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Interactions of polysaccharide stabilisers with casein aggregates in stirred skim-milk yoghurt
ARTICLE IN PRESS
International Dairy Journal 15 (2005) 1175–1183 www.elsevier.com/locate/idairyj
Interactions of polysaccharide stabilisers with casein aggregates in stirred skim-milk yoghurt David W. Everett, Rosalind E. McLeod Department of Food Science, University of Otago, Dunedin 9001 New Zealand Received 18 February 2004; accepted 8 December 2004
Abstract The stabilisation mechanisms of low-methoxy pectin, l-carrageenan, guar gum, locust bean gum and xanthan in stirred yoghurt were investigated using dynamic oscillatory rheometry and water holding capacity measurements. As the level of low-methoxy pectin and l-carrageenan increased, the stabilisation mechanism was consistent with the model of casein aggregates passing through a region of bridging flocculation, followed by partial steric stabilisation. For guar gum, locust bean gum and xanthan, the results were consistent with depletion flocculation at a low concentration of stabiliser, and trapped aggregated casein within a viscous polysaccharide solution at higher concentrations. A peak was observed for the phase angle of guar-stabilised yoghurt as a function of frequency, from 4.5 to 0.2 Hz as guar increased from 0.5 to 5 g L1 for 1-day yoghurt, and from 4.5 to 2.2 Hz as the yoghurt containing 0.5 g L1 guar gum was aged from 1 to 42 days. Results are interpreted with an understanding that the rheological behaviour is affected by the pre-sharing conditions of the stirred yogurt. r 2005 Elsevier Ltd. All rights reserved. Keywords: Yoghurt; Stabilisers; Rheology; Syneresis
1. Introduction Yoghurt contains a network of three-dimensional casein strands aggregated through iso-electric precipitation brought about by the action of lactic acid bacteria (Tamime & Robinson, 1985). The casein strands can be broken apart by shearing, and the size of the aggregates decreases as the rate of shearing increases (Afonso & Joao, 1999). Rearrangement and syneresis of the acidinduced casein network in yoghurt occurs during storage (van Vliet, Lucey, Grolle, & Walstra, 1997), although this process is far from being completely understood. Syneresis can be reduced by increasing the casein content of the milk (Fiszman, Lluch, & Salvador, 1999), by reducing the incubation temperature and rate of acidification (Lucey, 2002), or by adding stabilisers that interact with the casein network.
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E-mail address: [email protected] (D.W. Everett). 0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.12.004
The firmness of yoghurt is affected by homogenisation, pH, processing parameters (stirred or set yoghurt), and heat treatment of milk that denatures b-lactoglobulin and that subsequently may bridge adjacent casein micelles (Lucey & Singh, 1997; Lucey, Teo, Munro, & Singh, 1997). Yoghurt is usually prepared from homogenised milk to improve stability. This process coats the increased surface of fat globules with casein, enabling the fat globules to participate as a copolymer with casein to strengthen the gel network and reduce syneresis (Keogh & O’Kennedy, 1998). Stabilisers are used in yoghurt to improve consistency (increase viscosity) and reduce syneresis (Lucey, 2002). These may include gelatin, starch, pectin, alginate, carrageenan, derivatives of methylcellulose, gum arabic, tragacanth, karaya, locust bean gum (LBG) or guar (Tamime & Robinson, 1985). Xanthan, k-carrageenan and LBG have been used to improve the consistency and dispersion properties of spray-dried yoghurt powder in water (Ramirez-Figueroa, Salgado-Cervantes, Rodriguez, & Garcia, 2002). A primary stabiliser such as
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carboxymethyl cellulose (CMC), LBG, alginate, or guar gum can be used as a thickener in conjunction with a secondary stabiliser such as carrageenan to reduce syneresis (Hansen, 1993). Anionic hydrocolloids (e.g. CMC, pectin, l-carrageenan) interact with the positive charges on the surface of casein micelles to strengthen the casein network and reduce syneresis, and will be classified as adsorbing polysaccharides for the purposes of this study. Neutral hydrocolloids (e.g. xanthan, guar, LBG) do so through a different mechanism by increasing the viscosity of the continuous phase (Hansen, 1993), and will be classified as non-adsorbing polysaccharides. At low concentration of some types of stabilisers, destabilisation can occur by the bridging of casein micelles (Langendorff et al., 1999; Langendorff, Cuvelier, Launay, & Parker, 1997; Maroziene & de Kruif, 2000; Syrbe, Bauer, & Klostermeyer, 1998). Non-adsorbing or an excess of adsorbing polysaccharides can cause depletion flocculation of suspended colloids (Bourriot, Garnier, & Doublier, 1999; Dickinson, Semenova, Antipova, & Pelan, 1998; Hemar, Tamehana, Munro, & Singh, 2001; Langendorff et al., 1997, 1999; Maroziene & de Kruif, 2000; Schorsch, Jones, & Norton, 1999), and also increase the viscosity of the continuous phase and form an entangled polymeric network (Syrbe et al., 1998). The quality of the solvent will affect the ability of adsorbed polymers to stabilise casein micelles. In good solvents such as water, the polymer will dissolve in the water phase and casein micelles will be prevented from aggregating. In poor solvents such as ethanol, the adsorbed polymer will not dissolve in the solvent phase and will lie compact on the surface of the micelle. In this case, the steric repulsive mechanism is minimised (Syrbe et al., 1998). To examine the effect of stabilisers on yoghurt viscosity, elasticity and water holding capacity, five polysaccharides were added to cultured yoghurt at concentrations ranging from 0.5 to 5 g L1. Two adsorbing polysaccharides, low-methoxy (LM) pectin and l-carrageenan, and three non-adsorbing polysaccharides, guar gum, LBG and xanthan were selected and the mechanisms of stability examined as the concentration of stabiliser increased. This study provides evidence for structural models of casein aggregates as the concentrations of adsorbing and non-adsorbing polysaccharides increase.
