Control of microgel particle growth in fresh cheese (concentrated fermented milk) with an exopolysaccharide-producing starter culture

International Dairy Journal 36 (2014) 46e54

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Control of microgel particle growth in fresh cheese (concentrated fermented milk) with an exopolysaccharide-producing starter culture Christian Hahn a, Esther Müller a, Susanne Wille a, Jochen Weiss b, Zeynep Atamer a, *, Jörg Hinrichs a a

University of Hohenheim, Institute of Food Science and Biotechnology, Department of Dairy Science and Technology, Garbenstrasse 21, D-70599 Stuttgart, Germany University of Hohenheim, Institute of Food Science and Biotechnology, Department of Food Structure and Functionality, Garbenstrasse 25, D-70599 Stuttgart, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 October 2013 Received in revised form 17 December 2013 Accepted 17 December 2013

The hypothesis whether or not the microgel particle growth in fresh cheese (pH ¼ 4.50) during postprocessing is mainly a diffusion-limited process was tested by increasing the shear viscosity by adding polyethylene glycol and, in a natural way, by using an exopolysaccharide (EPS)-producing adjunct culture (0e0.1%, w/w) in fresh cheese production. EPS increased water-binding capacity and storage modulus. Subsequent tempering (23e54  C for 1e300 min) generally increased particle size whereas EPS caused a decrease. Based on the activation energies (EA w 60 kJ mol1), and as EPS was mainly localised at the proteineserum interface, a reaction-limited particle growth was proposed. The EPS may interact with proteins protecting the core from aggregation and acting as an active filler. Concentrations of EPSproducing culture <0.005% (w/w) may be applied to reduce the formation of rough particles, which decrease in-mouth creaminess, providing potential to compensate for temperature variations, e.g., during process stops, and to reduce hydrocolloid addition. Ó 2014 Published by Elsevier Ltd.

1. Introduction For the production of fermented milk products, the aggregation of structural elements in milk is achieved by reducing the natural pH of milk using starter cultures (Lucey & Singh, 1997; Mellema, Walstra, Van Opheusden, & Van Vliet, 2002). In the case of fresh cheese small amounts of chymosin may be added (Lucey, 2004). After stirring the acidified milk gel, the generated microgel particles (1e100 mm) are concentrated, e.g., by membrane filtration (Bäurle, Walenta, & Kessler, 1984; Weidendorfer & Hinrichs, 2010). Thus, the aggregation process of structural elements is needed to provide the consumer the desired textural properties, i.e., usually a smooth and homogeneous texture. However, the process parameters have to be controlled precisely to avoid an excessive aggregation leading to the formation of larger aggregates finally resulting in textural defects, e.g., graininess or syneresis (Lucey, 2004). Several process parameters affect the texture of fermented milk products (Sodini, Remeuf, Haddad, & Corrieu, 2004), where the temperature was shown to have a marked effect in all process steps

* Corresponding author. Tel.: þ49 711 459 23792. E-mail address: [email protected] (Z. Atamer). 0958-6946/$ e see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.idairyj.2013.12.011

(Hahn, Sramek, Nöbel, & Hinrichs, 2012a; Hahn et al., 2012b; Küçükçetin, 2008; Küçükçetin, Weidendorfer, & Hinrichs, 2009; Sainani, Vyas, & Tong, 2004). Recently, we have shown for fresh cheese, fermented by solely using an acidifying culture to pH 4.50, that based on the calculated activation energy (w26 kJ mol1) the temperature-driven microgel particle growth is a mainly diffusionlimited process (Hahn et al., 2012a). For industrial production of fresh cheese, sometimes higher fermentation temperatures are preferred as this leads to higher acidification rates (Lucey, 2002). However, increasing the temperature also modifies textural properties (Van Vliet, Van Dijk, Zoon, & Walstra, 1991) which, depending on the product, is accepted by the consumer in a certain range. Nevertheless, if the actual process conditions differ from the scheduled process, e.g., during a process stop, where semi-finished or finished products are exposed to process temperatures that may be clearly over the storage temperature (w6  C), higher temperatures more quickly lead to textural defects. Thus, it is beneficial to have ingredients that are capable of compensating for small variations from the scheduled process, especially regarding higher temperature loads. Furthermore, it would be of interest if these ingredients do not have to be added in a separate process step. Microbial exopolysaccharides (EPS) belong to the class of hydrocolloids, which are defined as long-chain, high-molecular-mass

