state diode pumped Nd:YAG (Yttrium Aluminium Garnet) laser (VERDI) 532 nm, maximum output power 2 W (Coherent, Santa Clara, CA, USA). Two glass windows of the water tank allowed incident and scattered light beam to pass. A single fibre optic, that collects the transmitted scattered light, was bifurcated and fed to two matched photomultipliers (HC120-03, Hamamatsu, Loveland, OH, USA) and a correlator (FLEX2K-12 2, Correlator.com, Bridgewater, NJ, USA). The intensity of laser was calibrated daily by means of standard latex spheres with a diameter of 260 nm (Portland Duke Scientific, Palo Alto, CA, USA) dispersed in MilliQ water. Correlation functions and intensity of the transmitted scattered light were measured at intervals of 3 min until the end of fermentation. Data were analysed using specialised software (DWS-Fit, Mediavention Inc., Guelph, ON, Canada). Each experiment was repeated at least 3 times, starting from different batches of fresh skim milk. Transmission DWS detects the intensity of the light scattered multiple times from the dispersed particles and collected after it has travelled the length (L) of the scattering cell. The fluctuation of the scattered light that happens due to the Brownian motion of particles can be characterised by means of the correlation function (Weitz & Pine, 1993). The value of l*, the photon transport mean free path, can be calculated from the total intensity of the light scattered by the sample and the laser intensity measured using 260 nm latex spheres dispersed in water. The diffusion coefficient D (m2 s1) is calculated from the correlation function using a value of 1.34 for n, the refractive index of milk serum. From the diffusion coefficient D, the radius of the particle can be calculated using the StokeseEinstein equation (Weitz & Pine, 1993). The pH of aggregation was determined as the pH value denoting the rapid growth of particle size compared to the original particle size. It was extrapolated from the curves of particle radii as a function of pH using the tangent graphical method at a radius scale of 2 mm.
2.2. Production of fermented milk
2.4. Rheological measurements
Aliquots of 97 mL of pasteurised skim milk (Crown Dairy Ltd., Guelph, ON, Canada) were distributed in sterilised 100 mL glass bottles. Milk was heated at 90 C for 20 min (not including come up time, w10 min) in a water bath to ensure a nearly complete heat denaturation of the whey proteins. The bottles were cooled by placing them into ice water. Three millilitres from the tube containing the inoculum were added into the bottle under aseptic conditions to prevent contamination. Twenty millilitres of inoculated milk was added in the cup of the rheometer for rheological measurements. Also, a few millilitres were added into a tube and the DWS cuvette for pH and DWS measurements, respectively. The cup and bob of the rheometer, the cuvette of DWS, the tube used in pH measurement and the glass electrode were cleaned with ethanol before use. Milk bottles were then incubated at 30 C until pH 4.6 was reached, when the bottles were cooled fast by immersing them into ice water and then stored in the refrigerator at 4 C. The pH of samples was monitored as a function of time with a pH meter AR15 (Fisher Scientific, Mississauga, ON, Canada). The changes of pH as a function of time were analysed by curve fitting using SigmaPlot version 10 (Systat Software, Inc., San Jose, CA, USA)
A Physica MCR 301 rheometer (Anton Paar Germany GmbH, Ostfildern, Germany) with a measuring cell C-PTD200 equipped with a Peltier temperature controller and a concentric cylinder geometry (diameter of cup and bob: 28.92 and 26.66 mm, respectively) was used for rheological measurements. A Julabo circulator (Julabo West, Inc., CA, USA) was used as counter cooling system for the Peltier element. Twenty millilitres of inoculated sample were added into the cup and a solvent trap was used to cover the cup and prevent sample evaporation. Oscillatory measurements were performed at 0.1% strain and frequency of 1 Hz at 30 C. Storage modulus (G0 ), loss modulus (G00 ), and loss tangent (tan d) were tracked for as long as required for the sample to reach pH 4.6. Gelation point was arbitrarily defined as the pH or time when G0 ¼ G00 . Once the samples reached pH 4.6, the temperature in the rheometer cell was decreased to 4 C in 30 min and an oscillatory time sweep measurement was performed for 10 min at 4 C, at 0.1% strain and frequency of 1 Hz, followed by a steady state viscosity measurement over the range of shear rates of 0.1e500 s1. After reaching 500 s1, this shear rate was maintained for 20 min to disrupt the gel, as previously reported (Girard & Schaffer-Lequart, 2007b) and the recovery of structure was then followed by