2. Materials and methods LM-pectin, l-carrageenan, LBG (Sigma Chemical Company, St. Louis, Missouri, USA), guar gum (Tic Gums, Belcamp, Maryland, USA) and xanthan (Jungbunzlauer, Vienna, Austria) were purchased and used without further purification. LM-pectin, with a 9.4%
methoxy and an 83% galacturonic acid content, was derived from citrus peel. This particular pectin preparation comprised 0.91% sodium and 0.57% calcium. No low molecular weight polysaccharides were present. Non-gelling l-carrageenan was derived from Gigartina aciculaire and G. pistillata seaweed and comprised 0.65% calcium, 4.72% potassium and 4.71% sodium, with less than 40% ester sulphate content. LBG, a straight chain polymer of mannose with one galactose branch on every fourth mannose and a molecular weight of approximately 310,000 was derived from Ceratonia siliqua seeds. Guar gum, consisting mainly of non-ionic galactomannan and with a molecular weight of approximately 250,000 was derived from the seeds of Cyamopsis tetragonolobus. Unpasteurised skim milk was obtained fresh (Meadowfresh Dairies, Dunedin, New Zealand) and used to make yoghurt within 4 days. 2.1. Yoghurt manufacture The casein contents of skim milk and skim milk powder were measured using a modified micro-Kjeldahl technique. Casein was first precipitated at the isoelectric point and filtered to remove undenatured whey proteins prior to nitrogen determination (Everett & Jameson, 1993). Skim milk powder was mixed with the skim milk to an initial casein content of 50 g L1 and standardised to a final casein content of 45 g L1 1 day after manufacture. The milk was heated at 85 1C for 20 min, dry powdered stabiliser added, and the mixture heated for a further 10 min at 85 1C. The types and amounts of stabilisers (by volume) were as follows: LM-pectin (0.5, 1, 2 3, 4 and 5 g L1), l-carrageenan (0.5, 1, 3 and 5 g L1), guar gum (0.5, 1 and 5 g L1), LBG (0.5, 1 and 5 g L1) and xanthan (0.5, 1, 3 and 5 g L1). Yoghurt manufacture was adapted from a standard technique (Kosikowski & Mistry, 1999). The mixture of milk and added stabiliser was cooled to 30 1C and a Streptococcus thermophilus and Lactobacillus bulgaricus direct vat set culture (YC-380, Chr. Hansen, Hamilton, New Zealand) was mixed into the milk and allowed to ferment for 16 h at 30 1C in autoclaved glass jars. Yoghurt samples were stored at 4 1C for 1, 21 and 42 days until required for analysis. The pH of the yoghurt was adjusted to 4.25 on day 1 after manufacture using 1M-NaOH or 1M-HCl as appropriate, and the volume of added acid or base solution kept constant (100 mL L1 of yoghurt volume) by further addition of deionised water such that the final casein content of the milk was 45 g L1. All utensils and equipment were washed with 200 mg L1 sodium hypochlorite solution before use. 2.2. Water holding capacity Water holding capacity measurements of yoghurt were made in triplicate (Keogh & O’Kennedy, 1998).
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Samples (30 g) were centrifuged at 200 g for 10 min at 4 1C and the extent of trapped serum phase quantified and expressed as a percentage of yoghurt volume by measuring the volume of supernatant. Water holding capacity measurements were made in triplicate and the average and absolute error range calculated.