C. Hahn et al. / International Dairy Journal 36 (2014) 46e54

polymers that dissolve or disperse in water (De Vuyst & Degeest, 1999), and are used to increase the shear viscosity and to reduce syneresis (De Vuyst & Degeest, 1999; Küçükçetin et al., 2009). Furthermore, EPS seems to control textural defects like graininess in fermented milk products (Lucey & Singh, 1997; Sodini et al., 2004). Depending on their chemical structure and their potential to form intermolecular associations, hydrocolloids differently increase the product viscosity and interact with milk constituents (De Vuyst & Degeest, 1999; Sodini et al., 2004). EPS are built in-situ during fermentation and represent extracellular polysaccharides that are either attached to the cell surface as capsules or found unattached in the extracellular medium (De Vuyst & Degeest, 1999). A great variety of bacteria including lactic acid bacteria produce EPS which can be subdivided into the groups of homopolysaccharides and heteropolysaccharides (Broadbent, McMahon, Welker, Oberg, & Moineau, 2003; De Vuyst & Degeest, 1999; Monsan et al., 2001). While homopolysaccharides are composed of just one repeating unit, e.g., glucose or fructose, heteropolysaccharides are composed of more repeating monosaccharide units. For the physical properties of fermented milk, the molecular mass, the three-dimensional structure, and the potential to form intermolecular associations were shown to be more relevant than the actual amount of EPS produced (De Vuyst & Degeest, 1999; Hassan, Frank, & Qvist, 2002; Sodini et al., 2004). For yoghurt, many data are available showing that EPS is capable to reduce graininess, generate a creamy and smooth texture, enhance structural stability, and to decrease syneresis, giving the potential to reduce the addition of other ingredients (De Vuyst & Degeest, 1999; Hassan, Frank, Schmidt, & Shalabi, 1996b; Hassan, Ipsen, Janzen, & Qvist, 2003; Hess, Roberts, & Ziegler, 1997; Küçükçetin et al., 2009; Sodini et al., 2004). Data for concentrated fermented milk products like fresh cheese mainly focus on the fermentation conditions, and data concerning the effect on textural properties are lacking, which is probably due to the fact that EPS increases the water holding capacity. For instance, Champagne, Barrette, Roy, and Rodrigue (2006) used EPS-producing cultures for the fermentation of a so-called concentrated milk formulation (protein content 5%, w/w, fat content 4%, w/w) and studied the fermentation dynamics. Likewise, Grattepanche, Audet, and Lacroix (2007) proposed the fermentation of ultra-high temperature (UHT) skim milk using mixed cultures containing EPS-producing strains supposed to obtain improved functional properties of fresh cheese. In the present work, it was the aim, firstly, to corroborate the hypothesis that the microgel particle growth is a mainly diffusionlimited process. Therefore, polyethylene glycol (PEG) was added to fresh cheese to increase the shear viscosity of the serum, which is the suspending medium for the microgel particles. Secondly, the potential of an EPS-producing adjunct starter culture to reduce the microgel particle growth was investigated. To do this, different concentrations of the EPS-producing culture were used in order to see its effect on the centrifugal force needed to produce fresh cheese in lab scale and the resulting textural properties, especially the possibility to compensate small variances in the scheduled process. 2. Materials and methods To study the effect of PEG on the aggregation rate of microgel particles and the effect of EPS on textural properties of fresh cheese, the amount of an acidifying culture was always kept constant, while, for the EPS trials, the amount of an EPS-producing adjunct culture was increased. Thereby, higher concentrations of the EPSproducing culture than the ones proposed by the manufacturer were applied to check the effect on textural properties, e.g., the presence of large protein aggregates due to an incompatibility of the EPS with proteins (Hassan et al., 2003).

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2.1. Fresh cheese samples 2.1.1. Fresh cheese production in lab scale Fresh cheese (protein content 7.80  0.36%, w/w; number of measurements, n ¼ 16) containing an EPS-producing starter culture (0e0.1%, w/w) was produced in lab scale (Hahn, Müller, Nöbel, & Hinrichs, 2011) based on the process earlier described by Hahn et al. (2012a). Skim milk (fat content <0.1%, w/w) was pasteurised (74  C for 30 s), and a permeate solution (5.2%, w/w), which was reconstituted from permeate powder Bayolan PT (BMI eG, Landshut, Germany; dry matter 96.1%, w/w, protein content 3.5%, w/w, fat content 0.1%, w/w, lactose 83.0e87.0%, w/w, ash 8.2%, w/w) and distilled water, was added to standardise the protein content (3.39  0.12%, w/w, n ¼ 16). The protein and fat content in the milk samples was analysed with a LactoScopeÔ FTIR Advanced (Delta Instruments B.V., Drachten, The Netherlands). Afterwards, the milk was continuously heated to 95  C in a tubular pilot plant (150 Lh1, ASEPTO-Therm, Asepto GmbH, Dinkelscherben, Germany), held at 95  C for 4.3 min, cooled to 20  C, filled into stainless steel vats (5 L), and subsequently equilibrated in a water bath (20  0.3  C) for 1.5 h. Fermentation to a pH of 4.50  0.03 (n ¼ 16) was started by suspending 0.02% (w/w) of an acidifying culture (F-DVS CC06, Chr. Hansen GmbH, Nienburg, Germany), which contained Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris. Additionally, a mainly EPS-producing starter culture (F-DVS HB-3, Chr. Hansen GmbH) was added in different concentrations ranging between 0 and 0.1% (w/w). The HB-3 culture represented a mesophilic adjunct culture (type O) consisting of L. lactis subsp. cremoris that builds up texture but does not produce diacetyl or CO2. Thus, according to the manufacturer, the HB-3 culture is proposed to be used in combination with other lactic acid cultures, where an inoculation rate of 0.0025 and 0.005% (w/v) provides a high and a very high viscosity, respectively. At a pH of 6.50, 1 mL Chy-MaxÔ (Chr. Hansen GmbH; minimum activity 190 IMCU mL1) was added and the fermentation proceeded until the final pH of 4.50 was reached. Independent of the HB-3 dosage, the time required to reach the final pH was approximately 15 h. The gel was then broken down by moving a perforated plunger 20 times up and down and, afterwards, by blending with an Ultra-Turrax T25 digital (IKA-Werke GmbH & Co. KG) at 10,000 rpm while stirring with the perforated plunger until, visually, a homogeneous texture without any larger aggregates was obtained. As it was the aim to keep the shear impact as small as possible, it is important to note that the time required for the UltraTurrax treatment (1e3 min) was decreased with increasing concentration of EPS-producing culture. Similar observations have also been described by other authors (e.g., Hassan et al., 2003). The microgel suspension (45  0.1 g) was then filled into centrifuge tubes (50 mL), and, analogous to the process established in pilot scale (Hahn et al., 2012a), tempered in a water bath (38  0.3  C) for 15 min before the centrifugation was carried out with a centrifuge (Heraeus BioFuge S tratos, Thermo Electron Corporation, Waltham, MA, USA). To keep the compression of the microgel particles as small as possible, the conditions of centrifugation were adapted to the added concentration of EPS-producing culture (0e0.1%, w/w). Therefore, the centrifugal force (1050e2500  g) was always adjusted in such a way that after 5 min of centrifugation 27.5  0.1 g of whey were expelled. After thoroughly removing 27.0 g of the supernatant with a pipette, the remaining mass was mixed carefully with a spatula. 2.1.2. Tempering of the produced fresh cheese samples To check whether or not the microgel particle growth is a mainly diffusion-limited process, PEG (chain length w200,

