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 (2008) 1109–1118

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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 ˜ a a,1, M. Corredig a, * I. Ayala-Herna´ndez a, A. Hassan b, H.D. Goff a, R. Mira de Ordun a b

Food Science Department, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Dairy Science Department, South Dakota State University, Brookings, SD 57007, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 November 2007 Received in revised form 5 April 2008 Accepted 25 June 2008

The aim of this work was to study exopolysaccharides (EPS) produced by Lactococcus lactis subsp. cremoris (JFR1), and their interactions with dairy proteins. The highest viscosities were obtained in media (permeate from filtration with polyvinylidene fluoride (PVDF) 0.1 mm) with added protein at pH 5.8 and 5.5. Isolated EPS showed a rheological behaviour different from that of the whole fermented media. The size of the EPS was measured using size exclusion chromatography coupled with light scattering detection. Results suggested that molecules with higher molecular mass and a more compact structure were produced from fermentation at pH 5.5 than molecules produced from fermentation at pH 6.5. Studies using dynamic light scattering demonstrated that EPS molecules are negatively charged and interact with whey proteins at acidic pH. It was concluded that the impact of EPS on textural properties is determined not only by the molecular characteristics but also by the ability to interact with proteins. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Exopolysaccharides (EPS) produced by some strains of lactic acid bacteria (LAB) improve textural properties and decrease susceptibility to syneresis in fermented dairy products (Ruas-Madiedo, Hugenholtz, & Zoon, 2002). In addition, some reports have associated the consumption of EPS with health benefits (Chabot et al., 2001; Dal Bello, Walter, Hertela, & Hammesa, 2001; Kitazawa et al., 1998). A major limitation for the development of EPS as an ingredient is the low yields obtained from the fermentations; reported yields are in the range of 2 g L1 for the highest producers (Macedo, Lacroix, Gardner, & Champagne, 2002). Nevertheless, the high viscosity and unique rheological properties that EPS molecules confer to fermented milks, even at such small concentrations, make EPS a potential ingredient in food products. Several attempts have been made to control the fermentation conditions in order to increase the EPS production, but there are still many unresolved questions. One of the major problems is that strain-specific behaviour prevails (De Vuyst & Degeest, 1999; De Vuyst, De Vin, Vaningelgem, & Degeest, 2001; Jolly, Vincent, Duboc,

* Corresponding author. Tel.: þ1 519 824 4120x56101; fax: þ1 519 824 6631. E-mail address: [email protected] (M. Corredig). 1 Present address: Department of Food Science and Technology, Cornell University, NYSAES, Geneva, NY 14456, USA. 0958-6946/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2008.06.008

& Neeser, 2002; Laws, Gu, & Marshall, 2001) rendering the identification of general principles challenging. Studies with different strains (e.g., Grobben et al., 1997; Mozzi, Giori, Oliver, & Valdez, 1996) demonstrated that the production of EPS under continuously controlled pH is, in general, significantly higher than in batch fermentations with uncontrolled pH. In some cases, the effect of pH control on final EPS yield was found to be greater than that of supplementation with nutrients (De Vuyst & Degeest, 1999). There have been reports that the molecular structure and sugar composition of the EPS depend on fermentation conditions (Grobben et al., 1997; Petry et al., 2003). The sugar components of EPS from LAB are most commonly galactose, glucose and rhamnose (Cerning, 1990). The EPS from some strains of Lactococcus lactis subsp. cremoris contain rhamnose, glucose, galactose and phosphates (Higashimura, Mulder-Bosman, Reich, Iwasaki, & Robijn, 2000; Nakajima, Hirota, Toba, Itoh, & Adachi, 1992; Yang, Huttunen, Staaf, Widmalm, & Heikki, 1999), while others contain only glucose and galactose. Marshall, Cowie, and Moreton (1995) found that Lc. lactis subsp. cremoris LC330 produced two EPS with different sugar composition and molecular mass: a neutral EPS of 1 106 Da and a smaller negatively charged EPS (containing phosphate groups) of about 1 104 Da. The viscosity of fermented milks seems to be related to the intrinsic viscosity (and to molecular properties such as molar mass and polymer stiffness) of the strain used (Ruas-Madiedo, Tuinier, Kanning, & Zoon, 2002). As the composition and the molecular properties of the EPS molecules will influence their rheological

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behaviour, this aspect needs to be better addressed before a full understanding of their potential utilization as ingredients in foods. Oba et al. (1999) studied the viscoelastic properties of the phosphopolysaccharide produced by Lc. lactis subsp. cremoris SBT 0495 and indicated that this EPS in aqueous solution behaved as an entangled polyelectrolyte. The solutions with EPS from several strains of Lc. lactis subsp. cremoris showed a shear thinning behaviour at concentrations >1% w/v. The objective of this research was to study fermentation conditions favouring the production of EPS by Lc. lactis subsp. cremoris (a highly ropy strain) in ultrafiltration permeate media. The reasons for the changes in rheological properties of EPS-containing media during fermentation were also investigated. Milk permeate was chosen as the fermentation medium as its fermentation with LAB could represent an opportunity for the creation of value-added ingredients from a by-product of the dairy industry. Most of the studies reported so far have focused on isolated analysis of production conditions (one pH or no pH control), rheological properties or molecular characteristics. This research presents a novel approach to the study of EPS by combining the molecular characterization with an analysis of the functionality and viscosity of EPS produced under different conditions: fermentations were conducted at controlled pH values (6.5, 5.8 and 5.5) and bacterial growth and changes in medium viscosity were measured. The EPS samples were then isolated from the media and characterized to determine composition, molecular weight and size. 2. Materials and methods 2.1. Bacterial strain Lc. lactis subsp. cremoris (JFR1), a highly ropy strain (Hassan, Frank, & Elsoda, 2003), was obtained from Dr. Hassan’s collection at South Dakota State University. The strain was received in milk and propagated twice in M17 medium (Terzaghi & Sandine, 1975) with lactose as a carbon source, before being stored at 80  C in reconstituted skim milk (10% w/v) with 25% glycerol. Before inoculation, the stock culture was propagated three times using 1% (v/ v) inoculum each time, for 12–16 h at 32  C in M17 containing 0.5% (w/v) lactose. The fermentation medium was inoculated with 2% (v/v) of this culture. 2.2. Fermentation media Ultrafiltration permeate was used as a fermentation medium. Permeate was produced with a pilot scale ultrafiltration unit (Koch Membrane Filtration Systems, Inc., Wilmington, MA, USA) equipped with a 0.1 mm spiral-wound polyvinylidene fluoride (PVDF) membrane (Snyder Filtration, Vacaville, CA, USA). Low heat skim milk powder (Cloverdale, Parmalat Food Inc., Toronto, Canada) was reconstituted in deionized water (11% w/v), dissolved for 1 h and left overnight at 4  C to ensure complete hydration. The permeate was obtained concentrating the milk four times. The temperature during microfiltration was maintained between 20 and 30  C. The resulting permeate was stored frozen at 20  C in batches until further use. The nitrogen content of two different batches of this permeate was determined by Kjehldal method and ranged from 0.33 to 0.48%, whereas the lactose content (determined using an enzymatic kit, Lactose/D-Galactose UV method for enzymatic analysis, Roche r-Biopharm, Darmstadt, Germany) was about 4.2%. When supplementation was required, research grade whey protein isolate (BiPro, Davisco Foods International, Le Sueur, MN, USA) containing 95% protein (dry basis, N 6.38) was added to the permeate at 2% (w/v). Batch pasteurization of the media at 63  C for 30 min was carried out in bottles, to control the level of denaturation of the