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3. Results Increasing the concentration of the two adsorbing stabilisers (LM-pectin and l-carrageenan) up to 1 g L1 resulted in no change in apparent viscosity, and a decrease in apparent viscosity at higher concentrations (Fig. 1A, B). For two of the non-adsorbing stabilisers
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Apparent viscosity and elasticity of yoghurt samples were measured 1, 21 and 42 days after manufacture at 10 1C using a cup and spindle configuration (RheoStress 1, Gebru¨der Haake GmbH, Karlsruhe, Germany) surrounded by a circulating water bath. For apparent viscosity measurements, samples were pre-sheared at 1000 s1 until the shear stress reached a constant value over a 20 s period (with a change of less than 0.05 Pa), typically taking less than 150 s to achieve. Shear stress was then measured as a function of decreasing shear rate from 1000 to 0.03 s1, allowing sufficient time at each shear rate step for the shear stress to attain a new equilibrium value. Elastic parameters were measured using dynamic oscillatory rheometry (Velez-Ruiz & Barbosa Canovas, 1997). The pre-measurement shear history of each sample was minimised by allowing time for the structure to be regained after carefully pouring the sample into the rheometer cup. To determine the region of linear viscoelasticity, samples were sheared under oscillatory conditions at 1 Hz and at an increasing strain (from 0.001 to 1 dimensionless units), and the storage modulus, G0 (in Pa, defined as the ratio of in-phase stress to strain) and the loss modulus, G00 (defined as the ratio of out-of-phase stress to strain) measured as a function of strain (Barnes, Hutton, & Walters, 1989). A subsequent sample of yoghurt was then sheared under oscillatory conditions at a strain of 0.003 units, determined to be within the linear viscoelastic region, and G0 and G00 measured as a function of frequency from 0.1 to 10 Hz. The ratio of G00 to G0 (equal to the tangent of the phase angle, d; between the shear stress and strain) was also calculated. A characteristic relaxation time was calculated by taking the inverse of the frequency of oscillation at the point where the dynamic moduli (storage and loss) showed peak values. Rheological measurements were made in triplicate and the average and absolute error range calculated.
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Stabiliser (g L−1) Fig. 1. Apparent viscosity of yoghurt at different concentrations of two adsorbing stabilisers (A) low-methoxy pectin and (B) l-carrageenan, and three non-adsorbing stabilisers (C) guar gum, (D) locust bean gum and (E) xanthan (pH 4.25). Measurements were made 1 day after manufacture at a shear rate of 10 s1 and a temperature of 10 1C (n ¼ 3; error bars are absolute range).
(LBG and guar gum), the apparent viscosity decreased up to 1 g L1 stabiliser concentration, then increased at
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5 g L1 (Fig. 1C, D). For the other non-adsorbing stabiliser (xanthan), the apparent viscosity remained unchanged up to 3 g L1, followed by an increase at higher concentrations (Fig. 1E), consistent with previously reported results for xanthan mixed with reconstituted skim milk powder or milk protein concentrate (Hemar et al., 2001). Two types of behaviour were evident for G0 and G00 at 1 Hz as a function of stabiliser concentration. For the two non-adsorbing stabilisers (Fig. 2A, B), a decrease in the two moduli occurred as the concentration of stabiliser increased above 1 g L1 for LM-pectin and between 0 and 1 g L1 for l-carrageenan. For the three non-adsorbing stabilisers, LBG, guar gum and xanthan, a decrease occurred up to 1 g L1 followed by an increase at 5 g L1 stabiliser, indicating a transition between two stabilising mechanisms at around 1 g L1 (Fig. 2C, D, E). The pH of 1-day yoghurt was adjusted to 4.25; however, the pH values after 21 and 42 days were not standardised. These were recorded above the appropriate set of data for 21-and 42-day-old yoghurt (Figs. 3 and 4). In most cases the change in pH over the 42-day storage period decreased by less than 0.2 pH units. The phase angle of the yoghurt samples at 1 Hz was calculated from G0 and G00 as a function of stabiliser concentration after 1, 21 and 42 days storage (Fig. 3). For the adsorbing stabilisers LM-pectin and l-carrageenan, the phase angle was constant up to 1 g L1 stabiliser. Phase angle increased above 2 g L1 LMpectin but remained constant up to 5 g L1 l-carrageenan. For two of the non-adsorbing stabilisers, LBG and guar gum, d increased up to 1 g L1, then remained unchanged at 5 g L1. The phase angle of the other nonadsorbing stabiliser, xanthan, remained constant over the concentration range examined (0.5 to 5 g L1). In most cases, for all five stabilisers, phase angle was not affected by the length of storage time of the yoghurt, although at higher concentrations of adsorbing stabiliser (X3 g L1), the 42-day yoghurt was more liquid as shown by the higher value for d (Fig. 3A, B). The water holding capacity decreased up to 1 g L1 then increased up to 5 g L1 for LM-pectin to an amount more than for the unstabilised yoghurt (Fig. 4A). This occurred for both 1- and 42-day yoghurt, and also for 21-day yoghurt with the exception of 5 g L1 LM-pectin. For the other adsorbing stabiliser, the water holding capacity increased up to 3 g L1 l-carrageenan, then decreased at 5 g L1 (Fig. 4B). A similar trend occurred for LBG where a decrease in water holding capacity took place between 1 and 5 g L1 stabiliser (Fig. 4C). There was no consistent trend for guar gum (Fig. 4D). Water holding capacity of xanthan yoghurt remained relatively unchanged compared to the other four stabilisers (Fig. 4E).