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Sigma-Aldrich, Steinheim, Germany) was used (Trgo, Koxholt, & Kessler, 1999). Thereby, it was intended to decrease the diffusion rate of the dispersed microgel particles by increasing the shear viscosity of the continuous phase. According to Hinrichs (1994), the shear viscosity of skim milk at 10  C with a PEG content of 0 and 10% (w/w) is increased logarithmically from approximately 3 to 16 mPa s, respectively. PEG was added to fresh cheese systems which were solely fermented by using the acidifying culture (EPS-producing culture 0%, w/w) to give 0, 1, 5, and 10% (w/w) of PEG in the final samples. After blending, the fresh cheeseePEG-mixture was tempered at 38.0  0.5  C for 1, 30, 60, 100, and 300 min. To study the effect of EPS, the concentrated microgel suspensions were tempered at 23.0  0.5, 38.0  0.5, or 54.0  0.5  C for 1, 30, 60, 100, or 300 min. Independent of the trials, all samples were subsequently cooled in a water bath (10.0  0.3  C) and stored in a refrigerator (10  C) until the measurements were carried out within the next two days. The experiments were conducted in duplicate or triplicate at each temperature for the samples with PEG or EPS, respectively. 2.2. Particle size measurement Using static laser light scattering, the size of the particle clusters was analysed with a Small Volume Module Plus LS230 (BeckmanCoulter Inc., Miami, FL, USA) (Hahn et al., 2012a). Samples were suspended in distilled water, and the particle size distribution was analysed at an obscuration of 14e16% (average of three consecutive runs, each run 60 s, protein and water refractive index 1.75 and 1.33, respectively). For the PEG samples, the Sauter mean diameter, d32, was analysed as this parameter describes an average particle size that is representative for all microgel particles in a sample. For the EPS samples, where mainly the behaviour of large microgel particle clusters was of interest, the volume-weighted diameter dv (0.75) was evaluated, which represents the diameter below which 75% and above which 25% of the volume of the particles is found. All samples were analysed in duplicate. 2.3. Rheological measurements

2.3.2. Permeate The permeate was obtained during fresh cheese production by centrifuging the microgel suspension. As a low shear viscosity was expected, a double gap cylinder geometry was used (stator inner radius 20 mm, rotor inner radius 20.38 mm, and rotor outer radius 21.96 mm); the gap was 500 mm, and the temperature was adjusted to 10  C. For the measurement, approximately 20 g permeate was filled into the geometry. After an equilibration step (10  C for 2 min), the sample was initially sheared (200 s1 for 20 s), and, afterwards, the shear rate decreased logarithmically from 200 to 5 s1. All samples showed Newtonian behaviour. Samples were measured in duplicate. 2.4. Confocal laser scanning microscopy Fresh cheese samples containing different concentrations of EPS-producing culture were visualised undiluted. A microscope Eclipse-C1 (Nikon GmbH, Düsseldorf, Germany) was used. The protein phase in the samples was stained with Rhodamine B (Merck, Darmstadt, Germany) according to the method described by Hahn et al. (2012a) and Heilig, Göggerle, and Hinrichs (2009). The produced EPS was labelled covalently with an Alexa Fluor 488Wheat Germ Agglutinin (WGA) conjugate. An Alexa Fluor 488 solution (Invitrogen GmbH, Darmstadt, Germany) was prepared according to Hassan et al. (2002), and 30 mL of each Rhodamine B and Alexa Fluor 488-WGA-solution was transferred with a pipette onto the sample located in a custom-made plexiglass specimen holder. The sample was covered with a cover glass and fixed with nail polish. The prepared specimens were kept for at least 1 h at 10  C before visualisation. The images were taken with a resolution of 1024  1024 pixels and the average mode of two pictures. 3. Results and discussion 3.1. Effect of PEG on the particle cluster formation Fig. 1 shows the effect of the PEG concentration on the microgel particle growth in the fresh cheese systems during tempering at 38  C. In the samples without PEG (0%, w/w), it was observed that the relative particle growth (ratio of the Sauter mean diameter in