whey protein. The permeate was then transferred aseptically to the bioreactor, which was autoclaved separately at 121  C for 45 min. All other solutions and materials used in the fermentations (for pH control and others) were autoclaved at 121  C for 1 h. 2.3. Fermentation conditions All fermentations were performed in a 15 L MF-114 (New Brunswick Scientific, Edison, NJ, USA) fermentation unit or in a 14 L BIOFLO 3000 fermentation unit (New Brunswick Scientific), both with adaptations for pH and temperature control, agitation and nitrogen addition (N2 gas, added to generate anaerobiosis). Fermentation volumes were generally 6 L. Fermentations were performed at 30  C, under nitrogen flow. Agitation was maintained between 70 and 100 rpm to provide adequate dispersion of the base solution used to control pH without excessive shear. Fermentations were carried out for at least 24 h, prolonging up to 48 h or more if ropiness was produced, to observe changes in ropiness at long fermentation times. Samples were taken at different intervals to monitor bacterial growth and changes in viscosity. Duplicate fermentations (one fermentation per batch) were carried out for all fermentation conditions reported (medium composition or pH of fermentation). Fermentations were carried out under controlled pH at 5.5, 5.8, or 6.5. The pH was adjusted to the desired value before inoculation, with the addition of citric acid. The pH of the pasteurized media (pH 6.3) was initially adjusted with sterile 20% (w/v) citric acid solution and 3 M NaOH. Both solutions were also used to maintain the pH during the fermentation process. An automated pH controller (2100e pH Transmitter, Mettler Toledo, Mississauga, ON, Canada) equipped with peristaltic pumps (Model P-3, Pharmacia, Stockholm, Sweden) for base and acid addition was used to maintain the pH at the desired value. 2.4. Sampling Samples were aseptically withdrawn before inoculation, immediately after inoculation (0 h), and at different time intervals (generally every 5–8 h), cooled with ice and stored at 4  C. For statistical analysis, the sampling times were grouped according to growth stages into time 0 h, 15–22 h, 25–33 h, 40–50 h and 51– 65 h. Samples were immediately analyzed for viable cell counts. The viscosity of the culture medium at various times of fermentations was tested after cooling at 4  C for at least 6 h. The remainder of the fermentation medium was frozen for isolation of EPS. 2.5. Bacterial enumeration Bacterial growth was followed during the fermentation, tracking three indicators: enumeration of viable bacterial cells with the spread plate technique in M17-agar plates; turbidity measurements; and rate of acidification. The spread plate technique was used to determine viable cell counts (cfu mL1). Serial dilutions were prepared in sterile buffered dilution blanks and the bacteria were enumerated using M17 (Difco Laboratories, Detroit, MI, USA) agar (15 g L1) containing 0.5% (w/v) lactose as a carbon source (Terzaghi & Sandine, 1975). Plates were incubated for 72 h at 32  C. All the plating was performed in duplicate. For rapid measurement of bacterial growth, the turbidity of the culture media at 600 nm was measured as previously reported by Kanasaki, Breheny, Hillier, and Jago (1975), with slight modifications, as the original method was developed for milk, which contains casein micelles. The fermented medium (permeate) was diluted to appropriate concentrations with an ice-cold 0.2% (w/v) EDTA solution. Several trials were performed to determine the appropriate dilution rate, and dilutions from 1:1 to 1:9 showed

