- Email: [email protected]
Effect of exopolysaccharide-producing Lactococcus lactis strains and temperature on the permeability of skim milk gels.
Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253 www.elsevier.com/locate/colsurfa
Effect of exopolysaccharide-producing Lactococcus lactis strains and temperature on the permeability of skim milk gels. Patricia Ruas-Madiedo, Pieternella Zoon * NIZO Food Research, Kernhemseseweg 2, PO Box 20, 6710 BA Ede, The Netherlands
Abstract In fermented milks such as yoghurt, lactic acid bacteria (LAB) produce lactic acid, which induces aggregation of the milk proteins, leading to gel formation. Exopolysaccharide (EPS)-producing LAB may improve the texture of fermented milks, depending on the strain. In this study the effect of EPS-producing strains on the permeability of fermented milk gels was investigated. Permeability is related to the roughness and serum separation in fermented milk products, which are important quality characteristics. Milk was fermented at 20, 25 and 30 8C with one non-EPSproducing Lactococcus lactis strain, coded SK110, and with four EPS-producing L. lactis strains, coded B35, B39, B40 and B891. Permeability of the gels was measured by permeation of serum through the gels and for the calculation of the permeability coefficient differences in kinematic viscosity of the various sera were taken into account. The permeability of the gels made with the EPS-producing strains was higher than the permeability of the gels made with SK110, although no clear differences could be observed in the protein network structure as visualised with confocal scanning laser microscopy (CSLM). Therefore the flow properties of the sera were studied in more detail. A new method was used: the flow of serum through a porous glass medium (glass filter). It appeared that the relative differences in flow time through the glass filter of the various sera were much larger than the relative differences in kinematic viscosity. This implies that the effect of EPS on the permeability coefficient originated mainly from the flow behaviour of the sera and not from an effect on the structure of the protein network. At higher fermentation temperature, the permeability coefficient of the gels was higher due to larger pores in the protein network, especially at 30 8C. The only exception was strain B891. The permeability coefficients of the gels made with this strain were almost the same at 20, 25 and 30 8C. The low permeability coefficient at 30 8C is probably due to the high resistance against flow of the serum and the formation of EPS relatively late during the fermentation (high concentration in the pores). At 20 8C clusters of bacteria were seen on CSLM pictures, which might have caused a relatively high permeability. This effect will counteract the temperature effect on the protein network structure (smaller pores at lower temperature). # 2002 Elsevier Science B.V. All rights reserved. Keywords: Fermented milks; Structure; Permeability; Exopolysaccharides; Lactic acid bacteria
1. Introduction
* Corresponding author. Tel.: /31-318-659-511; fax: /31318-650-400. E-mail address: [email protected] (P. Zoon).
Dairy products are generally considered as healthy and tasteful foods. Many of these products are fermented with lactic acid bacteria (LAB), e.g.
0927-7757/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 2 ) 0 0 5 1 7 - 4
246
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
cheeses, butter, yoghurt and yoghurt drinks. Both mesophilic and thermophilic LAB strains are used. Mesophilic strains, such as Lactococcus lactis species, are used for semi-hard cheese manufacture and in the Scandinavian countries for fermented milk products, for example la˚ngfil. Thermophilic strains, such as Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus , are commonly used for yoghurt. The optimal growth for mesophilic strains occurs around 30 /35 8C, and for thermophilic strains around 40/45 8C. A common feature of the LAB is the conversion of the milk sugar, lactose, into lactic acid. In yoghurt and other fermented milks, the lactic acid causes gel formation due to aggregation of the milk proteins. The most abundant protein, the casein, is present as associates named casein micelles. These micelles are sterically stabilised by a layer of k-casein, which have a net negative charge [1]. During acidification, this ‘hairy’ layer collapses, thereby diminishing the stabilisation, and around the iso-electric point the casein micelles start to aggregate and form a gel. The gel structure has been mainly studied by electron microscopy [2] and more recently by confocal scanning laser microscopy (CSLM) [3 /5]. Another useful technique involves the measurement of the gel permeability [6,7]. Stirred yoghurt is made by gently stirring the (acidified) milk gel, cooling and subsequent packaging. Good-quality yoghurt has a fresh acidic taste, a typical yoghurt aroma originating mainly from acetaldehyde, and has a smooth appearance and a thick, creamy texture. Besides lactic acid, LAB also produce flavour components such as acetaldehyde and proteolytic and, to a lesser extent, lipolytic enzymes. Most LAB also form exopolysaccharides (EPS), which enhances the viscosity of stirred yoghurt. Recently, the mechanism by which EPS-producing LAB control the viscosity of fermented milks has been studied by various PhD students and a Postdoctorate at NIZO food research in co-operation with the Universities of Wageningen, Twente and Utrecht [5,8 /11]. The key factors for viscosity enhancement appear to be the concentration, molar mass and stiffness of the EPS. The latter is related to the constituent sugar residues, the
linkages between the sugar residues and the presence of side groups [12]. From the work of Van Marle [5] it appeared that besides these EPS characteristics also the permeability of the yoghurt gel is able to affect the viscosity of stirred yoghurt. Furthermore, a strong relation was found between the permeability of yoghurt gels and the ‘roughness’ or the particle size of the stirred yoghurts. The variation in permeability was obtained by using different thermophilic starter cultures and by variation in temperature (32 /45 8C) during fermentation. At higher temperature, stress relaxation is enhanced as measured by a higher loss angle in dynamic rheological testing [13] and also the attraction between the casein micelles is stronger [14], leading to a more compact network with larger pores and thus a higher permeability coefficient. The variation in permeability among the gels produced by the starters in the work of Van Marle [5] was mainly due to the way in which the starters were growing at the moment of casein aggregation. The Lactobacillus cells of culture LL grew in colonies when casein aggregation started, whereas only slight association of the cells of the culture ISt and no association of the culture RR was observed at the start of the casein gel formation. The colony-wise growth caused large pores and thereby a high permeability coefficient. The role of EPS in permeability was not investigated by Van Marle, although the difference in permeability coefficient between gels made with the starter culture RR and gels made by chemical acidification with glucono-d-lactone (GDL) were assumed to originate at least partly from the presence of EPS or LAB cells in the larger pores of the casein network. However, not only was the presence of EPS or LAB different between the gels made with LAB or with GDL, but also the pH decrease with GDL was much faster and gluconic acid was formed instead of lactic acid; these factors also might affect the protein structure of the gel. In the present study, the effect of EPS on the permeability coefficient of fermented milk gels was further investigated in the temperature range between 20 and 30 8C. Special attention was given to the flow properties of the serum obtained from the fermented milk products in relation to the
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
permeability coefficient. The flow properties were measured by capillary and low-shear viscometry as used by Tuinier [15], but also by a new method: the flow of serum through a fixed porous medium (a glass filter). Four EPS-forming (mesophilic) Lactococcus lactis strains of the NIZO culture collection, coded B35, B39, B40 and B891, were used and one non-EPS-forming L. lactis strain coded SK110. The chemical composition of the EPS of these strains was known [10] as well as the physical properties of the EPS in aqueous solution [8] and the viscosity-enhancing effect in fermented milk products [11].