For all experiments, a stress-controlled rheometer AR2000ex (TA Instruments, Eschborn, Germany) was used, and rheological data were evaluated with the software Rheology Advantage V5.7.0 (TA Instruments). All measurements were conducted in duplicate. 2.3.1. Fresh cheese Frequency sweep measurements of all fresh cheese samples were performed at 10  C after carefully stirring the samples in the centrifugal tubes and filling them into the cup (sample weight approximately 17 g). A concentric cylinder system was used. Prior to an equilibration step (10  C for 15 min), the bob was lowered and the frequency sweep measurements (angular frequency, u ¼ 0.05e 628 rad s1, seven points per decade) were performed at 10  C in the predetermined linear viscoelastic region (shear deformation, g ¼ 0.0025). Storage modulus (G0 ), loss modulus (G00 ), and phase angle (d) at u ¼ 10 rad s1 were evaluated. Additionally, flow curves were obtained in separate trials at 10  C after an equilibration step (10  C for 15 min). The shear rate was increased from 0 to 500 s1, then held at 500 s1, and decreased from 500 to 0 s1 within 3 min for each step, and the shear stress was evaluated. The shear viscosity, h, was taken at 100 s1 ðh100 s1 Þ in the first step; the hysteresis loop was calculated as the area between the upward (first step) and downward (third step) curve, which is a measure for the extent of structural breakdown during shearing (Hassan, Frank, Schmidt, & Shalabi, 1996a).

Fig. 1. Influence of the polyethylene glycol (PEG) concentration on the relative particle growth at 38  C in fresh cheese samples. Fresh cheese was produced by fermentation at 20  C with an acidifying culture (0.02%, w/w) to a pH of 4.50 and subsequently followed by concentration. By adding PEG to fresh cheese a PEG concentration of (C) 0, (V) 1, (,) 5, and (>) 10% (w/w) was adjusted in the fresh cheeseePEG-mixture. Each data point represents the average of duplicate determinations.

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the tempered samples, d32, related to the Sauter mean diameter in the sample tempered for 1 min, d32 t ¼ 1 min) increased rapidly at the beginning and levelled off at higher holding times (>100 min). Generally, this was also the case for the samples having higher PEG concentrations. The obtained relative particle growth at 300 min was 2.1, 1.6, 1.4, and 1.2 at PEG concentrations of 0, 1, 5, and 10% (w/w), respectively. Thus, the results showed that increasing the PEG concentration increased the shear viscosity of the serum, which, in turn, decreased the temperature-driven particle growth supporting the hypothesis that the microgel particle growth in fresh cheese (pH 4.50) is a mainly diffusion-limited process. However, a solely diffusion-limited process should eventually lead to the same particle size independent of the PEG concentration in the fresh cheeseePEG-systems, where, at least after 300 min, this trend is not straightforward to predict (Fig. 1). For the interaction of PEG and different proteins, it was shown that, depending on the physico-chemical properties of the different components in suspension, the interactions change (Arakawa, & Timasheff, 1985; Lee, & Lee, 1981). For instance, depending on the pH, the net charge number was modified, where, as a result, PEG may be excluded from or bound to the proteins (Lee & Lee, 1981). Thus, as PEG is essentially a nonpolar polymer, the binding to hydrophobic sites on proteins was proposed, which, additionally, may change the physico-chemical properties of the proteins (Arakawa & Timasheff, 1985). As the fresh cheese used to prepare the fresh cheeseePEGsystems had a pH of 4.50, where mainly hydrophobic interactions are present (Keim, 2005), the interaction between proteins and PEG may have reduced the diffusion-limiting impact of the pH. Furthermore, PEG was added to fresh cheese to simulate the effect of an EPS-producing culture. In accordance with the results presented in Fig. 1, it was already shown for EPS-producing cultures that, as the formation of EPS increased, both the diameter and the number of particles (>1 mm) decreased (Küçükçetin et al., 2009). This was ascribed to the fact that EPS-producing cultures interfere with the number and strength of bonds between the casein particles and the spatial distribution of the aggregates. 3.2. Effect of EPS on textural properties 3.2.1. Centrifugation conditions To adjust to a constant protein content in the concentrated microgel suspensions independent of the concentration of EPSproducing culture (0e0.1%, w/w), the centrifugal force was adjusted to remove 27.5  0.1 g whey within 5 min from the microgel suspensions (Table 1). The required centrifugal force increased as the concentration of EPS-producing culture was increased. As seen in Table 1, higher centrifugal forces were required for concentrations of the EPS-producing culture >0.005% (w/w). This was ascribed to the molecular structure of exopolysaccharides usually leading to a high water-binding capacity (Hassan et al., 1996b). Centrifuging the microgel suspensions at constant conditions confirmed the results. As the highest centrifugal force needed to expel 27.5  0.1 g whey was 2500  g, a slightly higher centrifugal force (3500  g) was applied and again centrifuged for 5 min. With increasing concentration of EPS-producing culture the amount of expelled whey decreased. Additionally, an increase was observed in the shear viscosity ðh100 s1 Þ of both the microgel suspension and the concentrated microgel suspension. As expected, h100 s1 was 5e10 times higher in the concentrated microgel suspensions. However, a marked increase in h100 s1 was only detected at higher concentrations of EPS-producing culture. As a measure for the extent of structural breakdown during shearing, the hysteresis loop was calculated depending on the concentration of EPS-producing culture. Similar to h100 s1 , the hysteresis loop increased with increasing concentration of EPS-