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good correlation with cell growth. In most cases, a 1:1 dilution was chosen, because of the low turbidity of the fermentation medium. The turbidity results were always confirmed with analysis of bacterial growth, since concerns have been reported in the literature regarding the lack of reliability of this technique when dealing with complex media (De Vuyst & Degeest, 1999). The acidification rate was also monitored to follow bacterial growth by tracking the rate of NaOH consumption. 2.6. Isolation of the polysaccharide EPS was purified from the various fermentation media based on published methods (Ruas-Madiedo & de los Reyes-Gavilan, 2005), with modifications. In brief, 20% trichloroacetic acid (TCA) was added at a 1:1 volume ratio to the fermentation medium. The precipitate was removed by centrifugation at 17,000  g for 40 min at 4  C (J2-21 with JA-10 rotor, Beckman Coulter Inc., Fullerton, CA, USA). The EPS was then precipitated by addition of an isovolume of 95% ethanol to the supernatant, and the mixture was kept at 4  C for at least 12 h, and then filtered through a 1.2 mm pore size polycarbonate filter (Isopore, Millipore, Billerica, MA, USA). The filter was washed with ultrapure water (ASTM type I reagent water, used for all the experiments) to recover the precipitate. The TCA and ethanol precipitations were then repeated and the final EPS dispersion was extensively dialyzed against ultrapure water at 4  C (8–10 kDa cutoff, Fisherbrand, Fisher Scientific Corporation, Ottawa, ON, Canada). The EPS solution was then frozen at 80  C and freeze-dried. The protein content of the samples obtained after purification was determined to be <2% (w/v), using the colorimetric Bradford method (DC Protein Assay, BioRad Laboratories, Hercules, CA, USA). 2.7. Rheological measurements 2.7.1. Viscosity of the permeate during fermentation The effect of EPS production on the rheological properties of the fermentation medium was tested by measuring the apparent viscosity at various fermentation times, using a controlled stress rheometer (AR1000, TA Instrument Ltd., New Castle, MA, USA) equipped with a conical concentric cylinder geometry with the following specifications: 5920 mm fixed gap, 15 mm radius, 14 mm rotor outer radius and 42 mm cylinder immersed height. The sample volume used was approximately 20 mL. The temperature was kept at 4  C with an external water bath (Isotemp 3016, Fisher Scientific, Whitby, ON, Canada). The inoculated culture medium, taken immediately after inoculation, was also measured. Samples were equilibrated at 4  C before the measurements and softly mixed by inversion to ensure homogeneity of the sample. A shear rate ramp was performed from 0.1 to 100 s1. 2.7.2. Viscosity of solutions of purified exopolysaccharides Selected samples of isolated EPS were dissolved in milk permeate prepared in the laboratory by ultrafiltration of reconstituted skim milk (11% solids) at two different pH values, 6.7 or 5.5 (using HCl). Milk permeate was chosen as the solvent in order to minimize variations in solvent composition. The ultrafiltration was performed using a laboratory scale ultrafiltration cartridge (PGC 10k, regenerated cellulose cartridge, Millipore Corporation, Bedford, MA, USA). EPS solutions were mixed overnight in permeate at pH 5.5 or 6.7 to ensure adequate solubilization and the viscosity was measured at four different concentrations of EPS (0.0625, 1.25, 2.5 and 5 mg mL1) at 4  C using the controlled stress rheometer (AR 1000 TA Instruments) with a cone and plate geometry with the following specifications: 40 mm diameter standard steel cone, 2 angle, fixed gap 51 mm. A peltier plate system was employed for temperature control. The samples were equilibrated for 10 min and then a shear rate ramp was imposed from 0.1 to 200 s-1 in 5 min.

1111

2.8. Monomer composition of the exopolysaccharides To determine the monomer composition of the isolated EPS, sugar analysis was performed after acid hydrolysis, following the methods of Vaningelgem et al. (2004) and Goh, Hemar, and Singh (2005). Acid hydrolysis was performed with 6 M trifluoroacetic acid at 100  C for 3 h. Each sample was measured in duplicate and the analysis was performed twice on a high performance liquid chromatography/capillary electrophoresis (HPLC/CE) system (Dionex, Sunnyvale, CA, USA) using an anion exchange chromatography column (Carbopac PA1, 4  250 mm, Dionex) and guard column (3  25 mm). Monosaccharide detection was carried out by pulsed amperometry with a gold electrode. The pulse potentials (E, volts) and durations (t, ms) were E1 ¼ 0.05, t1 ¼ 480, E2 ¼ 0.6, t2 ¼ 180; E2 ¼ 0.6, t2 ¼ 180, E3 ¼ 0.6, t3 ¼ 60 with a 1.0 s detector response time. The elution was carried out at 1 mL min1 with 15 min equilibration with 8 mM NaOH before the injection of each sample, using 8 mM NaOH for 7 min and 28 min with 300 mM NaOH followed by rinsing with ultrapure water. The column temperature was maintained at 35  C. Standard solutions of glucose, galactose, and rhamnose were prepared in ultrapure water (in a range of concentrations from 10 to 100 mg mL1). All samples were filtered through 0.45 mm filters (Millipore) prior to sample injection. 2.9. Molecular characterization of the exopolysaccharides using size exclusion chromatography Size exclusion chromatography (SEC) coupled with a multiangle laser light scattering (MALLS) detector was used to determine the absolute molecular masses and radius of gyration of selected samples of isolated EPS produced under different fermentation conditions. Freeze-dried EPS samples were thoroughly dissolved in water by overnight mixing at concentrations ranging from 1 to 3 mg mL1. The polysaccharide samples were eluted on two gel filtration columns connected in series (Biosep 4000 and 3000, Phenomenex, Torrance, CA, USA). These columns have a nominal exclusion volume of 2000 and 700 kDa, respectively. The separation system was connected to HPLC (SpectraSystems, ThemoFinnigan, San Jose, CA, USA), consisting of a degasser, a pump (P-2000) an autosampler and a UV detector. Radius and molecular mass parameters were determined by on line detection with a refractive index (RI, Optilab Rex, Wyatt Tech., Santa Barbara, CA, USA) and multi-angle light scattering detector (Dawn EOS, Wyatt Tech.). EPS solutions (100 mL) were injected using an autosampler and elution was carried out at room temperature (23  C) using 50 mM NaNO3 as mobile phase eluting at 0.5 mL min1. The mobile phase was triple filtered (0.2 and 0.1 mm filters, Millipore) and degassed using an inline degassing system. Prior to the injection, the EPS solutions were filtered through 0.8 and 0.45 mm PVDF filters (Millipore). Absolute molecular masses and radius of gyration (Rg) were determined using the ASTRA software (version 5.1.9.1, Wyatt Tech.) with the RI as a concentration detector. The refractive index increment value used in the calculations was 0.135 (Yang et al., 1999). Molecular mass averages were calculated as

Mn ¼

Mw ¼

hX

ci =

hX

and Mz ¼

i X ðci =Mi Þ ;

ci Mi =

h X

X i ci ;

ci Mi2

.X

i ci Mi :

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D E0:5 h X D E0:5 . X i R2g ðci Mi Þ ; ¼ ci Mi R2g z