247
Microprocessor pH/ION meter (WTW, Weilheim, Germany). 2.3. Milk gel formation analysis (gelation point) The formation of the protein-network gel during milk fermentation was determined by light scattering (diffusing wave spectroscopy) using RheoLight equipment (NIZO food research, Ede, The Netherlands) in back-scattering mode. From the correlation function the half-decay time was determined. Aggregation of casein micelles leads to an increase in half-decay time. 2.4. EPS analysis
2. Material and methods 2.1. Bacterial strains and culture conditions Strains of L. lactis subsp. cremoris of the NIZO food research culture collection were used. Four of these strains (B35, B39, B40 and B891) were EPSproducing. The EPS concentration in the fermented milks varied from 33 to 79 mg l 1 and the viscosity of the stirred fermented milks was high (/100 Posthumus s) [11]. Strain SK110 produced no detectable amount of EPS (less than 5 mg l 1) and the viscosity of stirred fermented milks was low (:/15 Posthumus s). This strain was considered as a non-EPS-producing strain. All bacteria were stored at /40 8C in litmus milk and these stocks were directly grown overnight at 30 8C. 2.2. Manufacture of fermented milk gels The manufacture of fermented milk gels was carried out at 20, 25 or 30 8C according to the procedure described by Ruas-Madiedo et al. [11]. Pasteurised reconstituted (11.8% dry matter) skim milk was used. Yeast extract (0.1%) was added to milk in fermentations at 20 8C to enhance the growth and acidification. For fermentations with B39, yeast extract (0.1%) was used at all fermentation temperatures, because this strain did not grow well in milk. It took 169/1 h at 30 8C and 189/1 h at 20 or 25 8C to reach a final pH values of 4.459/ 0.05. The pH was continuously monitored using a
The EPS production was determined around the gelation point and at the end of the fermentation period (pH about 4.45) as described by RuasMadiedo et al. [11]. 2.5. Permeability measurements of gels The method described by van Marle and Zoon [6] was used to determine the permeability of milk gels acidified with the five LAB strains. Sterile glass tubes, open at both ends, were placed in the inoculated milk and the gel formation took place inside the tubes during the incubation period at 20, 25 or 30 8C. The tubes containing milk-gels were placed into a vat filled with serum obtained after centrifugation (10 000 /g, 4 8C, 30 min) of the fermented milks made under identical conditions (same strain and incubation temperature). The permeability measurement was always performed at 20 8C. The amount of serum permeated through the gel was measured at subsequent intervals of 45 min and the permeability coefficient was calculated: (h ht2 ) Bt ln nH=g(t2 t1 ) (1) (h ht1 ) where Bt is the permeability coefficient, h is the height of the serum in the reference tube, ht 1 is the height of the serum in the gel tube at time t1, ht 2 is the height of the serum in the gel tube at time t2, n is the kinematic viscosity of serum, H is the length
248
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
of the milk gel and g is the acceleration due to gravity. 2.6. Viscosity measurements of fermented milk serum Serum samples were obtained by centrifugation (10 000 /g , 4 8C, 30 min) from milk gels obtained at 20, 25 and 30 8C after fermentation to pH 4.45. The flow properties of serum samples were measured at 20 8C by three methods, as follows. 2.6.1. Ubbelohde capillary viscometer The kinematic viscosity was measured with an Ubbelohde viscometer (AVS350, Schott-Gera¨te, Germany) employing a capillary with diameter of 0.63 mm. 2.6.2. Low shear viscometer The dynamic shear viscosity (h) was measured with a Contraves LS30 rheometer (Contraves AG, Zu¨rich, Austria) at a shear rate of 0.1498 s 1. The apparatus was equipped with a concentric cylinder geometry Couette type (outer and inner diameter 12 and 11 mm, respectively, and bob length of 8 mm). 2.6.3. Filtration time through porous glass membrane A sample of serum (25 ml) was poured into a glass cylinder with a glass filter at the bottom (23 mm diameter, 3 mm thickness and 100/160 mm pore size). The glass filter was placed on the top of a measuring cylinder and the time (s) needed to permeate the first 10 ml through the glass filter was registered as flow time. 2.7. Serum separation from fermented milks Milk gels made with strain B35 or B891 at 20 or 30 8C were centrifuged at 5 8C for 10 min at speeds ranging from 3000 to 9000 rpm in a Sorvall centrifuge (Du Pont, Delawere, USA) employing a SLA-600 TC rotor (Du Pont). The amount of released serum was weighed and expressed as percentage of the weight of the initial fermented milk sample.
2.8. Confocal scanning laser microscopy The microstructure of fermented milks incubated at 20 and 30 8C with strains B35, B891 and SK110 were analysed by confocal scanning laser microscopy (CSLM). Samples were prepared according to the method described by van Marle [5]. The milk was centrifuged (10 000 /g, 4 8C, 30 min) to remove non-dissolved powder particles. The fluorescent dye Rhodamine B (0.001% w/v) (Sigma Chemical Co., St. Louis, MO) was added to the milk before fermentation to stain protein and Acridine Orange (0.002% w/v) (BDH Laboratory Supplies, Poole, England) was added to stain microorganisms and protein. Fermentation took place in special cuvettes with thin cover microscopy-slips (24 /60 mm) as bottom and top sides. A Leica TCS NT/SP (Leica Microsystems GmbH, Heidelberg, Germany) microscope with an ArKr-laser was used to analyse the fermented milk gels. The numerical aperture of the objective (63 /) was 1.2 and gels were studied at a depth between 5 and 25 mm above the bottom. The excitation wavelength of Rhodamine and Acridine dyes was l /488 and 568 nm, respectively. Two CSLM micrographs (area: 1024 /1024 pixels) of each gel type were taken in two replicated experiments.