49

Table 1 Process conditions and shear viscosity ðh100 s1 Þ of microgel and concentrated microgel suspensions depending on the concentration of EPS-producing culture.a Concentration of EPS-producing culture (%, w/w)

Centrifugal force (g)

Expelled whey (g)

0 0.001 0.003 0.005 0.050 0.100

1050e1150 1150e1250 1200e1300 1400e1600 1700e1900 2300e2500

32.3 32.0 31.7 29.5 21.3 16.8

     

0.3 0.6 0.7 1.0 3.8 4.4

h100 s1 ðPa sÞ Microgel suspension 0.25 0.25 0.23 0.29 0.36 0.55

     

0.01 0.01 0.01 0.01 0.01 0.02

Concentrated microgel suspension 2.38 2.23 2.57 2.55 2.52 2.88

     

0.01 0.33 0.28 0.35 0.14 0.36

a Values are means and standard deviation. Centrifugal force is the range applied to expel 27.5  0.1 g whey within 5 min from the microgel suspensions (45.0  0.1 g) depending on the concentration of the EPS-producing culture (n ¼ 3). Expelled whey is from 45.0  0.1 g microgel suspension after centrifugation at 3500  g for 5 min (n ¼ 2). Experiments were conducted in duplicate.

producing culture (0, 0.001, 0.003, 0.005, 0.050 and 0.100%, w/w) for both the microgel (5.83  0.04, 6.27  0.18, 5.76  0.30, 7.15  0.58, 12.6  0.86, and 17.6  2.00 kPa) and the concentrated microgel suspensions (63.0  1.5, 59.5  8.8, 67.5  5.8, 70.0  7.7, 71.0  5.2, and 81.0  9.3 kPa) showing the effect of EPS on the structure formation of the protein matrix. 3.2.2. Particle cluster formation In Fig. 2, the relative particle growth (ratio of dv (0.75) of the tempered samples related to the particle size of the sample tempered for 1 min, dv (0.75)t ¼ 1 min) is given for different concentrations of the EPS-producing culture and holding times at 38  C. For a given concentration of EPS-producing culture, e.g., 0%, (w/w), the relative dv (0.75) increased asymptotically with the holding time. This was in accordance with results reported earlier by Hahn et al. (2012a). After a holding time of 300 min, the relative dv (0.75) was approx. 2.2 times the value of the sample tempered for 1 min. The increase of the relative particle growth was also seen at higher concentrations of EPS-producing culture where, however, the measured dv (0.75)t ¼ 1 min decreased with increasing concentration of EPS-producing culture. The dv (0.75)t ¼ 1 min was

Fig. 2. Influence of the concentration of an exopolysaccharide (EPS)-producing adjunct starter culture on the relative particle growth at 38  C in fresh cheese samples. Fresh cheese was obtained by fermentation at 20  C with an acidifying culture (0.02%, w/w) and an EPS-producing culture (0e0.1%, w/w) to a pH of 4.50 and subsequently followed by concentration. Fresh cheese samples containing (C) 0, (D) 0.001, (,) 0.003, (>) 0.005, (l) 0.050, and (V) 0.1% (w/w) of EPS-producing culture are illustrated. Each data point represents the averages of triplicate determinations.