i

The radius of gyration Rg can be defined as the distribution of the volume elements of the molecule with respect to the square of the distance from its center of gravity. Values of Rn, Rw and Rz were calculated as for molecular mass averages (see above). 2.10. Size and zeta potential measurements of exopolysaccharides in a mixture with colloidal particles To further characterize the behaviour of the purified EPS, the charge and its interaction behaviour with colloidal particles were also determined by carrying out light scattering measurements on selected samples of EPS adsorbed to colloidal particles (latex) or emulsion droplets stabilized with whey proteins at pH 3.5. Selected samples of freeze-dried EPS were dissolved in ultrapure water by mixing overnight at a concentration of 1.5 mg mL1. A suspension of latex particles (60 and 350 nm diameter polystyrene nanospheres, Duke Scientific Corporation, Palo Alto, CA, USA) was diluted to approximately 0.01 mg mL1 latex in three concentrations of EPS (0.15, 0.375 and 0.75 mg mL1). The mixtures were incubated for 2 h and the apparent diameter of the suspensions with or without EPS was then measured using dynamic light scattering (DLS) after extensive dilution (approximately 1:2000) in 5 mM NaNO3 (Nanosizer, Malvern Instruments Ltd., Malvern, UK). The adsorption of EPS on the latex sphere was determined by measuring the original bare latex particle, and the same particle after incubation with EPS. The difference in the size was taken as an indication of the hydrodynamic radius of the polymer once adsorbed on the particle. Zeta potential measurements were then carried out by diluting the suspensions in 5 mM NaNO3 (Nanosizer, Malvern Instruments). With these experiments, the change in charge of the latex particle caused by the adsorption of the EPS was measured. Additionally, a similar experiment was performed by mixing EPS solution with a 1% whey protein-stabilized emulsion at pH 3.5. The emulsion was prepared by mixing a whey protein isolate (WPI) solution (BiPro, Davisco Foods Int.) at pH 7 with 20% soy oil, and homogenizing at 40 MPa with four passes using a high pressure valve homogenizer (Avestin Emulsiflex C-5, Avestin, Ottawa, Canada). The emulsion was then brought to pH 3.5 using HCl, to obtain oil droplets coated with positively charged whey proteins (Gancz, Alexander, & Corredig, 2005). In these experiments the isolated EPS samples were dissolved in permeate at pH 6.7 to a final concentration of 5 mg mL1 and then adjusted to pH 3.5 before mixing with the emulsion droplets. The final concentration of the mixture was 0.5% oil droplets and 0.08, 0.2, 0.32 and 0.4 mg mL1of EPS. The interaction of EPS with the positively charged WPI-stabilized emulsions was studied as described above using DLS and zeta potential measurements (Nanosizer, Malvern Instruments). 2.11. Statistical analysis Fermentations were all carried out in duplicate (different batches). EPS extractions were conducted from different media. Data shown are the average of at least two independent experiments. Significant differences were determined by using the general linear model procedure in the statistical software package SAS (version 8.1, Cary, NC, USA). For significant effects, the least square means were contrasted and differences were considered significant at p < 0.01.

3. Results and discussion 3.1. Fermentation conditions The growth of Lc. lactis subsp. cremoris JFR1 at different pH values in media with/without additional protein was followed over time. Bacterial counts reached about 109 cfu mL1 (from initial bacterial counts about 107), indicating that milk permeate was a suitable medium for the growth of Lc. lactis subsp. cremoris JFR1. The growth curves were similar for all fermentations, with a lag phase of around 4–6 h, followed by an exponential phase of accelerated growth, which could be tracked by viable cell counts, turbidity and acidification rate. The exponential phase generally lasted 16–20 h. All fermentation experiments were duplicated. Fig. 1 represents an example of the bacterial growth in milk permeate containing 2% additional whey protein at a controlled pH of 5.5. Neither the pH within the range tested nor 2% whey protein addition seemed to affect bacterial growth since the maximum counts of viable bacteria remained in all cases in the range of 109 cfu mL1, even for media at pH 5.5. This is an interesting point to notice, considering that previous studies on the growth characteristics of Lc. lactis subsp. cremoris generally have been conducted at higher pH values (5.8–7.0) (Gruter, Leeflang, Kuiper, Kamerling, & Vliegenthart, 1992; Higashimura et al., 2000; Looijesteijn et al., 2000). Previous studies have reported the difficult task of quantifying the change in ‘ropiness’ (‘mucoidness’) that is generated during EPS production, since rotational viscosity measurements do not reflect the level of ropiness. Visual observations such as assessing the formation of long strands during fall from a pipette are often used as a criterion to measure ropiness (Cerning, 1990). Rawson and Marshall (1997) performed large deformation textural studies (using a rotational viscometer and a texture analyzer) in stirred yoghurts produced with different combinations of ropy and nonropy LAB strains to investigate the contribution of ropiness to the overall texture and concluded that the ropiness contributes directly to adhesiveness, whereas the parameters firmness and elasticity are more related to the properties of the protein matrix. Therefore, it is important to note that although in our study the rheological measurements of viscosity were sensitive enough to show differences among various fermentations and fermentation times (Fig. 2), the visual differences observed in the ropiness of the media were much more dramatic. A decrease in ropiness and viscosity could be observed in some instances after prolonged fermentation times (>30 h), in agreement with previous reports on the presence of glycohydrolases in

1.2 1.1

2.00

1 0.9

1.50

0.8 1.00

0.7 0.6

0.50 0.00

OD 600nm

where ci is the concentration of polymer at a elution volume i and Mi is the mass at i. In addition, the root mean square radius was calculated as

Bacterial count (cfu ml-1) x 109

1112

0.5 0

10

20

30

40

0.4 50

Time (h) Fig. 1. Microbial growth determined as viable cell counts (-, ,) and optical density at 600 nm (:, 6) for two replicate fermentations (replicate 1, solid symbols, replicate 2, open symbols) conducted at pH 5.5 with permeate containing 2% additional WPI.

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the fermentation media at prolonged times of fermentation (Cerning, Bouillanne, Landon, & Desmazeaud, 1992; Crescenzi, 1995; De Vuyst, Vanderveken, Van de Ven, & Degeest, 1998; Gancel & Novel, 1994; Mozzi et al., 1996; Pham, Dupont, Roy, Lapointe, & Cerning, 2000). During the fermentations performed in the present study, a loss of ropiness in the media at extended times could be visually appreciated, and was in some cases further detected by the measurement of viscosity. It is important to note that EPS degradation by glycohydrolases may be facilitated at certain pH values (close to pH 6.0) where enzymes are at their optimum activity (Petry et al., 2003); and this would explain why the loss of ropiness could only be seen under certain fermentation conditions (at pH 6.5 but not at pH 5.5). Table 1 indicates that the fermentation pH and the composition of the culture media affected the final viscosity obtained. Visually, high ropiness and viscosity were shown only at pH 5.8 and 5.5, and in the presence of whey proteins. However, it must be mentioned that the changes in pH and composition of the media may have affected not only the extent of the EPS production, but also the physicochemical complexes that may form between EPS and proteins in the medium. The presence of whey proteins in the media resulted in a marked increase in the viscosity after about 20 h of fermentation. In both permeate media, with and without whey proteins, pH 5.8 produced the highest viscosity followed by pH 5.5. The fermentation at pH 6.5 produced a significantly lower viscosity than at pH 5.5 or 5.8, even though the maximum bacterial counts did not differ. The addition of protein seemed to affect not only the total increase in viscosity that could be achieved after 15 h of