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measurement of G0 , G00 and tan d for 10 h at 4 C using 0.1% strain and frequency of 1 Hz. Flow curves were fitted with the rheological models of HerscheleBulkley and Carreau (Lee & Lucey, 2006). 2.5. Confocal laser scanning microscopy The microstructure of set and stirred fermented milk was observed using a confocal laser scanning inverted microscope (Leica TCS SP2, model Leica DM IRE2, Leica Microsystems CMS GmbH, Mannheim, Germany) with an Ar/Kr visible light laser and 40 (oil) objective. The fluorescent dye rhodamine B (excitation and emission wavelengths 543 and 625 nm, respectively) was used to stain the protein network. Resolution of the acquired digital images was 1024 1024 pixels. Twenty microlitres of 0.2% (w/v) aqueous solution of rhodamine B were mixed with 5 mL inoculated milk; a few drops of the mixture were transferred to a slide with cavity and covered with a cover slip. The slide was placed in an incubator at 30 C until the pH of the sample dropped to pH 4.6 (as indicated from the measurements in pH meter). The slide was subsequently stored at 4 C overnight and used next day to take images of the set fermented milk. Preparation of stirred sample for confocal laser scanning microscopy (CLSM) testing was done by adding a few drops of 0.2% (w/v) of rhodamine B solution on a small portion of fermented milk that was stirred in the rheometer at 4 C, 500 s1 for 20 min, and transferred in a glass bottom dish. The sample was kept at 4 C for 1 h to allow diffusion of the fluorescent dye before observation in the microscope. 2.6. Statistical analysis All experiments were repeated at least three times. The results reported in the tables are means standard deviations. Statistical analysis was carried out using SigmaPlot version 10 (Systat Software, Inc., San Jose, CA, USA). Statistical significance at P < 0.05 was determined using Student’s t-test. Confocal images are representative of at least three observations of each separate replicate. 3. Results and discussion 3.1. Gelation properties The aggregation of casein micelles during fermentation was followed using DWS, and the development of apparent particle radius (diffusion coefficient in inset Fig. 1) and turbidity parameter 1/l* are shown as a function of pH in Fig. 1. The turbidity parameter 1/l* of the control sample increased continuously throughout fermentation. Such behaviour of 1/l* has been previously reported for acidification of heated milk with glucono-d-lactone (GDL) (Alexander & Dalgleish, 2004; Azim, Corredig, Koxholt, & Alexander, 2010) or fermentation of heated milk with a commercial culture (Azim et al., 2010). The increase in the turbidity parameter 1/l* of milk fermented with the EPSþ strain was comparable with that of EPS-free fermented milk, implying that similar changes occurred in the casein micelles during fermentation. The value of 1/l* depends on the physical properties of the micelles as well as particleeparticle interactions, and the results shown in Fig. 1 suggest that the presence of EPSþ culture did not affect physical parameters such as the refractive index contrast or the structural organisation of the micelles. It is important to note that previous work has demonstrated that in the presence of a phase separating polysaccharide, the value of 1/l* decreases, because of structuring of casein micelles in microphase separated clusters (Acero-Lopez, Alexander, & Corredig, 2010). In the present work, no differences were noted in the values of 1/l* between EPSþ and EPS fermented
Fig. 1. Light scattering parameters 1/l* (B, 7), apparent particle radius (C, ;) and (insert) diffusion coefficient (C, ;) measured by diffusing wave spectroscopy as a function of pH during fermentation of milk with the exopolysaccharide- (EPS-) producing (C, B) and EPS-non producing strain (;, 7).