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calculated as 41.9  3.8, 32.7  1.6, 32.2  3.5, 33.0  2.8, 26.9  0.7 and 26.1  1.3 mm for the concentrations of 0, 0.001, 0.003, 0.005, 0.050 and 0.100% (w/w), respectively. This may be ascribed to the fact that by the presence of EPS either the aggregation of the proteins during fermentation was reduced, or the gel was broken down more easily into smaller microgel particles upon stirring (Hassan et al., 2003). Thus, a dv (0.75) of 40 mm was reached approx. after holding times of 1 min (0%, w/w), 30 min (0.001e0.005%, w/w), or 100 min (0.050e0.100%, w/w) showing that the presence of EPS was capable to reduce the rate of particle cluster formation. To study the underlying process of particle cluster formation, the obtained data were linearised by applying a power law function according to Hahn et al. (2012a). The temperature-dependent velocity constants (kT) and the activation energy (EA) were determined. The obtained values depending on the concentration of EPSproducing culture are given in Table 2. For the samples containing no EPS (0%, w/w), an activation energy of 29.4  3.2 kJ mol1 was calculated. This is in accordance with the mainly diffusion-limited particle growth close to the isoelectric point (pH w 4.5) reported for concentrated microgel suspensions without EPS (Hahn et al., 2012a). However, by adding the EPS-producing culture, the obtained EA was higher and varied between 60 and 70 kJ mol1. It has to be noted that the activation energy calculated for the samples at a concentration of 0.050 and 0.100% (w/w) has to be taken carefully, as at a temperature of 23  C (296 K) a low correlation coefficient was observed. This is probably because the low temperature and the high concentration of EPS-producing culture led to a low particle growth. According to literature, several levels of EA exist to characterise the underlying process of a temperature-dependent process (Kessler, 2002). Thereby, an EA of 50e150 kJ mol1 indicates that a chemical reaction limits the reaction rate. To apply this concept to the concentrated microgel suspensions produced with the EPSproducing culture, the microgel particles, in a first step, have to diffuse to get in contact while, in a second step, a reaction between the microgel particles leads to particle growth (Smoluchowski, 1917; Wagner, 1961). While diffusion-limited particle cluster formation occurs when only negligible repulsive forces are present between the particles, e.g., in the case of fresh cheese without EPS Table 2 Velocity constants (kT) and activation energy (EA) depending on the concentration of EPS-producing culture in concentrated microgel suspensions.a Concentration of EPS-producing culture (%, w/w)

Temperature (K)

kT (e)

R2

EA (kJ mol1)

R2

0

296 311 327 296 311 327 296 311 327 296 311 327 296 311 327 296 311 327

0.095 0.152 0.295 0.029 0.149 0.303 0.034 0.153 0.356 0.022 0.128 0.353 (0.006) 0.097 0.308 0.025 0.115 0.290

0.931 0.912 0.974 0.940 0.903 0.932 0.955 0.974 0.943 0.871 0.967 0.967 (0.268) 0.821 0.972 0.613 0.891 0.977

29.4  3.2

0.988

61.1  13.3

0.955

61.1  9.3

0.978

72.2  10.5

0.979

(61.1)

e

63.7  8.4

0.983

0.001

0.003

0.005

0.050

0.100

a Values are means and standard deviation of triplicate determinations. Fermentation to a pH of 4.50 was carried out by always suspending an acidifying culture (0.02%, w/w) and, additionally, adding the EPS-producing adjunct starter culture. R2: correlation coefficient.

Table 3 Rheological properties of concentrated microgel suspensions.a Concentration of EPS-producing culture (%, w/w)

G010 rad s1 ðkPaÞ

0 0.001 0.003 0.005 0.050 0.100

1.5 1.6 1.8 2.0 1.6 1.6

     

0.2 0.1 0.2 0.2 0.2 0.1

G0010 rad s1 ðkPaÞ

0.4 0.4 0.5 0.5 0.4 0.4

     

0.1 0.1 0.1 0.1 0.1 0.1

d10 rad s1 ð Þ

15.3 14.4 14.6 14.3 14.1 14.2

     

0.1 0.2 0.1 0.2 0.1 0.1

a G0 (storage modulus), G00 (loss modulus) and d (phase angle) were obtained at 10 rad s1 for concentrated microgel suspensions tempered at 38  C for 1 min depending on the concentration of EPS-producing culture. Values are means and standard deviation of triplicate determinations. Fermentation to a pH of 4.50 was carried out by always suspending an acidifying culture (0.02%, w/w) and, additionally, adding the EPS-producing adjunct starter culture.

(0%, w/w), reaction-limited particle growth is present when after diffusion the particle interaction is hindered, and time is needed to overcome these repulsive forces. As, in contrast to the (concentrated) microgel suspensions (Table 1), the shear viscosity of the permeate was always between 1.62  0.02 and 1.72  0.08, and, thus, independent of the EPS-concentration, the EPS may be retained to a large extend in the concentrated microgel suspensions. Therefore, based on the calculated activation energy (60e 70 kJ mol1), we suggest that the EPS used may be capable to interact with the microgel particles. Thereby, on the one hand, their aggregation rate is decreased as the EPS layer at the proteineserum interface protects the core, i.e., the microgel particles, with a kind of steric hindering from aggregation. 3.2.3. Rheological properties Besides protecting the core, the EPS may, on the other hand, act as an active filler having the potential to increase the dynamic moduli (G0 , G00 ) (Purohit, Hassan, Bhatia, Zhang, & Dwivedi, 2009). Table 3 shows the rheological properties (G0 , G00 , and d), which were obtained at 10 rad s1, of the concentrated microgel suspensions tempered at 38  C for 1 min depending on the concentration of EPSproducing culture. While G0 (10 rad s1) was in a comparatively small range (1.5e2.0 kPa), there was a clear trend that increasing the concentration of EPS-producing culture at first increased and then decreased G0 (10 rad s1), where the sample with a concentration of 0.005% (w/w) showed the highest value (2.0 kPa). This was also the case for G00 (10 rad s1). As the dynamic moduli represent the energy stored (G0 ) and dissipated (G00 ) per oscillation cycle, these parameters give an information about the strength and number of bonds in a network indicating the contribution of stronger (G0 ) and weaker (G00 ) proteineprotein bonds (Keim, 2005; Lankes, Ozer, & Robinson, 1998; Lucey, 2001; Lucey & Singh, 1997; Lucey, Teo, Munro, & Singh, 1997a). Thus, the data suggest that adding the EPS-producing culture led to a higher number and strength and, hence, stronger interactions in the gel network that, in turn, may be ascribed to the potential of the used EPS to form interactions with the microgel particles at a pH < 4.6. However, at higher concentrations (>0.005%, w/w) the potential to act as active filler enhancing the structure was reduced (Table 3). There is literature describing that EPS in fermented milk products led to a decrease of the dynamic moduli probably due to an incompatibility of EPS with protein in the product (Hassan et al., 2003; Hess et al., 1997; Küçükçetin et al., 2009). Supported by confocal laser scanning microscopy (CLSM) images, Hassan et al. (2003) found denser and larger protein aggregates in yoghurt, and an agglomeration of EPS in the continuous phase thereby leading to fewer proteineprotein interactions. Likewise, Purohit