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fermentation, but also influenced the contribution of pH to the medium viscosity. In milk permeate (without the whey protein added) the viscosity results obtained at pH 5.5 and 5.8 were very similar, whereas in the media with 2% added protein the viscosity increased after 15 h of fermentation at pH 5.8 by 4.52 mPa s, while it increased by only 2.3 mPa s at pH 5.5 (Fig. 2B). Analysis of variance of the viscosity data showed that pH of fermentation, protein supplementation and time of fermentation had a significant effect on the viscosity. As previously mentioned, these results need to be interpreted with caution, especially with regards to making a direct relationship with the total amount of EPS produced in each case. This is because it is important to consider that the thickening effect might be generated by protein interactions with the EPS and the different biopolymers’ interactions occurring at different environmental pH, as previously suggested by other authors (Tuinier, 1999). It is important to point out that statistical analysis has been reported seldom for such systems, due to the challenges caused by the inherent variability of the EPS produced in these fermentation processes. This further enforces the conclusion that different fermentation pH generate different rheological properties (viscosity and ropiness) in the media.

3.2. Molecular characterization of exopolysaccharides 3.2.1. Monosaccharide composition The monosaccharide composition of the EPS isolated from selected fermentations is presented in Table 2. The monosaccharide

0.02

A

0.018

Viscosity (Pa s)

0.016 0.014 0.012 0.01 0.008 0.006 0.004 0.002

0

0

10

20

30

40

50

60

70

80

90

100

Shear rate (s-1)

0.013

B

Viscosity (Pa s)

0.011 0.009 0.007 0.005 0.003 0.001

0

10

20

3

40

50

60

70

80

90

100

Shear rate (s-1) Fig. 2. Viscosity of (A) milk permeate with 2% whey protein fermented at a controlled pH of 5.8 at 0 h (A), 14 h (,), 19 h (:) and 39 h (B) and (B) different media at the optimal time of fermentation: WPI at 2%, pH 5.8, 19 h (:); WPI at 2%, pH 5.5, 22 h (C); WPI at 2%, pH 6.5, 25 h (-); no WPI, pH 6.5, 22 h (,); no WPI, pH 5.8, 22 h (6); no WPI, pH 5.5, 20 h (B).

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Table 1 Least square means of viscosities (Pa s) measured at 100 s1 for permeates fermented at various pH valuesa Fermentation pH

WPI added

0h

15–22 h

25–33 h

40–50 h

51–65 h

6.5 6.5 6.2 5.8 5.8 5.5 5.5

0 2% 0 0 2% 0 2%

0.00233a 0.00301a 0.00140a 0.00204a 0.00239a 0.00201a 0.00290a

0.00266a n/d 0.00169a 0.00320a 0.00691b 0.00296a 0.00513c

0.00264a 0.00340a n/d 0.00318a 0.00661b n/d 0.00465c

n/d 0.00346a n/d n/d 0.00455a n/d 0.00409c

n/d n/d n/d n/d 0.00619b 0.00245a n/d

0.04

Viscosity (Pa s)

1114

A

0.03 0.02 0.01 0

0

1

Values with a different superscript letter are significantly different (p < 0.01); n/d, not determined.

3.2.2. Rheological behaviour of purified exopolysaccharides To better understand the effect of EPS on the rheological properties of milk systems, the viscosity of isolated EPS was measured at 4  C after preparing solutions of EPS in milk permeate adjusted to pH 5.6 or 6.7, at concentrations ranging from 0.625 to 5 mg mL1. Results from EPS isolated from the fermentations at pH 5.5 and 6.5 are shown in Fig. 3. As expected, the viscosity of the solutions increased with concentration of EPS. At low concentrations, no difference could be noted between pH or protein supplementation. At higher concentration (5 mg mL1) there was a significant difference in the viscosity of the EPS solutions as shown in Fig. 3. Statistical analysis showed that the pH of the solution (5.5 or 6.7) had no significant Table 2 Relative monosaccharide composition of EPS produced by Lactococcus lactis subsp. cremoris JFR1 isolated from various fermentationsa Culture medium

pH Fermentation Relative percentages time (h) Rha Gal Glu

Ratio

Milk permeate, no protein added

5.5 20 5.8 22

Milk permeate with 2% 5.5 added protein 5.8 5.8 5.8 6.5

30 21 39 51 25

Rha Gal Glu

25.55 24.56

13.22 15.40

61.23 60.04

1.9 1.6

1.0 1.0

4.6 3.9

24.00 26.41 26.46 25.99 27.10

16.26 13.63 13.44 13.03 11.89

59.73 59.95 60.10 60.97 61.01

1.5 1.9 2.0 2.0 2.3

1.0 1.0 1.0 1.0 1.0

3.7 4.4 4.5 4.7 5.1

a Each value represents the average of two separate EPS samples purified from two duplicate fermentations. All averages presented a standard deviation of less than 10%. Rha, rhamnose; Gal, galactose; Glu, glucose.

3

4

6

5

0.04

Viscosity (Pa s)

ratios were not affected by the differences in the culture medium, pH, or the duration of the fermentation. The EPS produced by Lc. lactis subsp. cremoris JFR1 was composed mainly of glucose, rhamnose and galactose in an average ratio of approximately 4:2:1, respectively. These monomer compositions agree with previous reports for other Lc. lactis strains (Deveau, Van Calsteren, & Moineau, 2002; Higashimura et al., 2000; Looijesteijn et al., 2000). The importance of the ratio of glucose to galactose for the generation of viscosity has been emphasized previously by Petry et al. (2003), who reported that a higher proportion of glucose in EPS seemed to be associated with higher viscosities. Variations in the monosaccharide composition of EPS produced by a particular strain have been reported only in studies where the carbon source was changed (Grobben et al., 1997) or when the same strain produced two different EPS (Marshall et al., 1995). Glucose is the dominant monosaccharide in the EPS produced by the Lactococcus strain used in this study under all conditions. This is not surprising, as the same carbon source (lactose) was used in all fermentations. This result is also in agreement with studies where no differences were shown in the monosaccharide composition of EPS produced in milk or chemically defined media (Petry et al., 2003).