3. Results and discussion 3.1. Effect of EPS on permeability Milk gels were made with the EPS-forming strains B35, B39, B40 and B891 and with the non-EPS-producing strain SK110 at 20, 25 and 30 8C. After a pH of 4.459/0.05 was reached, the permeability of the gels was measured at 20 8C. The permeability coefficient (Fig. 1) was calculated with Eq. (1) using the kinematic viscosity of the sera measured with an Ubbelohde capillary viscometer (Table 1). The permeability coefficients of gels made with the non-EPS-producing strain SK110 were much higher (factor of about 2) than those of the gels made with the EPS-forming strains. If the flow properties of the serum in the gel were described well by the kinematic viscosity,
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
Fig. 1. Mean permeability coefficient (Bt) of milk gels fermented with five strains of L. lactis subsp. cremoris at 20 (j), 25 (I) and 30 8C (b). The kinematic viscosity obtained by Ubbelohde measurement was used to calculate the permeability coefficient.
this would imply that the structure of the protein gel was more inhomogeneous for the gel made with SK110 than for the gels made with the EPSforming strains. The structure of the gels was visualised by CSLM (Fig. 2). The number of bacteria in the pictures was high, probably due to some sedimentation of bacteria. The cells of SK110 were homogeneously spread in the protein gel, whereas the cells of B35 and B891 were
249
clustered, especially at 20 8C. These clusters will probably cause an increase in permeability [5]. Based on the CSLM pictures it seems unlikely that the lower permeability of gels made with B35 and B891 originates from a less coarse protein network containing smaller pores compared to the gels made with SK110. Another reason for the lower permeability coefficient of the gels made with EPS-producing cultures could be a higher resistance against flow of the EPS-containing sera than that measured with the Ubbelhode capillary viscometer. Therefore two other methods were used to measure the flow properties of the serum: dynamic shear viscosity measured at low shear (ca. 0.15 s 1) and flow through a glass filter. The results of the low shear measurements are given in Table 1. On average, the viscosity values measured with the low shear viscometer were higher than those measured with the Ubbelohde viscometer; also the differences between the strains were larger for the low-shear viscosity, e.g. the low-shear viscosity of B40 is about twice as high as the low-shear viscosity of SK110, whereas the kinematic viscosity measured with the Ubbelohde viscometer is only 1.5 times higher for B40 than for SK110. The lower values obtained with the Ubbelohde visc-
Table 1 Kinematic viscosity (n ) of serum obtained from milks fermented with five strains of L. lactis subsp. cremoris at 20, 25 or 30 8C Strain
Incubation T . (8C)
nubbelohde (10 6 m2 s 1)
nlow
B35
30 25 20 30 25 20 30 25 20 30 25 20 30 25 20
1.49/0.12 1.59/0.06 1.79/0.32 1.49/0.02 1.49/0.01 1.49/0.02 1.89/0.05 1.89/0.01 1.89/0.01 1.49/0.08 1.59/0.01 1.59/0.08 1.29/0.01 1.29/0.00 1.29/0.01
1.89/0.31 2.89/0.01 2.29/0.74 1.8 2.0 1.6 2.69/0.26 3.09/0.05 3.59/0.85 1.59/0.09 1.89/0.21 1.89/0.25 1.69/0.02 1.4 1.3
B39
B40
B891
SK110
shear
(10 6 m2 s 1)
Measurements were carried out at 20 8C by two methods: Ubbelohde capillary viscometer and low-shear viscometer. The kinematic viscosity from low-shear measurements was calculated from the formula: n /hr 1, where h is the shear viscosity and r is the density /1026 kg m 3 for all sera.
250
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
Table 2 Permeability coefficients (Bt) of gels fermented with EPSproducing strains at 30 8C calculated with values of kinematic viscosity measured by Ubbelohde capillary viscometer and obtained from low-shear viscometer (at 0.1498 s 1) Strain
B35 B39 B40 B891
Fig. 2. CSLM micrographs of milk gels fermented by SK110 (a: 20 8C; and b: 30 8C), B35 (c: 20 8C; and d: 30 8C) and B891 (e: 20 8C; and f: 30 8C) strains. Scanning depth: 15 mm. Bar / 20 mm. Milk proteins are stained in yellow and bacteria are coloured in green.
ometer will be due to the high shear rate [15]. If the low-shear viscosity is used in Eq. (1), the permeability coefficient of the gels made with the EPSforming strains increases somewhat (Table 2), but still remains a factor of about 2 lower than the permeability coefficient of the gel made with SK110. Possibly, also the low-shear viscosity is not a good measure of the flow properties of EPScontaining serum through the pores of the protein network. Therefore, the time needed for a given volume of serum of B35, B891 and SK110 to flow through a glass filter was measured. The glass filter can be regarded as a ‘model network structure’. The flow times are given in Fig. 3. The flow times
Bt (10 14 m2) Ubbelohde
Low-shear
8.89/0.92 11.09/0.15 9.19/1.30 5.19/0.27
9.89/1.03 11.89/0.16 8.79/0.88 5.29/0.28
of the EPS-containing sera are much longer than the flow time of SK110 serum: for the 30 8C fermentation, the flow time of B35 was about five times higher and that of B891 even 11 times higher than that of SK110. Furthermore, the flow time of B891 serum obtained at 30 8C was about twice that of B35, whereas the kinematic viscosity of the same sera measured with the Ubbelohde viscometer was equal. This implies that the type of EPS might affect its flow properties in a porous system. A limited number of experiments was done with a different glass filter to check whether (qualitatively) the same results would be obtained (Table 3, the data of filter 1 are those shown in Fig. 3). Although the absolute values of the flow time were different, the relative differences between the sera were fairly similar with both glass filters. If the flow time is divided by the flow time of water and multiplied by the serum density (1026 kg m 3) a kind of ‘kinematic viscosity’ is calculated. If this value is used in Eq. (1), a much higher permeability is obtained for the EPS-containing gels (53 /10 14 m2 for B35 and 64 /10 14 m2 for B891), whereas the value of SK110 remains almost the same (25 /10 14 m2). Although the flow of serum through this porous glass medium is different from the serum flow through the milk gels, the much higher values of the ‘kinematic viscosity’ obtained from the glass filter measurement imply that the flow of the serum through the milk gel is not well described by the simple shear viscosity. Probably elongational effects play a large part in the flow behaviour through the gel: the pores do
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
251
Fig. 3. Serum viscosity of B35 (j), B891 (I) and SK110 (b) strains of L. lactis subsp. cremoris measured at 20 8C by filtration through a porous glass membrane (a) and ‘kinematic viscosity’ calculated from these values (b). The fermentation temperatures were 20 and 30 8C.
Table 3 Flow time of water and serum samples obtained from milk fermented at 30 8C with strains B35, B891 and SK110 through two different glass filters Strain
B35 B891 SK110 H2O
Flow time (s) Filter 1
Filter 2
1089/4 2369/6 219/1 149/1
429/7 1519/3 199/2 99/1
not consist of long tubes with a constant diameter, but they are interconnected openings with varying diameters, thereby causing continuous compression and tension of the permeated liquid. In addition to the shear viscosity, which is a good measure of the viscosity-enhancing effect of EPS in stirred fermented products, the new ‘glass filter method’ could be a good tool to ‘semi-quantitatively’ evaluate the flow of EPS-containing serum in the milk gel. Due to the different structure of the pores in the glass filters compared to that in the milk gels and due to the different interaction of EPS with glass or with proteins, it will be difficult to obtain correct absolute values of the serum viscosity from the glass filtration experiments.