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Fig. 3. Influence of the concentration of EPS-producing culture and tempering on the microstructure in (concentrated) microgel suspensions. Fermentation to a pH of 4.50 within approximately 15 h was carried out by always suspending an acidifying culture (0.02%, w/w) and, additionally, an EPS-producing adjunct starter culture (0.001e0.1%, w/w). Concentrated microgel suspensions were tempered at 38  C for 1 and 300 min. Rhodamine B and an Alexa Fluor 488-WGA conjugate was used to stain the protein phase (red) and to covalently label EPS (green), respectively. Magnification 63, scale bar 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

et al. (2009) used five different EPS-producing cultures for yoghurt production where in four cases the EPS-producing cultures reduced the storage and loss modulus as compared to the control sample produced without EPS. However, it was observed that one EPS culture increased the dynamic moduli. It was proposed that interactions between EPS and protein were what increased the number of bonds. Thus, we suggest that the higher storage and loss moduli and the lower loss tangent in the present work is a result of the capability of the EPS to interact with milk proteins leading to an EPS layer around the microgel particle core, where, depending on the concentration of EPS-producing culture, these particles may respond (de Kruif & Tuinier, 2001) and result in a stronger network. However, as d (10 rad s1) was only slightly lower in the concentrated microgel suspensions with EPS (Table 3), the effect of

different concentrations of EPS-producing cultures may be low (De Vuyst & Degeest, 1999; Purohit et al., 2009). 3.2.4. Confocal laser scanning microscopy images Fig. 3 shows the CLSM images of the microgel suspensions and the concentrated microgel suspensions tempered for 1 and 300 min with different concentrations of EPS-producing culture. The protein phase and EPS are visualised in red and green, respectively; black areas represent the serum phase and voids. At constant concentration of EPS-producing culture (e.g., 0.001%, w/w) the microgel suspension showed a dense and compact protein matrix consisting of mainly irregular shaped, uniformly distributed microgel particles with a size ranging from 10 to 40 mm. EPS was mainly localised at the proteineserum interface and not mainly in the voids as

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Fig. 4. Schematic drawing showing the effect of different concentrations of an EPS-producing adjunct culture on textural properties of fresh cheese. Fresh cheese samples were produced with a constant concentration of an acidifying culture (0.02%, w/w) and low, medium and high concentrations of an EPS-producing adjunct culture. All fresh cheese samples were tempered at 23, 38 and 54  C for holding times ranging from 1 to 300 min. CP, protein content; CEPS, concentration of EPS-producing culture; dv (0.75)t ¼ 1 min: diameter of the sample tempered for 1 min; relative particle growth: ratio of the dv (0.75) in the tempered samples, dv (0.75), related to the dv (0.75)t ¼ 1 min; G0 : storage modulus; G00 : loss modulus. Qualitative levels of textural properties ranging from 1 (low), 2 (medium) to 3 (high) are given. For fresh cheese with 0% (w/w) EPS-producing culture the qualitative levels for the parameters were: water-binding capacity, 1; shear viscosity, 1; dv (0.75)t ¼ 1 min, 3; relative particle growth (300 min), 3; G0 (10 rad s1), 1; G00 (10 rad s1), 1.