2

Concentration (mg mL-1)

a

B

0.03 0.02 0.01 0

0

1

2

3

4

5

6

Concentration (mg mL-1) Fig. 3. Viscosity measured at 100 s1 for purified EPS solutions in permeate at pH 5.5 or 6.7, as a function of EPS concentration. (A) EPS extracted from fermentations at a controlled pH of 5.5 in milk permeate (-, ,) or milk permeate with additional 2% whey protein (:, 6). Sample tested at pH 5.5 (-, :) and 6.7 (,, 6). (B) EPS extracted from fermentations of permeate containing 2% WPI at pH 5.5 (:, 6) and pH 6.5 (-, ,), tested at pH 5.5 (-, :) and 6.7 (,, 6). Values are average of two separate EPS samples purified from two duplicate fermentations.

effect on the behaviour of the EPS; however, for the EPS isolated from the media fermented at pH 5.5 (Fig. 3A) the concentration and the presence of whey proteins in the fermentation medium had a significant effect on the viscosity generated by the isolated polysaccharide (viscosity measured at 100 s1). Differences were clearly noted in 5 mg mL1 EPS solutions. The data contrast with those shown in Fig. 2B on the viscosity of the fermented media. The viscosity of the purified EPS isolated from permeate containing 2% whey protein was lower than that isolated from permeate without whey protein added. On the other hand, in the fermented media the viscosity of the supplemented permeates was markedly higher than that in the unsupplemented permeates. This might indicate that the amount of EPS produced under each condition might be different, but it could also show that the increased viscosity and ropiness in the fermented medium containing whey proteins were generated mainly by interactions between the EPS and the protein. The viscosity of the 5 mg mL1 solution of purified EPS produced at pH 6.5 was also significantly higher than that produced at pH 5.5 (Fig. 3B), although the ropiness and viscosity of the fermentation medium were much higher at pH 5.5 than at pH 6.5. It is important to note that the isolation process may have affected the structure, charge and functionality of the EPS. When EPS is in the fermented medium it is highly hydrated and is interacting with proteins. The pH of the medium may also have an effect on these interactions, although it was shown that there was no effect of the environmental pH of the purified EPS solution (in permeate at pH 5.5 and 6.7) on the viscosity (Fig. 3). 3.3. Molecular parameters 3.3.1. Multi-angle light scattering The differences in molecular mass and conformation of the isolated EPS can further explain the rheological behaviour of the

I. Ayala-Herna´ndez et al. / International Dairy Journal 18 (2008) 1109–1118

polysaccharides. Selected EPS samples were analyzed using size exclusion chromatography coupled with a multi-angle laser light scattering system (SEC–MALLS). The light scattering (A) and refractive index (B) traces for two isolated EPS are shown in Fig. 4. When looking at the chromatographic elution, it is clear that the polymers extracted from media fermented at pH 6.5 are considerably smaller that those extracted from media fermented at pH 5.5. Table 3 summarizes the results of determinations of molecular mass and root mean square radius for the various EPS isolated from media fermented at different pH. The purified EPS produced at pH 6.5 showed a smaller molecular mass than those produced at pH 5.5: whereas the EPS produced at pH 6.5 had an average molecular mass of 9  105 Da, the EPS produced at pH 5.5 had a molecular mass of about 3  106 Da. On the other hand, the estimated average radius of the EPS produced at pH 5.5 and 6.5 were about 68 nm and 86 nm, respectively. These results suggested that the EPS produced at pH 5.5 is a more compact molecule than the EPS produced at pH 6.5. This might explain the higher viscosity of the solution of EPS produced at pH 6.5. The more extended the EPS molecule, the higher the viscosity in solution. The compact EPS indicates more intramolecular interactions and more aggregation. Such interactions reduce the ability of the EPS to cause entanglements in solutions at low concentrations. The molecular mass values for EPS extracted from media at pH 5.8 are smaller compared with those extracted at the other pH values but show a very large standard deviation, indicating a high variability in the measurements. These samples could not be measured accurately, as the polysaccharide recovery was very low after filtration, indicating that the majority of the polysaccharide was present in an aggregated form. For this reason, the values at pH

1.6

A

1.4 1.2

LS (V)

1 0.8 0.6 0.4 0.2 0

5

10

15

20

25

30

35

40

Elution time (min) 1.2E-05

B 1.0E-05

RI

8.0E-06 6.0E-06 4.0E-06 2.0E-06 0.0E+00

5

10

15

20

25

30

35

40

Elution time (min) Fig. 4. Light scattering (90 angle) (A) and refractive index (B) traces for two EPS samples extracted from permeate fermented at pH 6.5 (for 25 h; grey line) and pH 5.5 (for 30 h; black line).

1115

Table 3 Molecular parameters determined by size exclusion chromatography coupled with light scattering detection for EPS purified from fermentations at different pH values, with milk permeate supplemented with 2% whey protein isolatea pH

Mn (105 Da)

Mw (105 Da)

Mz (105 Da)

Rn (nm)

Rw (nm)

Rz (nm)

6.5 5.5 5.8

8.75  0.4 24.6  7.6 6.58  2.3

9.18  0.2 29.2  6.8 7.59  2.72

9.56  0.2 33.0  6.4 8.34  3.22

86.6  6.4 68.4  3.8 68.0  5.2

87.5  6.3 69.4  3.1 69.0  4.7

88.66  6.1 67.4  2.8 69.9  4.3

a Parameters are molecular mass and radius averages Mn, Mw, Mz and Rn Rw and Rz, as defined in Section 2: values presented are the average of three EPS samples, extracted from three independent fermentation runs.