milk samples, suggesting the lack of depletion flocculation in the early stages of acidification. In addition, it is clear from Fig. 1 that up to pH 5.5 no changes occurred in the apparent radius of the casein micelles. When comparing the development of apparent radius (Fig. 1) and diffusion coefficient (Fig. 1 inset) of casein micelles during fermentation of milk with the EPSþ and EPS culture, no significant differences were found between the two treatments. The aggregation of casein micelles was marked by a sharp increase of the apparent particle radius that allowed for determination of a pH of aggregation. This value was not significantly different for EPSþ and EPS fermented milk (5.21 0.07 and 5.23 0.11, respectively). These findings indicate that the production of EPS from L. lactis subsp. cremoris JFR1 does not have a significant effect on the casein micelles in the stages preceding aggregation. An average radius of gyration of 68 and 86 nm has been previously reported (Ayala-Hernández et al., 2008) for the EPS produced by L. lactis subsp. cremoris JFR1 during fermentation of milk permeate supplemented with 2% whey protein isolate at the constant pH of 6.5 and 5.5, respectively. It can be hypothesised that if EPS was produced and interacted with the casein micelles, a thick layer of EPS of the order of the radius of gyration would form, and this would significantly increase the radius of the casein micelles by at least 60e80 nm. However, no changes were noted in the apparent radius and diffusion coefficient of the caseins. It has also been hypothesised that the EPS produced by L. lactis subsp. cremoris JFR1 is negatively charged (Ayala-Hernández et al., 2008), and at pH > 5.6 this should cause microphase separation of the casein micelles by means of a depletion flocculation mechanism (Tuinier et al., 1999); again, the present work showed no differences in the turbidity parameter and the diffusion coefficient between the control and the milk fermented with EPSþ culture. It is also important to point out that an increase of the viscosity of the continuous phase due to the presence of EPS should also result in a lower diffusivity of the casein micelles compared to the control. Previous authors (Ruas-Madiedo & Zoon, 2003) reported a range of viscosity values from 2 to 3.6 mPa s for serums obtained from milk fermented with four strains of EPSþ L. lactis subsp. cremoris. It can be assumed that the EPS produced in the stages before gelation caused an increase in the viscosity of the serum. For example, if the viscosity of the serum due to the presence of EPS were 3 mPa s, i.e., three times greater than the background viscosity value used in calculations of particle radii from DWS, the diffusion coefficient
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would be significantly lower (about three times) than the one measured in the present study. Again, as no such changes in diffusion coefficient were observed in the EPSþ fermented milk, it can be concluded that if any EPS was produced by L. lactis subsp. cremoris JFR1 during fermentation, it was at levels below detection by DWS, which, on the other hand, is very sensitive to changes in background viscosity, refractive index contrast, particleeparticle interactions and mobility of the casein micelles. The fermentation of skim milk inoculated with either EPSþ or EPS strain was performed in situ in the rheometer and the development of gel structure was followed using small amplitude shear strain oscillatory testing. Table 1 summarises the rheological parameters measured during acidification of milk fermented with EPSþ and EPS strains. Milk fermented with EPSþ strain required more time to reach gelation point (Tgel) as well as pH 4.6 (TpH 4.6) than milk fermented with the EPS-free culture. However, the rate of pH drop below pH w6.2 was similar for both samples (data not shown). The production of EPS did not have a significant effect (P > 0.05) on the pH of gelation (pHgel) (Table 1), which is in agreement with previously published results (Van Marle & Zoon, 1995). It is important to note that, as mentioned above, the values of diffusion coefficient of the micelles did not vary between EPSþ and EPS samples; therefore, a similar behaviour between the two systems at the pH of gelation was expected. The values of pH of gelation determined from rheological data (5.30 and 5.29 for EPSþ and EPS samples, respectively) were in agreement with the pH of aggregation measured by DWS (5.21 and 5.23 for milk gels fermented with EPSþ and EPS strains, respectively). Fig. 2 shows representative profiles of G0 , G00 , and tan d of milk samples fermented with EPSþ or EPS strains as a function of pH. Although EPS did not show any impact on the fermentation process before and during aggregation of casein micelles, differences were noted at the later stages of the gelation process. The presence of EPS contributed to a stiffer gel network with significantly higher (P < 0.05) G0 values at the end of fermentation (Table 1 and Fig. 2). Previous authors (Girard & Schaffer-Lequart, 2007b) correlated the higher firmness of some of the EPS containing milk gels to stronger caseinecasein interactions due to depletion flocculation caused by a combination of negative charge, sulphate groups and high molecular weight of these EPS. However, stiffer gels could also derive from bridging of the polysaccharide molecules with whey proteins and casein particles within the network (Acero-Lopez et al., 2010). Other studies have reported lower G0 values (Doleyres et al., 2005; Hassan, Corredig, & Frank, 2001; Hassan, Ipsen, Janzen, & Qvist, 2003b; Hess, Roberts, & Ziegler, 1997) or no difference (Girard & Schaffer-Lequart, 2007a; Van Marle & Zoon, 1995) on the G0 values when milk was fermented with an EPSproducing culture compared with a control. Table 1 Effect of exopolysaccharide (EPS) production on the rheological parameters measured during fermentation of milk with EPS-producing (EPSþ) and EPS-non producing (EPS) cultures. Parametera
EPSþ
EPS
Tgel (min) TpH 4.6 (min) pHgel G0 pH 4.6 (Pa) Tan dmax Tan dpH 4.6 pHtand max
370 8a 544 20a 5.30 0.07a 324 48a 0.409 0.002a 0.319 0.012a 4.82 0.09a
279 17b 402 35b 5.29 0.05a 249 39b 0.388 0.003b 0.297 0.011b 4.88 0.07a
a Definitions are: Tgel, time (min) at which G0 ¼ G00 ; TpH 4.6, time (min) from inoculation to pH 4.6; pHgel, pH at which G0 ¼ G00 ; G0 pH 4.6, storage modulus at pH 4.6; Tan dmax, maximum in tan d; Tan dpH 4.6, tan d at pH 4.6; pHtand max, pH at which the maximum in tan d is observed. Different superscript letters in the same row indicate significant differences determined by Student’s t-test (P < 0.05); standard deviation is also shown.
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Fig. 2. Rheological parameters G0 (C, ;), G00 ( , ) and tan d (B, 7) measured during fermentation of milk containing exopolysaccharide- (EPS-) producing (C, , B) and EPS-non producing (;, , 7) strain at 30 C as a function of pH. Curves are representative runs (see Table 1 for statistical analysis).
A maximum in tan d was observed in both samples at pH 4.8 (Fig. 2 and Table 1). The maximum in tan d can be attributed to a partial loosening of the caseinecasein interactions due to solubilisation of colloidal calcium phosphate, which causes an increase in charge repulsion between micelles, before the enhancement of mutual casein interactions as the pH decreases towards the isoelectric point of caseins and the repulsion between casein particles is reduced (Lucey et al., 1997). Other authors (Van Marle & Zoon, 1995) reported also no significant difference in the pH of tan dmax during gel formation of milk acidified with different EPS-producing cultures and GDL. As was shown from DWS measurements, no effect of EPS on the caseins in the stages preceding gelation was observed. However, the values of tan dmax and tan d plateau at pH 4.6 were significantly higher for the samples containing EPS compared with the control samples (Table 1), implying a stronger contribution of the viscous component to the overall viscoelastic character of the protein network of gels made with the EPSþ strain. A higher tan d of casein gels indicates that a higher number of proteineprotein bonds relax, allowing more rearrangements of the network structure (van Vliet, van Dijk, Zoon, & Walstra, 1991). Furthermore, the time at which EPS production takes place may have a significant contribution on the formation and rearrangements of casein network (Ruas-Madiedo & Zoon, 2003). The EPS that may be synthesised after the aggregation of the casein micelles commences, will be limited mainly in pockets around bacteria. Such separate domains of protein aggregates and pockets with EPS will be under continuous dynamic changes as the pH drops towards the isoelectric point of caseins (pH 4.6) and more EPS is produced in the pores. This dynamic environment would allow for thicker strands (higher G0 ) and fast and intense rearrangements to occur in the range of pH 5.3e4.6, which could count for the higher tan dmax and tan d at pH 4.6 values for EPSþ gels. The viscosity of fermented milk as a function of shear rate measured in steady state after cooling of samples at 4 C is depicted in Fig. 3. As the shear rate increased the happ of both samples decreased, a relationship that shows shear thinning characteristics of these gels. The presence of EPS brought about significantly higher apparent viscosity of fermented milk compared with the sample without EPS (Table 2), which is in agreement with previous published work (Doleyres et al., 2005; Haque, Richardson, & Morris, 2001; Hassan et al., 2003b). Furthermore, the gels showed different viscosity profiles; when the gel containing EPS was sheared, the
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Fig. 3. Apparent viscosity as a function of shear rate measured at 4 C for gels fermented with exopolysaccharide- (EPS-) producing (C) and EPS-non producing (;) strains. The data are average of three replicate experiments. Error bars indicate standard deviations. The Carreau fit is indicated by a solid line.