3.2. Effect of temperature Gels were made at 20, 25 and 30 8C. In accordance with the work of Van Marle [5] and of Lucey et al. [4], the permeability coefficient of the gels was higher at higher temperature for all strains, with only small differences between 20 and 25 8C and larger differences between 25 and 30 8C (Fig. 1). The only exception was the permeability coefficient of the gels made with B891. At all temperatures the permeability coefficient of these gels was about equal. The exceptional behaviour of strain B891 at 30 8C was further investigated. Milk gels of B891 and B35 made at 20 and 30 8C were centrifuged (at 5 8C) at several speeds and the amount of serum release was measured (Fig. 4). The serum release of gels made with B891 was equal at both temperatures, whereas the serum release of gels made with B35 at 30 8C was larger than that of gels made at 20 8C, which will be due to the higher permeability coefficient at 30 8C. The network structure was visualised with CSLM (Fig. 2). In both gels (B35 and B891) made at 20 8C some of the bacteria were present in clusters and the clusters of B891 were larger than those of B35. This might increase the permeability due to the presence of large pores, which are induced by these clusters. At 30 8C the pores in the protein network will be larger than at
252
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
Fig. 4. Amount of serum separation (% w/w) as a function of centrifugal speed of milk gels fermented with strains B35 and B891 at 20 8C (m) and 30 8C (^).
20 8C due to enhanced re-arrangements of the casein micelles, but on the other hand the very large pores will disappear due to the disappearance of the clusters of bacteria. For B891, the latter effect seems to be larger than for B35. The serum viscosity (Table 1, Fig. 3) did not show much dependence on the fermentation tem-
perature. Only for B35 was a higher viscosity found at 20 8C, which was due to the higher EPS production at that temperature [11]. The other strains produced about the same amount of EPS at all temperatures and also the molar mass and stiffness of the EPS were not affected clearly by temperature in the range between 20 and 30 8C
Fig. 5. Diffusing wave spectroscopy measurements of milk during fermentation with strain B891 at 20 and 30 8C. The half decay time is depicted as a function of pH.
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
[11]. In the previous work of Ruas-Madiedo et al. [11] only the total EPS production was measured. The time at which EPS formation occurs might also be important for its effect on permeability. One might suppose that if EPS is synthesised before the casein micelle aggregation, the EPS would be homogeneously spread over the total gel system. If the EPS were synthesised after the start of the casein micelle aggregation the EPS, due to diffusional limitations, will be predominantly present in the pore in which the EPS-producing bacteria are present and will decrease the permeability coefficient. The amount of EPS produced just before casein micelle coagulation was measured for strains B35 and B891 at 20 and at 30 8C. At 20 8C casein micelle aggregation started for both strains around pH 5.4 and at 30 8C around pH 5.2 (Fig. 5). Strain B35 produced about 64% of the EPS before gelation at 20 8C and about 67% at 30 8C. For strain B891 these figures were 63% at 20 8C and 55% at 30 8C, so B891 produced somewhat more EPS after the start of casein micelle coagulation. In conclusion, the low permeability of gels made with strain B891 at 30 8C is probably due to the high resistance against flow of the serum in the pores, partially caused by the high concentration of EPS in the pores (late EPS formation). Possibly, also other factors affect the flow properties of the serum, such as proteolysis of milk proteins by proteinases excreted by LAB. The proteolytic activity varies among strains [16] resulting in various levels and various types of protein fragments (peptides) in the serum. Another factor to take into account is the interaction between the EPS molecules and other components (mainly proteins) present in the milk gel. A special feature of the B891 EPS is the presence of an acetyl group bound to the glucose residue in the side chains of the EPS polymer backbone [10]. For another EPS, xanthan, it is suggested that the degree of acylation may affect its interaction with other food ingredients such as proteins [17]. It would be worthwhile to further investigate in model systems
253
the interaction of this EPS and other EPSs with casein at the pH of the gel (4.5).
Acknowledgements P. Ruas-Madiedo thanks the European Commission for its postdoctoral fellowship (MCFI1999-01449). Saskia de Jong is acknowledged for the performance of the CSLM pictures.
References [1] C.G. de Kruif, Int. Dairy J. 9 (1999) 183 /188. [2] M. Kalab, P. Allan-Wojtas, E. Phipps-Todd, Food Microstruct. 2 (1983) 51 /66. [3] A.N. Hassan, J.F. Frank, M.A. Farmer, K.A. Schmidt, S.I. Shalabi, J. Dairy Sci. 78 (1995) 2624 /2628. [4] J.A. Lucey, T. van Vliet, K. Grolle, T. Geurts, P. Walstra, Int. Dairy J. 7 (1997) 389 /397. [5] M.E. van Marle., Structure and rheological properties of yoghurt gels and stirred yoghurts, PhD thesis, Twente University, The Netherlands, 1998. [6] M.E. van Marle, P. Zoon, Neth. Milk Dairy J. 49 (1995) 47 /65. [7] J.A. Lucey, H. Singh, Food Res. Int. 7 (1998) 529 /542. [8] R. Tuinier, An exocellular polysaccharide and its interactions with proteins, PhD thesis, Wageningen University, The Netherlands, 1999. [9] E.J. Faber, Investigation of the structure of exopolysaccharides produces by lactic acid bacteria, PhD thesis, Utrecht University, The Netherlands, 2000. [10] W.H.M. Van Casteren, Structural characterisation and enzymic modification of exopolysaccharides from Lactococcus lactis , PhD thesis, Wageningen University, The Netherlands, 2000. [11] P. Ruas-Madiedo, R. Tuinier, M. Kanning, P. Zoon, Int. Dairy J. 12 (2002) 689 /695. [12] R. Tuinier, W.H.M. van Casteren, P.J. Looijesteijn, H.A. Schols, A.G.J. Voragen, P. Zoon, Biopolymers 59 (2001) 160 /166. [13] P. Zoon, T. van Vliet, P. Walstra, Neth. Milk Dairy J. 42 (1988) 271 /294. [14] D.S. Horne, Int. Dairy J. 8 (1998) 171 /177. [15] R. Tuinier, P. Zoon, M.A. Cohen Stuart, G.J. Fleer, C.G. de Kruif, Biopolymers 50 (1999) 641 /646. [16] A.P. Laws, V.M. Marshall, Int. Dairy J. 11 (2001) 709 / 721. [17] I.W. Sutherland, Food Biotechnol. 6 (1992) 75 /86.