5.8 will not be further discussed, since they could not be tested reproducibly, while those at pH 5.5 will be compared with those at pH 6.5. Previous studies of molecular characteristics of EPS produced by Lc. lactis subsp. cremoris have reported apparent molecular masses that ranged from 1 105 to 4  106 Da (De Vuyst & Degeest, 1999; Ruas-Madiedo, Hugenholtz, et al., 2002; Ruas-Madiedo & de los Reyes-Gavilan, 2005; Ruas-Madiedo, Tuinier, et al., 2002). The results of molecular mass of EPS of the present work agree with the range of previous reports. The pH of the fermentation medium affected the molecular mass of the isolated EPS as shown in Fig. 5A for EPS extracted from media fermented at pH 5.5 than from media fermented at pH 6.5. Looijesteijn et al. (2000) and Petry et al. (2003) also reported that fermentation conditions affect the molecular mass of EPS. They reported that the lower the molecular mass of the polysaccharide, the lower the intrinsic viscosity. In the current study a direct relationship could not be established between molecular mass and viscosity as fermentation conditions changed not only the molecular mass but also the structure of EPS. Ruas-Madiedo, Hugenholtz, et al. (2002) and Ruas-Madiedo, Tuinier, et al. (2002) on the other hand, studying different EPS from different strains, could clearly conclude that there is a positive correlation between the molecular parameters linked to viscosifying effects (chain stiffness, molecular mass) and the bulk viscosity of the fermented milk. In the present work, although EPS isolated from the fermentations at pH 5.5 had higher molecular mass, it showed lower viscosity as it had a more compact structure than those produced at pH 6.5. The results of radii of gyration shown in Table 3 agree with those previously reported for Lc. lactis subsp. cremoris. Looijesteijn et al. (2000) reported radii of around 74 nm for EPS NIZO B40 and 52 nm for EPS NIZO B891 produced in different media. In addition, Tuinier (1999) previously reported a number-average radius of gyration of 86  2 nm for the EPS NIZO B40. In contrast, Ruas-Madiedo, Hugenholtz, et al. (2002) and Ruas-Madiedo, Tuinier, et al. (2002) measured radii of about 130 nm for different strains of Lc. lactis subsp. cremoris. Radii of gyration for EPS obtained from other LAB strains also have been reported and are generally larger than those found in this study and as previously reported for Lc. lactis subsp. cremoris. For example, the radius of gyration of EPS produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483 was around 150 nm (Goh et al., 2005). This emphasizes the differences in the molecular characteristics of EPS produced by different strains, although differences may also derive from variations in the methods of purification of the EPS. A higher amount of whey protein added to the milk permeate also seemed to affect the molecular mass of the resulting EPS. Fig. 5B shows the difference in the molecular mass between two EPS isolated from fermentations at pH 5.5 with or without 2% whey protein added. The EPS isolated from media containing additional whey protein showed a larger molecular mass than the EPS isolated from fermented permeate. Petry et al. (2003) suggested that milk constituents modify the physicochemical characteristics of EPS.

I. Ayala-Herna´ndez et al. / International Dairy Journal 18 (2008) 1109–1118

140 130

slope = 0.39

90 80 70 60 50 40 30 20 10 1.0E+05

1.1E+06

2.1E+06

3.1E+06

Molar mass (g

4.1E+06

5.1E+06

8.10E+06

1.01E+07

mol-1)

100 90

B slope = 0.15

80 70 60

slope = 0.05

50 40

0.8

1.0E+06

0.6 0.4

1.0E+05

20

1

21

22

23

0

24

25

Elution time (min) 1.0E+07

B

1

2.10E+06

4.10E+06

6.10E+06

Molar mass (g mol-1) 100 90

C

80

RMS radius (nm)

0.2

20

10 1.00E+05

Refractive index

Molar mass (g mol -1)

100

30

A

slope = 0.32

70 60

slope = 0.21

50 40 30

0.8

1.0E+06

0.6 0.4

1.0E+05

Refractive index

Molar mass (g mol -1)

slope = 0.27

110

1.0E+07

1.0E+04 19

A

120

RMS radius (nm)

Earlier, Cerning, Bouillanne, Desmazeaud, and Landon (1986) suggested that caseins stimulated the production of EPS. However, in the present work, the behaviour of the isolated EPS in solution suggests that the dramatic increase in viscosity of the fermentation media at low pH and with 2% whey protein present is mostly caused by the interactions between the polysaccharide molecules and the whey proteins in the media. The conformation of the EPS isolated from the different fermentation experiments can be predicted from a plot of the molecular mass (g mol1) versus the root mean square radius (RMS) as shown in Fig. 6. The slope of the log–log plot gives information regarding the polysaccharide conformation. The smaller the slope, the more compact the molecule. The slope of the dependence of molecular mass on radius ranged from 0.05 for the EPS produced at pH 5.5–0.39 for the EPS produced at pH 6.5 suggesting that the bacterial polysaccharide molecule adopted a more compact structure in EPS extracted from permeate fermented at low pH. The EPS produced at pH 6.5 showed the most expanded structure, followed by the EPS produced at pH 5.5 in media without protein addition, whereas the EPS isolated from pH 5.5 with 2% whey proteins resulted in the most compact structure. These findings support the conclusions derived from the molecular mass calculations and the rheological properties of the isolated EPS solutions. It is possible to hypothesize that when measured in isolation, at concentrations >5 mg mL1, without the effect of the interactions with protein, the less compact, more extended structure as the one produced at pH 6.5 would generate higher viscosities than the more compact one produced at pH 5.5. The same explanation would apply when comparing the medium

RMS radius (nm)

1116

0.2 1.0E+04 19

20

21

22

23

24

0

20 10 1.00E+05

6.00E+05

1.10E+06

1.60E+06

2.10E+06

Molar mass (g mol-1) Fig. 6. Conformation plots (RMS versus molecular mass) for two replicate samples (replicate 1, grey symbols; replicate 2, black symbols; from separate fermentations and purifications) for: milk permeate þ 2% whey protein at pH 6.5 (A), milk permeate þ 2% whey protein at pH 5.5 (B) and milk permeate with no additional protein at pH 5.5 (C). The slope from the log–log relationship is also indicated.