Fig. 4. Storage modulus (G0 ) of milk gels fermented with the exopolysaccharide- (EPS-) producing (C) or EPS-non producing (;) before structural breakdown (interval I) and recovery of G0 after shearing (interval III). Interval II represents shearing of sample at 500 s1 for 20 min, 4 C.
viscosity at low shear rates showed a tendency to level off (Fig. 3); this was not the case for EPS samples, where the viscosity decreased continuously with shear rate from the beginning of the shear rate range studied. The rheological model of HerscheleBulkley, which includes a yield stress term, did not fit to stress/strain functions of fermented milk containing EPS, while it fitted well to the data of control samples (R2 ¼ 0.98) (data not shown). On the other hand, the Carreau model, which includes a zero shear and infinite shear viscosity term fitted well (R2 ¼ 0.98) the EPSþ flow data, but did not fit the experimental curves of milk fermented with the EPS-free culture (Fig. 3), once again confirming the difference in their flow behaviour.
contained EPS compared with the ones made with EPS strains. The ratios of G0 after 60 min or 600 min of recovery over G0 before shearing, expressed in percentage, were calculated and used as indicators of the capability of the structure of milk gels to recover after shearing. Milk gels containing EPS showed much lower structure recovery compared to the control gels. Thus, EPSþ gels, 1 h after shearing stopped, recovered 0.3 0.07% of their original G0 compared with 9.5 5.2% for EPS fermented milk (Fig. 4). After 10 h of recovery these figures were 0.7 0.07 and 13.2 6.4%, respectively. This is in agreement with the reports of other authors (Folkenberg et al., 2006; Girard & Schaffer-Lequart, 2007b), who also showed that yoghurts containing EPS have a reduced ability to recover the structure after shearing compared with EPS-free gels. In contrast, it has been previously suggested that EPS could, at least partially, protect yogurt against the breakdown of the protein network or impart some resistance of yoghurt structure to stirring (Folkenberg, Dejmek, Skriver, & Ipsen, 2005).
3.2. Structural breakdown and recovery of the gels at 4 C The recovery of the gel structure after shearing at 500 s1 for 20 min was followed for 10 h at 4 C using dynamic oscillatory measurements at 1 Hz and 0.1% strain. The development of storage modulus G0 with time before shearing (interval I) and during recovery after shearing (interval III) is shown in Fig. 4. There was a fast increase in G0 over the first few minutes after ceasing of shearing and a much slower, but steady increase of G0 thereafter for both samples (Fig. 4, interval III). Similar recovery profiles of stirred yogurts have been reported by others (Arshad et al., 1993; Haque et al., 2001). Stirring caused lower G0 values for samples containing EPS compared with EPS gels (Fig. 4), and this was in contrast with the results of the undisturbed gel, where G0 vas higher for EPSþ. Similarly, Doleyres et al. (2005) and Hassan et al. (2003b) reported lower storage modulus values of stirred yogurts that Table 2 Apparent viscosity at selected shear rates for gels fermented with exopolysaccharide- (EPS-) producing (EPSþ) and EPS-non producing (EPS) strains.a Shear rate (s1)
Apparent viscosity (Pa s) EPSþ
11 47 121
EPS a
1.75 0.48 0.46 0.04a 0.27 0.06a
0.45 0.05b 0.20 0.02b 0.13 0.01b
a The data are average of three replicate experiments and values are means standard deviation. Measurements were performed at 4 C; values within the same row, followed by different superscript letters are significantly different (P < 0.05) as determined by Student’s t-test.