Effect of exopolysaccharide-producing Lactococcus lactis strains and temperature on the permeability of skim milk gels. Patricia Ruas-Madiedo, Pieternella Zoon * NIZO Food Research, Kernhemseseweg 2, PO Box 20, 6710 BA Ede, The Netherlands
Abstract In fermented milks such as yoghurt, lactic acid bacteria (LAB) produce lactic acid, which induces aggregation of the milk proteins, leading to gel formation. Exopolysaccharide (EPS)-producing LAB may improve the texture of fermented milks, depending on the strain. In this study the effect of EPS-producing strains on the permeability of fermented milk gels was investigated. Permeability is related to the roughness and serum separation in fermented milk products, which are important quality characteristics. Milk was fermented at 20, 25 and 30 8C with one non-EPSproducing Lactococcus lactis strain, coded SK110, and with four EPS-producing L. lactis strains, coded B35, B39, B40 and B891. Permeability of the gels was measured by permeation of serum through the gels and for the calculation of the permeability coefficient differences in kinematic viscosity of the various sera were taken into account. The permeability of the gels made with the EPS-producing strains was higher than the permeability of the gels made with SK110, although no clear differences could be observed in the protein network structure as visualised with confocal scanning laser microscopy (CSLM). Therefore the flow properties of the sera were studied in more detail. A new method was used: the flow of serum through a porous glass medium (glass filter). It appeared that the relative differences in flow time through the glass filter of the various sera were much larger than the relative differences in kinematic viscosity. This implies that the effect of EPS on the permeability coefficient originated mainly from the flow behaviour of the sera and not from an effect on the structure of the protein network. At higher fermentation temperature, the permeability coefficient of the gels was higher due to larger pores in the protein network, especially at 30 8C. The only exception was strain B891. The permeability coefficients of the gels made with this strain were almost the same at 20, 25 and 30 8C. The low permeability coefficient at 30 8C is probably due to the high resistance against flow of the serum and the formation of EPS relatively late during the fermentation (high concentration in the pores). At 20 8C clusters of bacteria were seen on CSLM pictures, which might have caused a relatively high permeability. This effect will counteract the temperature effect on the protein network structure (smaller pores at lower temperature). # 2002 Elsevier Science B.V. All rights reserved. Keywords: Fermented milks; Structure; Permeability; Exopolysaccharides; Lactic acid bacteria
1. Introduction
* Corresponding author. Tel.: /31-318-659-511; fax: /31318-650-400. E-mail address: [email protected] (P. Zoon).
Dairy products are generally considered as healthy and tasteful foods. Many of these products are fermented with lactic acid bacteria (LAB), e.g.
0927-7757/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 2 ) 0 0 5 1 7 - 4
246
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
cheeses, butter, yoghurt and yoghurt drinks. Both mesophilic and thermophilic LAB strains are used. Mesophilic strains, such as Lactococcus lactis species, are used for semi-hard cheese manufacture and in the Scandinavian countries for fermented milk products, for example la˚ngfil. Thermophilic strains, such as Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus , are commonly used for yoghurt. The optimal growth for mesophilic strains occurs around 30 /35 8C, and for thermophilic strains around 40/45 8C. A common feature of the LAB is the conversion of the milk sugar, lactose, into lactic acid. In yoghurt and other fermented milks, the lactic acid causes gel formation due to aggregation of the milk proteins. The most abundant protein, the casein, is present as associates named casein micelles. These micelles are sterically stabilised by a layer of k-casein, which have a net negative charge [1]. During acidification, this ‘hairy’ layer collapses, thereby diminishing the stabilisation, and around the iso-electric point the casein micelles start to aggregate and form a gel. The gel structure has been mainly studied by electron microscopy [2] and more recently by confocal scanning laser microscopy (CSLM) [3 /5]. Another useful technique involves the measurement of the gel permeability [6,7]. Stirred yoghurt is made by gently stirring the (acidified) milk gel, cooling and subsequent packaging. Good-quality yoghurt has a fresh acidic taste, a typical yoghurt aroma originating mainly from acetaldehyde, and has a smooth appearance and a thick, creamy texture. Besides lactic acid, LAB also produce flavour components such as acetaldehyde and proteolytic and, to a lesser extent, lipolytic enzymes. Most LAB also form exopolysaccharides (EPS), which enhances the viscosity of stirred yoghurt. Recently, the mechanism by which EPS-producing LAB control the viscosity of fermented milks has been studied by various PhD students and a Postdoctorate at NIZO food research in co-operation with the Universities of Wageningen, Twente and Utrecht [5,8 /11]. The key factors for viscosity enhancement appear to be the concentration, molar mass and stiffness of the EPS. The latter is related to the constituent sugar residues, the
linkages between the sugar residues and the presence of side groups [12]. From the work of Van Marle [5] it appeared that besides these EPS characteristics also the permeability of the yoghurt gel is able to affect the viscosity of stirred yoghurt. Furthermore, a strong relation was found between the permeability of yoghurt gels and the ‘roughness’ or the particle size of the stirred yoghurts. The variation in permeability was obtained by using different thermophilic starter cultures and by variation in temperature (32 /45 8C) during fermentation. At higher temperature, stress relaxation is enhanced as measured by a higher loss angle in dynamic rheological testing [13] and also the attraction between the casein micelles is stronger [14], leading to a more compact network with larger pores and thus a higher permeability coefficient. The variation in permeability among the gels produced by the starters in the work of Van Marle [5] was mainly due to the way in which the starters were growing at the moment of casein aggregation. The Lactobacillus cells of culture LL grew in colonies when casein aggregation started, whereas only slight association of the cells of the culture ISt and no association of the culture RR was observed at the start of the casein gel formation. The colony-wise growth caused large pores and thereby a high permeability coefficient. The role of EPS in permeability was not investigated by Van Marle, although the difference in permeability coefficient between gels made with the starter culture RR and gels made by chemical acidification with glucono-d-lactone (GDL) were assumed to originate at least partly from the presence of EPS or LAB cells in the larger pores of the casein network. However, not only was the presence of EPS or LAB different between the gels made with LAB or with GDL, but also the pH decrease with GDL was much faster and gluconic acid was formed instead of lactic acid; these factors also might affect the protein structure of the gel. In the present study, the effect of EPS on the permeability coefficient of fermented milk gels was further investigated in the temperature range between 20 and 30 8C. Special attention was given to the flow properties of the serum obtained from the fermented milk products in relation to the
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
permeability coefficient. The flow properties were measured by capillary and low-shear viscometry as used by Tuinier [15], but also by a new method: the flow of serum through a fixed porous medium (a glass filter). Four EPS-forming (mesophilic) Lactococcus lactis strains of the NIZO culture collection, coded B35, B39, B40 and B891, were used and one non-EPS-forming L. lactis strain coded SK110. The chemical composition of the EPS of these strains was known [10] as well as the physical properties of the EPS in aqueous solution [8] and the viscosity-enhancing effect in fermented milk products [11].