25

Elution time (min) Fig. 5. Molar mass as a function of elution time for EPS purified from fermentations (A) at pH 5.5 (black line) or 6.5 (grey line); (B) fermentations at pH 5.5 with (black line) or without whey protein supplementation (grey line). The RI elution traces are also indicated.

supplemented with whey proteins versus unsupplemented medium. In this case the structure produced in the presence of whey proteins would be more compact, by itself, but when analyzed in the original fermentation media the interactions with

I. Ayala-Herna´ndez et al. / International Dairy Journal 18 (2008) 1109–1118

3.3.2. Dynamic light scattering To obtain additional information on the behaviour of EPS in colloidal systems, such as milk and dairy products, the interactions of the purified EPS with colloidal particles were studied. Firstly, it was important to determine if EPS could be adsorbed on polymer latex. Although latex is negatively charged, negatively charged polymers such as pectins or proteins at high pH often adsorb on the surface of these colloidal particles (Dalgleish & Leaver, 1991). The apparent diameter data demonstrate that the EPS was attached to latex particles (Fig. 7A). When 60 nm latex particles were used, changes in the apparent diameter were not noted, most likely because of the similarity in the size between the EPS aggregates and the latex particles. On the other hand, it was apparent that EPS adsorbed on 350 nm latex particles. With addition of EPS to the latex suspensions the apparent diameter of the latex increased, although EPS extracted from fermentations of permeate containing whey proteins at 5.5 and 5.8 showed an increase in size only when a high amount of EPS was added (0.75 mg mL1). The greatest increase in particle size was provided by the EPS extracted from the media without protein addition (5.5 or 5.8), followed closely by the EPS extracted from medium containing 2% whey protein but fermented at pH 6.5. These results can be explained by the structure of these polysaccharides compared to the EPS extracted at low pH from supplemented media. The conditions that caused the highest ropiness and viscosity in the fermentation media (pH 5.5 and 5.8, with 2% whey protein in the permeate) generated EPS that bound inefficiently to the polymer latex particles. This may be caused by their more aggregated structure, which might have less functional/ charged groups free to interact with the latex particles. The latex particles showed a zeta potential of about 60 mV and the charge decreased when EPS was adsorbed on the latex particle

Apparent diameter (nm)

550

A

500 450 400 350 300

0

0.2

0.4

0.6

0.8

Concentration of EPS (mg mL-1)

(Fig. 7B). The decrease in charge of the particles was not significantly affected with the increase in concentration of EPS. This indicated that the EPS is negatively charged, as further adsorption of the polymer on the surface maintained a negative zeta potential. If EPS molecules were neutral or positively charged, further adsorption on the surface of polymer latex would bring the particles closer to a neutral value of zeta potential. The highest extent of change in zeta potential was shown for EPS extracted from media fermented at pH 6.5 supplemented with whey proteins. The lowest changes were observed for samples of EPS isolated from fermentations at pH 5.5 and 5.8 in the presence of whey proteins, confirming the low adsorption of these molecules to the polymer latex particles, as indicated in Fig. 7A. These results provided further evidence that the EPS extracted under different fermentation conditions were quite different in their functional behaviour. To confirm the nature of the charge of the EPS extracted from the fermentation of Lc. lactis subsp. cremoris JFR1, a similar experiment was carried out observing the interactions between EPS and a different colloidal particle: a whey protein-stabilized emulsion droplet at pH 3.5. For this experiment, only EPS isolated from the fermentation of permeate containing 2% whey proteins at a controlled pH of 5.5 was employed. At pH 3.5 the whey protein emulsion droplets are positively charged and have been shown to interact with negatively charged polysaccharides (Gancz et al., 2005). Fig. 8 shows the average size of the whey protein-stabilized oil droplets as well as the zeta potential as a function of EPS concentration. The apparent diameter significantly increased with the addition of EPS (0.2 mg mL1), with a total increase of about 140 nm. This is in agreement with the estimated radius of gyration of the polysaccharide that was previously calculated (around 70 nm). Increasing concentrations of EPS > 0.2 mg mL1 did not show a further increase in size. With the adsorption of EPS molecules on the surface of WPI-stabilized emulsions, the charge of the emulsion droplets showed a significant decrease from þ25 to about 17 mV, confirming the negative charge of EPS and its interaction with whey proteins. A further increase in EPS concentration did not change the charge of the droplet. This is often the case during the adsorption of a polyelectrolyte on emulsion droplets. It is important to note that the EPS used in this experiment adsorbed the least amongst those tested in the adsorption experiments with latex shown in Fig. 7. This may suggest that the difference between the various EPS samples extracted from different fermentations may not be related only to the size and branching, but also to charge, with the EPS extracted from the low pH fermentation being the most negatively charged. This could also explain the varied extent of interaction of the EPS with negatively charged polymer latex (Fig. 7), and the differences in the EPS binding behaviour to latex compared to whey protein-stabilized emulsion droplets (Fig. 8).

-25 -30 -35 -40 -45 -50 -55 -60 -65

0

40

500

B

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Concentration of EPS (mg mL-1) Fig. 7. Apparent diameter (A) or zeta potential (B) of latex particles as a function of the amount of EPS present in suspension. EPS extracted from permeate without (B, ,) or with 2% whey protein added (C, -, :) fermented at a controlled pH of 5.5 (,, -), 5.8 (B, C) and 6.5 (:). Data are average of two independent experiments.

Apparent diameter (nm)

Zeta potential (mV)

-20

30

450

20

400

10

350

0 -10

300 250

-20

0

0.1

0.2

0.3

0.4

Zeta potential (mV)

the proteins generate a much stronger structuring effect that overshadows the viscosifying properties of the EPS alone.

1117

-30 0.5

Concentration of EPS (mg mL-1) Fig. 8. Effect of addition of EPS isolated from permeate with 2% whey protein added fermented at pH 5.5 to a positively charged whey protein emulsion. Apparent diameter (,) and zeta potential (B). Data are average of two independent experiments.

1118

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4. Conclusions The results of this research demonstrate that the pH of fermentation and the addition of protein to the media had an effect on the molecular characteristics and functionality of the EPS produced by Lc. lactis subsp. cremoris JFR1. Dynamic light scattering studies carried out to study the interactions of EPS with colloidal particles proved that the EPS molecules were negatively charged and they interacted with whey proteins at low pH. At pH 5.5 and 5.8 the whey proteins are still negatively charged overall, but it is possible to hypothesize that the EPS may interact with positively charged patches on the protein aggregates. The rheological behaviour of the crude EPS in the fermented media was different from that in the purified form, confirming the importance of both molecular characteristics and interaction with proteins on the viscosifying effect. The results of this work suggest that the impact of EPS on the textural properties of fermented products is determined not only by molecular parameters (molecular mass and conformation) but also by the ability of the EPS to interact with milk proteins.

Acknowledgements The authors would like to thank the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) for the funding provided for this project. Dr. Anton Korenevsy, Eduarda Bainy, Seddik Khalloufi and Edita Vesperej are greatly thanked for their invaluable help in the realization of these experiments. Davisco Foods is also gratefully acknowledged for donating the research grade whey protein isolate.

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