3.3. Microstructure of intact and stirred fermented milk Micrographs of set and stirred fermented milk prepared with EPSþ and EPS strains are shown in Fig. 5. The protein network was stained with rhodamine B and appears red in the images. Unstirred gels made with the EPSþ strain showed a network of thick, continuous protein aggregates, with large void spaces (Fig. 5a). On the other hand, control gels, fermented with EPS culture showed a network of fine protein strands and small pores (Fig. 5c). For the set-type gels (Fig. 5a and c) the micrographs are similar to those published by others (Hassan, Frank, & Qvist, 2002; Hassan et al., 2003b), who reported the formation of thick, dense protein strands in the presence of EPS with EPS being located in the channels of serum between protein aggregates. These results also would confirm the results shown in rheological testing (Fig. 2). Therefore, it can be concluded that the EPS was present in the serum pores and caused the formation of thicker strands. As discussed above, if a considerable amount of EPS was produced after the onset of aggregation of casein micelles, it would be entrapped in pockets between casein clusters and constrained around the bacteria cells. Such spatial constrain will reduce the probability of possible interactions of EPS with casein micelles and induce an intensification of the mutual interactions of casein particles in the
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Fig. 5. Confocal laser scanning micrographs of unstirred (a, c) and stirred (b, d) milk samples fermented with EPSþ (exopolysaccharide producing) strain (a, b) and EPS (EPS-non producing) strain (c, d). Bar ¼ 50 mm. (For interpretation of reference to color in this figure, please refer to the web version of this article.)
presence of highly concentrated regions of EPS. Other authors (Sanchez et al., 2000) have previously reported a very compact casein network with large pores in GDL acidified skim milk in the presence of 0.1% xanthan gum, which they attributed mainly to an incompatibility between casein particles and xanthan. The microstructure of stirred gels is shown in Fig. 5b and d. Shearing changed the microstructure of EPSþ fermented milk causing the formation of a larger number of pores and channels and less compact protein aggregates. It has been previously shown (Hassan et al., 2002, 2003b) that stirring results in formation of channels that contain EPS, concentrated in larger strands than those observed in the undisturbed gels. This was attributed to intense mutual interactions and aggregation of EPS molecules in the continuous serum phase due to incompatibility with casein micelles. The microstructure of EPSþ fermented milk after shearing (Fig. 5b) shows much less connectivity between protein aggregates, most likely because of pockets enriched with EPS that surround the casein aggregates. This type of microstructure would explain the lower ability to recover after structural breakdown due to shearing that was observed in Fig. 4. In EPS gels, stirring resulted in a more aggregated protein network with more connectivity between protein strands and smaller pores compared to the stirred gels containing EPS (Fig. 5d), in agreement with previous reports (Hassan et al., 2003b). It is important to note that the microstructure of the stirred control gels does not seem very different from that of the intact gel with the greatest noticeable difference being a more dense and aggregated
structure after stirring. This type of more spanning structure with greater connectivity is more likely to show better recovery after shearing compared with the structure of fermented milk gels containing EPS. 4. Conclusions The results obtained from DWS measurements of fermentation with EPSþ and EPS cultures suggested that EPS produced by L. lactis subsp. cremoris JFR1 during milk fermentation at 30 C did not affect the preceding stages of gelation as it did not change the properties of the casein micelles or their aggregation behaviour. While the presence of EPS did not change the pH of gelation, it affected the formation and rheological properties of the three dimensional network of casein micelles. The presence of EPS resulted in a stiffer gel, with a higher viscous component (higher tan dmax and tan dpH 4.6), which is translated into greater ability for rearrangements of the casein micelles during gel formation. Substantial effect of EPS was seen after cooling of the gel at 4 C, when a microstructure of large, dense protein clusters and large void pores was observed. After shearing, the microstructure of gels containing EPS changed and the gels showed a lower ability to recover than gels not containing EPS. It is hypothesised that there was little production of EPS up to the point of gelation, and the EPS produced after casein micelle aggregation concentrated in pockets around protein clusters, playing an important role in the formation and properties the EPSþ gel.
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Acknowledgements The authors would like to thank Dr. A. Hassan for donating the JFR1 culture. This work was funded by the Natural Sciences and Engineering Council of Canada and the Ontario Dairy Council through the Industrial Research Chair program.
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