247
Microprocessor pH/ION meter (WTW, Weilheim, Germany). 2.3. Milk gel formation analysis (gelation point) The formation of the protein-network gel during milk fermentation was determined by light scattering (diffusing wave spectroscopy) using RheoLight equipment (NIZO food research, Ede, The Netherlands) in back-scattering mode. From the correlation function the half-decay time was determined. Aggregation of casein micelles leads to an increase in half-decay time. 2.4. EPS analysis
2. Material and methods 2.1. Bacterial strains and culture conditions Strains of L. lactis subsp. cremoris of the NIZO food research culture collection were used. Four of these strains (B35, B39, B40 and B891) were EPSproducing. The EPS concentration in the fermented milks varied from 33 to 79 mg l 1 and the viscosity of the stirred fermented milks was high (/100 Posthumus s) [11]. Strain SK110 produced no detectable amount of EPS (less than 5 mg l 1) and the viscosity of stirred fermented milks was low (:/15 Posthumus s). This strain was considered as a non-EPS-producing strain. All bacteria were stored at /40 8C in litmus milk and these stocks were directly grown overnight at 30 8C. 2.2. Manufacture of fermented milk gels The manufacture of fermented milk gels was carried out at 20, 25 or 30 8C according to the procedure described by Ruas-Madiedo et al. [11]. Pasteurised reconstituted (11.8% dry matter) skim milk was used. Yeast extract (0.1%) was added to milk in fermentations at 20 8C to enhance the growth and acidification. For fermentations with B39, yeast extract (0.1%) was used at all fermentation temperatures, because this strain did not grow well in milk. It took 169/1 h at 30 8C and 189/1 h at 20 or 25 8C to reach a final pH values of 4.459/ 0.05. The pH was continuously monitored using a
The EPS production was determined around the gelation point and at the end of the fermentation period (pH about 4.45) as described by RuasMadiedo et al. [11]. 2.5. Permeability measurements of gels The method described by van Marle and Zoon [6] was used to determine the permeability of milk gels acidified with the five LAB strains. Sterile glass tubes, open at both ends, were placed in the inoculated milk and the gel formation took place inside the tubes during the incubation period at 20, 25 or 30 8C. The tubes containing milk-gels were placed into a vat filled with serum obtained after centrifugation (10 000 /g, 4 8C, 30 min) of the fermented milks made under identical conditions (same strain and incubation temperature). The permeability measurement was always performed at 20 8C. The amount of serum permeated through the gel was measured at subsequent intervals of 45 min and the permeability coefficient was calculated: (h ht2 ) Bt ln nH=g(t2 t1 ) (1) (h ht1 ) where Bt is the permeability coefficient, h is the height of the serum in the reference tube, ht 1 is the height of the serum in the gel tube at time t1, ht 2 is the height of the serum in the gel tube at time t2, n is the kinematic viscosity of serum, H is the length
248
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
of the milk gel and g is the acceleration due to gravity. 2.6. Viscosity measurements of fermented milk serum Serum samples were obtained by centrifugation (10 000 /g , 4 8C, 30 min) from milk gels obtained at 20, 25 and 30 8C after fermentation to pH 4.45. The flow properties of serum samples were measured at 20 8C by three methods, as follows. 2.6.1. Ubbelohde capillary viscometer The kinematic viscosity was measured with an Ubbelohde viscometer (AVS350, Schott-Gera¨te, Germany) employing a capillary with diameter of 0.63 mm. 2.6.2. Low shear viscometer The dynamic shear viscosity (h) was measured with a Contraves LS30 rheometer (Contraves AG, Zu¨rich, Austria) at a shear rate of 0.1498 s 1. The apparatus was equipped with a concentric cylinder geometry Couette type (outer and inner diameter 12 and 11 mm, respectively, and bob length of 8 mm). 2.6.3. Filtration time through porous glass membrane A sample of serum (25 ml) was poured into a glass cylinder with a glass filter at the bottom (23 mm diameter, 3 mm thickness and 100/160 mm pore size). The glass filter was placed on the top of a measuring cylinder and the time (s) needed to permeate the first 10 ml through the glass filter was registered as flow time. 2.7. Serum separation from fermented milks Milk gels made with strain B35 or B891 at 20 or 30 8C were centrifuged at 5 8C for 10 min at speeds ranging from 3000 to 9000 rpm in a Sorvall centrifuge (Du Pont, Delawere, USA) employing a SLA-600 TC rotor (Du Pont). The amount of released serum was weighed and expressed as percentage of the weight of the initial fermented milk sample.
2.8. Confocal scanning laser microscopy The microstructure of fermented milks incubated at 20 and 30 8C with strains B35, B891 and SK110 were analysed by confocal scanning laser microscopy (CSLM). Samples were prepared according to the method described by van Marle [5]. The milk was centrifuged (10 000 /g, 4 8C, 30 min) to remove non-dissolved powder particles. The fluorescent dye Rhodamine B (0.001% w/v) (Sigma Chemical Co., St. Louis, MO) was added to the milk before fermentation to stain protein and Acridine Orange (0.002% w/v) (BDH Laboratory Supplies, Poole, England) was added to stain microorganisms and protein. Fermentation took place in special cuvettes with thin cover microscopy-slips (24 /60 mm) as bottom and top sides. A Leica TCS NT/SP (Leica Microsystems GmbH, Heidelberg, Germany) microscope with an ArKr-laser was used to analyse the fermented milk gels. The numerical aperture of the objective (63 /) was 1.2 and gels were studied at a depth between 5 and 25 mm above the bottom. The excitation wavelength of Rhodamine and Acridine dyes was l /488 and 568 nm, respectively. Two CSLM micrographs (area: 1024 /1024 pixels) of each gel type were taken in two replicated experiments.