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Stabiliser (g L−1) Fig. 2. Dynamic moduli (storage, G0 , and loss, G00 ) of yoghurt (pH 4.25) at different concentrations of two adsorbing stabilisers (A) lowmethoxy pectin and (B) l-carrageenan, and three non-adsorbing stabilisers (C) guar gum, (D) locust bean gum and (E) xanthan. Measurements were made 1 day after manufacture at a strain of 0.003, frequency of 1 Hz and a temperature of 10 1C (n ¼ 3; error bars are absolute range). (’) G0 , (m) G00 .
The phase angle of 1-day yoghurt containing guar gum showed peak values that differed over the range in frequency of 0.1–10 Hz according to the amount of
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Stabiliser (g L−1) Fig. 3. Phase angle (d) of yoghurt at different concentrations of two adsorbing stabilisers (A) low-methoxy pectin and (B) l-carrageenan, and three non-adsorbing stabilisers (C) guar gum, (D) locust bean gum and (E) xanthan. Measurements were made at 1 (’), 21 (K) and 42 (m) days after manufacture at a strain of 0.003, frequency of 1 Hz and at a temperature of 10 1C (n ¼ 3; error bars are absolute range). The pH at day 1 was 4.25. Value for pH at days 21 (upper figure) and 42 (lower figure) are shown above each set of data. With no added stabiliser, the pH is 4.13 at day 21 and 4.14 at day 42 for all samples.
Fig. 4. Water holding capacity as a percentage of yoghurt volume at different concentrations of two adsorbing stabilisers (A) low-methoxy pectin and (B) l-carrageenan, and three non-adsorbing stabilisers (C) guar gum, (D) locust bean gum and (E) xanthan. Measurements were made at 1 (’), 21 (K) and 42 (m) days after manufacture at a temperature of 4 1C (n ¼ 3; error bars are absolute range). The pH at day 1 was 4.25. Value for pH at days 21 (upper figure) and 42 (lower figure) are shown above each set of data. With no added stabiliser, the pH is 4.13 at day 21 and 4.14 at day 42 for all samples.
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Fig. 5. Phase angle (d) of yoghurt (pH 4.25) at different concentrations of guar gum as a function of frequency of oscillation. Measurements were made 1 day after manufacture at a strain of 0.003 and a temperature of 10 1C (n ¼ 3; error bars are absolute range). Arrows indicate peaks.
Fig. 6. Phase angle (d) of yoghurt containing 0.5 g L1 guar gum as a function of frequency of oscillation. Measurements were made at 1 (pH 4.25), 21 (pH 3.96) and 42 days (pH 3.89) after manufacture at a strain of 0.003 and a temperature of 10 1C (n ¼ 3; error bars are absolute range). Arrows indicate peaks.
stabiliser (Fig. 5). Unstabilised yoghurt did not show a peak value. For increasing amounts of guar gum (0.5 and 1 g L1) the peak shifted to lower values, from 4.5 to 2.2 Hz, respectively. There was no clear peak for the 5 g L1 guar sample. The inverse of the frequency peak yielded characteristic relaxation times for each stabiliser concentration (0.22 and 0.46 s) for increasing concentration of guar. For the other stabilisers, peaks were not always evident (data not shown), possibly because the putative peak occurred outside the frequency range measured. Frequency dependency of phase angle has also been observed for acidified reconstituted skim milk (Lucey et al., 1997), where the phase angle decreased as frequency increased. A shift in the frequency peak occurred for yoghurt stabilised with 0.5 g L1 guar gum after different storage periods (Fig. 6). The peaks occurred at 4.5 Hz at 1 day, 2.5 Hz at 21 days, and 2.2 Hz at 42 days. The corresponding characteristic relaxation times were 0.22, 0.39 and 0.46 s for 0.5 g L1 guar gum sample aged for 1, 21 and 42 days, respectively.