described by Hassan et al. (2003). However, some larger agglomerates were visible (5e15 mm). In the concentrated microgel suspensions tempered for 1 min the protein matrix was more dense and compact due to the removal of whey. Additionally, there were fewer smaller (approximately 10 mm) and a higher number of larger microgel particles (20e50 mm). By tempering for 300 min, the size of the microgel particles consisting of aggregated particles (Lee & Lucey, 2004; Mellema et al., 2002) increased to approx. 100 mm, and a more heterogeneous and open structure showing larger pores evolved. Again, EPS seemed to be mainly localised at the proteineserum interface protecting the microgel particle core from aggregation. The observed particle cluster formation was attributed to extensive rearrangement leading to the aggregation of larger particles (Lucey, Van Vliet, Grolle, Geurts, & Walstra, 1997b; Lee & Lucey, 2006). The same characteristics were visible when the concentration of EPS-producing culture was increased. However, there were some differences. Firstly, the green visualised area increased, which indicates a higher amount of EPS produced and

agrees with the results presented in Table 1 (higher centrifugal force, less expelled whey). Secondly, the EPS was mainly localised at the proteineserum interface but with increasing concentration of EPS-producing culture (>0.005%, w/w) the produced EPS built agglomerates (5e50 mm) and successively filled the voids. Thirdly, in the samples tempered for 300 min the evolution of a heterogeneous structure showing larger particles and pores was reduced, and a more interconnected network was present. At first sight, this may be contradictory to the results presented in Fig. 2 showing that there was a lower dv (0.75) with increasing concentration of EPS-producing culture. However, Hess et al. (1997) reported that with increasing shear rate the gel network is disrupted slightly, and as the shear rate is increased the network is disrupted to a greater extend. Thus, we propose that the EPS used decreased the particle cluster formation by interacting with the proteins in fresh cheese and, thereby, diminished the temperaturedriven rearrangement of the microgel particles. By shearing, e.g., upon chewing or occurring during the sample preparation for the particle size measurement, the gel network was disrupted and

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microgel particles, which had been grown to a smaller size, were generated. 4. Conclusions The results of the study are summarised in Fig. 4. In detail, with increasing concentration of EPS-producing culture the centrifugal force required to adjust to a constant protein content increased that was ascribed to the molecular structure of EPS leading to a higher water-binding capacity. However, the increase in the shear viscosity was lower than expected, and was only detectable at higher concentrations (>0.005%, w/w). Nevertheless, analogous to PEG, EPS reduced the particle cluster formation. Based on both the determined activation energy (EA w 60 kJ mol1) and the CLSM images we propose that the EPS was capable to interact with milk proteins in fresh cheese (pH w 4.5), and, thereby (i) formed a layer around the microgel particles that protected the core from aggregation by reducing the aggregation rate, and (ii) acted as structure enhancer that increased the elastic nature (G0 ) of the samples; however, higher concentrations of EPS slightly reduced G0 probably because the EPS agglomerates interrupted the protein network. As the present state of the art usually requires a concentration step to produce fresh cheese, adding ingredients with a higher waterbinding capacity before the concentration step is challenging. Based on the presented results the addition of smaller concentrations of the EPS-producing culture (<0.005%, w/w) may be reasonable as there was only a slightly higher centrifugal force needed to adjust a constant protein content, and the particle cluster formation was reduced. Thus, there is potential to compensate variances in the process, e.g., during a process stop, and to reduce the amount of added hydrocolloids. Acknowledgements This research project was supported by the German Ministry of Economics and Technology (via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn). Project AiF 15584 N. The authors thank Giovanni Migliore, Luc Mertz, Stefanie Hotz, and Birgit Greif for their helping hands at the pilot plants and in the laboratories. References Arakawa, T., & Timasheff, S. N. (1985). Mechanism of poly(ethylene glycol) interaction with proteins. Biochemistry, 24, 6756e6762. Bäurle, H. W., Walenta, W., & Kessler, H. G. (1984). Herstellung von Magerquark mit Hilfe der Ultrafiltration [[Manufacture of low fat curd cheese by means of ultrafiltration]]. Deutsche Molkereizeitung, 105, 356e363. Broadbent, J. R., McMahon, D. J., Welker, D. L., Oberg, C. J., & Moineau, S. (2003). Biochemistry, genetics, and applications of exopolysaccharide production in Streptococcus thermophilus: a review. Journal of Dairy Science, 86, 407e423. Champagne, C. P., Barrette, J., Roy, D., & Rodrigue, N. (2006). Fresh-cheese milk formulation fermented by a combination of freeze-dried citrate-positive cultures and exopolysaccharide-producing lactobacilli with liquid lactococcal starters. Food Research International, 39, 651e659. De Vuyst, L., & Degeest, B. (1999). Heteropolysaccharides from lactic acid bacteria. FEMS Microbiology Reviews, 23, 153e177. Grattepanche, F., Audet, P., & Lacroix, C. (2007). Milk fermentation by functional mixed culture producing nisin Z and exopolysaccharides in a fresh cheese model. International Dairy Journal, 17, 123e132. Hahn, C., Müller, E., Nöbel, S., & Hinrichs, J. (2011). Reduziertes Partikelwachstum in Frischkäse durch EPS-bildende Kulturen [[Reduced particle cluster formation in fresh cheese by using EPS-producing cultures]]. Deutsche Molkereizeitung, 132, 26e29. Hahn, C., Sramek, M., Nöbel, S., & Hinrichs, J. (2012a). Post-processing of concentrated fermented milk: influence of temperature and holding time on the formation of particle clusters. Dairy Science and Technology, 92, 91e107. Hahn, C., Wachter, T., Nöbel, S., Weiss, J., Eibel, H., & Hinrichs, J. (2012b). Graininess in fresh cheese as affected by post-processing: influence of tempering and mechanical treatment. International Dairy Journal, 26, 73e77.

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