3. Results and discussion 3.1. Effect of EPS on permeability Milk gels were made with the EPS-forming strains B35, B39, B40 and B891 and with the non-EPS-producing strain SK110 at 20, 25 and 30 8C. After a pH of 4.459/0.05 was reached, the permeability of the gels was measured at 20 8C. The permeability coefficient (Fig. 1) was calculated with Eq. (1) using the kinematic viscosity of the sera measured with an Ubbelohde capillary viscometer (Table 1). The permeability coefficients of gels made with the non-EPS-producing strain SK110 were much higher (factor of about 2) than those of the gels made with the EPS-forming strains. If the flow properties of the serum in the gel were described well by the kinematic viscosity,
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
Fig. 1. Mean permeability coefficient (Bt) of milk gels fermented with five strains of L. lactis subsp. cremoris at 20 (j), 25 (I) and 30 8C (b). The kinematic viscosity obtained by Ubbelohde measurement was used to calculate the permeability coefficient.
this would imply that the structure of the protein gel was more inhomogeneous for the gel made with SK110 than for the gels made with the EPSforming strains. The structure of the gels was visualised by CSLM (Fig. 2). The number of bacteria in the pictures was high, probably due to some sedimentation of bacteria. The cells of SK110 were homogeneously spread in the protein gel, whereas the cells of B35 and B891 were
249
clustered, especially at 20 8C. These clusters will probably cause an increase in permeability [5]. Based on the CSLM pictures it seems unlikely that the lower permeability of gels made with B35 and B891 originates from a less coarse protein network containing smaller pores compared to the gels made with SK110. Another reason for the lower permeability coefficient of the gels made with EPS-producing cultures could be a higher resistance against flow of the EPS-containing sera than that measured with the Ubbelhode capillary viscometer. Therefore two other methods were used to measure the flow properties of the serum: dynamic shear viscosity measured at low shear (ca. 0.15 s 1) and flow through a glass filter. The results of the low shear measurements are given in Table 1. On average, the viscosity values measured with the low shear viscometer were higher than those measured with the Ubbelohde viscometer; also the differences between the strains were larger for the low-shear viscosity, e.g. the low-shear viscosity of B40 is about twice as high as the low-shear viscosity of SK110, whereas the kinematic viscosity measured with the Ubbelohde viscometer is only 1.5 times higher for B40 than for SK110. The lower values obtained with the Ubbelohde visc-
Table 1 Kinematic viscosity (n ) of serum obtained from milks fermented with five strains of L. lactis subsp. cremoris at 20, 25 or 30 8C Strain
Incubation T . (8C)
nubbelohde (10 6 m2 s 1)
nlow
B35
30 25 20 30 25 20 30 25 20 30 25 20 30 25 20
1.49/0.12 1.59/0.06 1.79/0.32 1.49/0.02 1.49/0.01 1.49/0.02 1.89/0.05 1.89/0.01 1.89/0.01 1.49/0.08 1.59/0.01 1.59/0.08 1.29/0.01 1.29/0.00 1.29/0.01
1.89/0.31 2.89/0.01 2.29/0.74 1.8 2.0 1.6 2.69/0.26 3.09/0.05 3.59/0.85 1.59/0.09 1.89/0.21 1.89/0.25 1.69/0.02 1.4 1.3
B39
B40
B891
SK110
shear
(10 6 m2 s 1)
Measurements were carried out at 20 8C by two methods: Ubbelohde capillary viscometer and low-shear viscometer. The kinematic viscosity from low-shear measurements was calculated from the formula: n /hr 1, where h is the shear viscosity and r is the density /1026 kg m 3 for all sera.
250
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
Table 2 Permeability coefficients (Bt) of gels fermented with EPSproducing strains at 30 8C calculated with values of kinematic viscosity measured by Ubbelohde capillary viscometer and obtained from low-shear viscometer (at 0.1498 s 1) Strain
B35 B39 B40 B891
Fig. 2. CSLM micrographs of milk gels fermented by SK110 (a: 20 8C; and b: 30 8C), B35 (c: 20 8C; and d: 30 8C) and B891 (e: 20 8C; and f: 30 8C) strains. Scanning depth: 15 mm. Bar / 20 mm. Milk proteins are stained in yellow and bacteria are coloured in green.
ometer will be due to the high shear rate [15]. If the low-shear viscosity is used in Eq. (1), the permeability coefficient of the gels made with the EPSforming strains increases somewhat (Table 2), but still remains a factor of about 2 lower than the permeability coefficient of the gel made with SK110. Possibly, also the low-shear viscosity is not a good measure of the flow properties of EPScontaining serum through the pores of the protein network. Therefore, the time needed for a given volume of serum of B35, B891 and SK110 to flow through a glass filter was measured. The glass filter can be regarded as a ‘model network structure’. The flow times are given in Fig. 3. The flow times
Bt (10 14 m2) Ubbelohde
Low-shear
8.89/0.92 11.09/0.15 9.19/1.30 5.19/0.27
9.89/1.03 11.89/0.16 8.79/0.88 5.29/0.28
of the EPS-containing sera are much longer than the flow time of SK110 serum: for the 30 8C fermentation, the flow time of B35 was about five times higher and that of B891 even 11 times higher than that of SK110. Furthermore, the flow time of B891 serum obtained at 30 8C was about twice that of B35, whereas the kinematic viscosity of the same sera measured with the Ubbelohde viscometer was equal. This implies that the type of EPS might affect its flow properties in a porous system. A limited number of experiments was done with a different glass filter to check whether (qualitatively) the same results would be obtained (Table 3, the data of filter 1 are those shown in Fig. 3). Although the absolute values of the flow time were different, the relative differences between the sera were fairly similar with both glass filters. If the flow time is divided by the flow time of water and multiplied by the serum density (1026 kg m 3) a kind of ‘kinematic viscosity’ is calculated. If this value is used in Eq. (1), a much higher permeability is obtained for the EPS-containing gels (53 /10 14 m2 for B35 and 64 /10 14 m2 for B891), whereas the value of SK110 remains almost the same (25 /10 14 m2). Although the flow of serum through this porous glass medium is different from the serum flow through the milk gels, the much higher values of the ‘kinematic viscosity’ obtained from the glass filter measurement imply that the flow of the serum through the milk gel is not well described by the simple shear viscosity. Probably elongational effects play a large part in the flow behaviour through the gel: the pores do
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
251
Fig. 3. Serum viscosity of B35 (j), B891 (I) and SK110 (b) strains of L. lactis subsp. cremoris measured at 20 8C by filtration through a porous glass membrane (a) and ‘kinematic viscosity’ calculated from these values (b). The fermentation temperatures were 20 and 30 8C.
Table 3 Flow time of water and serum samples obtained from milk fermented at 30 8C with strains B35, B891 and SK110 through two different glass filters Strain
B35 B891 SK110 H2O
Flow time (s) Filter 1
Filter 2
1089/4 2369/6 219/1 149/1
429/7 1519/3 199/2 99/1
not consist of long tubes with a constant diameter, but they are interconnected openings with varying diameters, thereby causing continuous compression and tension of the permeated liquid. In addition to the shear viscosity, which is a good measure of the viscosity-enhancing effect of EPS in stirred fermented products, the new ‘glass filter method’ could be a good tool to ‘semi-quantitatively’ evaluate the flow of EPS-containing serum in the milk gel. Due to the different structure of the pores in the glass filters compared to that in the milk gels and due to the different interaction of EPS with glass or with proteins, it will be difficult to obtain correct absolute values of the serum viscosity from the glass filtration experiments.
3.2. Effect of temperature Gels were made at 20, 25 and 30 8C. In accordance with the work of Van Marle [5] and of Lucey et al. [4], the permeability coefficient of the gels was higher at higher temperature for all strains, with only small differences between 20 and 25 8C and larger differences between 25 and 30 8C (Fig. 1). The only exception was the permeability coefficient of the gels made with B891. At all temperatures the permeability coefficient of these gels was about equal. The exceptional behaviour of strain B891 at 30 8C was further investigated. Milk gels of B891 and B35 made at 20 and 30 8C were centrifuged (at 5 8C) at several speeds and the amount of serum release was measured (Fig. 4). The serum release of gels made with B891 was equal at both temperatures, whereas the serum release of gels made with B35 at 30 8C was larger than that of gels made at 20 8C, which will be due to the higher permeability coefficient at 30 8C. The network structure was visualised with CSLM (Fig. 2). In both gels (B35 and B891) made at 20 8C some of the bacteria were present in clusters and the clusters of B891 were larger than those of B35. This might increase the permeability due to the presence of large pores, which are induced by these clusters. At 30 8C the pores in the protein network will be larger than at
252
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
Fig. 4. Amount of serum separation (% w/w) as a function of centrifugal speed of milk gels fermented with strains B35 and B891 at 20 8C (m) and 30 8C (^).
20 8C due to enhanced re-arrangements of the casein micelles, but on the other hand the very large pores will disappear due to the disappearance of the clusters of bacteria. For B891, the latter effect seems to be larger than for B35. The serum viscosity (Table 1, Fig. 3) did not show much dependence on the fermentation tem-
perature. Only for B35 was a higher viscosity found at 20 8C, which was due to the higher EPS production at that temperature [11]. The other strains produced about the same amount of EPS at all temperatures and also the molar mass and stiffness of the EPS were not affected clearly by temperature in the range between 20 and 30 8C
Fig. 5. Diffusing wave spectroscopy measurements of milk during fermentation with strain B891 at 20 and 30 8C. The half decay time is depicted as a function of pH.
P. Ruas-Madiedo, P. Zoon / Colloids and Surfaces A: Physicochem. Eng. Aspects 213 (2003) 245 /253
[11]. In the previous work of Ruas-Madiedo et al. [11] only the total EPS production was measured. The time at which EPS formation occurs might also be important for its effect on permeability. One might suppose that if EPS is synthesised before the casein micelle aggregation, the EPS would be homogeneously spread over the total gel system. If the EPS were synthesised after the start of the casein micelle aggregation the EPS, due to diffusional limitations, will be predominantly present in the pore in which the EPS-producing bacteria are present and will decrease the permeability coefficient. The amount of EPS produced just before casein micelle coagulation was measured for strains B35 and B891 at 20 and at 30 8C. At 20 8C casein micelle aggregation started for both strains around pH 5.4 and at 30 8C around pH 5.2 (Fig. 5). Strain B35 produced about 64% of the EPS before gelation at 20 8C and about 67% at 30 8C. For strain B891 these figures were 63% at 20 8C and 55% at 30 8C, so B891 produced somewhat more EPS after the start of casein micelle coagulation. In conclusion, the low permeability of gels made with strain B891 at 30 8C is probably due to the high resistance against flow of the serum in the pores, partially caused by the high concentration of EPS in the pores (late EPS formation). Possibly, also other factors affect the flow properties of the serum, such as proteolysis of milk proteins by proteinases excreted by LAB. The proteolytic activity varies among strains [16] resulting in various levels and various types of protein fragments (peptides) in the serum. Another factor to take into account is the interaction between the EPS molecules and other components (mainly proteins) present in the milk gel. A special feature of the B891 EPS is the presence of an acetyl group bound to the glucose residue in the side chains of the EPS polymer backbone [10]. For another EPS, xanthan, it is suggested that the degree of acylation may affect its interaction with other food ingredients such as proteins [17]. It would be worthwhile to further investigate in model systems
253
the interaction of this EPS and other EPSs with casein at the pH of the gel (4.5).
Acknowledgements P. Ruas-Madiedo thanks the European Commission for its postdoctoral fellowship (MCFI1999-01449). Saskia de Jong is acknowledged for the performance of the CSLM pictures.
References [1] C.G. de Kruif, Int. Dairy J. 9 (1999) 183 /188. [2] M. Kalab, P. Allan-Wojtas, E. Phipps-Todd, Food Microstruct. 2 (1983) 51 /66. [3] A.N. Hassan, J.F. Frank, M.A. Farmer, K.A. Schmidt, S.I. Shalabi, J. Dairy Sci. 78 (1995) 2624 /2628. [4] J.A. Lucey, T. van Vliet, K. Grolle, T. Geurts, P. Walstra, Int. Dairy J. 7 (1997) 389 /397. [5] M.E. van Marle., Structure and rheological properties of yoghurt gels and stirred yoghurts, PhD thesis, Twente University, The Netherlands, 1998. [6] M.E. van Marle, P. Zoon, Neth. Milk Dairy J. 49 (1995) 47 /65. [7] J.A. Lucey, H. Singh, Food Res. Int. 7 (1998) 529 /542. [8] R. Tuinier, An exocellular polysaccharide and its interactions with proteins, PhD thesis, Wageningen University, The Netherlands, 1999. [9] E.J. Faber, Investigation of the structure of exopolysaccharides produces by lactic acid bacteria, PhD thesis, Utrecht University, The Netherlands, 2000. [10] W.H.M. Van Casteren, Structural characterisation and enzymic modification of exopolysaccharides from Lactococcus lactis , PhD thesis, Wageningen University, The Netherlands, 2000. [11] P. Ruas-Madiedo, R. Tuinier, M. Kanning, P. Zoon, Int. Dairy J. 12 (2002) 689 /695. [12] R. Tuinier, W.H.M. van Casteren, P.J. Looijesteijn, H.A. Schols, A.G.J. Voragen, P. Zoon, Biopolymers 59 (2001) 160 /166. [13] P. Zoon, T. van Vliet, P. Walstra, Neth. Milk Dairy J. 42 (1988) 271 /294. [14] D.S. Horne, Int. Dairy J. 8 (1998) 171 /177. [15] R. Tuinier, P. Zoon, M.A. Cohen Stuart, G.J. Fleer, C.G. de Kruif, Biopolymers 50 (1999) 641 /646. [16] A.P. Laws, V.M. Marshall, Int. Dairy J. 11 (2001) 709 / 721. [17] I.W. Sutherland, Food Biotechnol. 6 (1992) 75 /86.