not trap serum phase, or in other words, the volume fraction is not changed. Increasing the LM-pectin concentration above 1 g L1 resulted in a decrease in apparent viscosity and dynamic moduli and an increase in water holding capacity. At the higher levels of LMpectin the flow units are increasingly covered by this polysaccharide and the aggregates are partially sterically stabilised. This will reduce the effective volume fraction, reducing viscosity, and increasing water holding capacity as the casein network begins to lose structural integrity and expel serum phase. Adsorption and bridging of casein micelles by LM-pectin at pH 5.3 occurs at low concentrations of pectin, leading to steric stabilisation at higher concentrations (Maroziene & de Kruif, 2000), although it must be pointed out that the micelles are non-interacting at this pH, unlike the aggregates in this present study. Pectin with 61% esterification adsorbs onto the surface of casein micelles at pH values of 5.0 and below, where the net charge on the aggregating micelles becomes positive (Tuinier, Rolin, & de Kruif, 2002). Bridging takes place at low pectin concentrations, with stabilisation at higher concentrations. These authors conclude that pectin electrostatically adsorbed to the micellar surface and formed multilayers at pH values at least between 3.5 and 4.8, possibly though a combination of electrostatic and hydrophobic interaction. The yoghurt cannot become completely dispersed at high stabiliser concentrations as micellar aggregates would not then form and syneresis could not occur. There must be some residual degree of micellar
4. Discussion 4.1. Low-methoxy pectin Bridging of casein aggregates by up to 1 g L1 LMpectin may strengthen the casein network, thus reducing water holding capacity (Fig. 4A), but not affect viscosity to a large extent (Fig. 1A) if the protein flow units do
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aggregation induced by either depletion flocculation by unadsorbed LM-pectin, or electrostatic interaction of micellar aggregates. Higher values of phase angle provide evidence that the network is becoming more liquid like at greater than 2 g L1 LM-pectin (Fig. 3A). An increasing tendency towards liquid-like behaviour, with concomitant increase in the phase angle, will make syneresis more likely (van Vliet, van Dijk, Zoon, & Walstra, 1991). An LM-pectin gel will eventually form in the continuous phase at concentrations above 6 g L1 providing there is enough Ca2+ present (Maroziene & de Kruif, 2000). This would increase the elasticity of yoghurt; however, the effect was not examined in this present study. 4.2. Carrageenan Similar behaviour for the other adsorbing polysaccharide, l-carrageenan, was found up to 3 g L1 stabiliser. This polysaccharide adopts a coil conformation under the ionic conditions and temperatures used in this present study (Dalgleish & Morris, 1988). It may adsorb to the surface of casein micelles (Syrbe et al., 1998); however, it does not gel unlike k- and i- variants. The reduction in apparent viscosity and dynamic moduli and the increase in water holding capacity up to 3 g L1 l-carrageenan may have occurred by a partial steric stabilisation mechanism with residual aggregation. In this case, the aggregate structure partially unfolds to reveal aggregated casein strands with a lower effective volume fraction of aggregated flow units, and a lower viscosity. The aggregates may be bridged by l-carrageenan at low concentrations, although this is not conclusive from the water holding capacity data, which did not decrease up to 1 g L1 l-carrageenan. Depletion flocculation of unadsorbed l-carrageenan above 3 g L1 may explain the higher dynamic moduli compared to LM-pectin. Adsorption of negatively charged l-carrageenan onto the casein micelle surface may take place by electrostatic interaction with the net positively charged moiety (f20–115) of k-casein (Snoeren, Payens, Jeurnink, & Both, 1975). It must be pointed out, however, that the positively charged part of the k-casein molecule is adsorbed close to the micelle surface and interaction with l-carrageenan may be hindered, and also that Snoeren et al. (1975) examined non-micellar k-casein. Tuinier et al. (2002) found that the adsorption of highmethoxy pectin does not occur above pH 5.0 in either the presence or absence of the k-casein brush layer, indicating an exclusively electrostatic interaction. Saturation coverage of casein micelles occurs at about 1 g L1 in equivalent unconcentrated skim milk (Dalgleish & Morris, 1988), indicating that depletion flocculation may take place above this concentration. Bridging of casein micelles by all three types of carrageenan is possible at concentrations less than around 1 g L1 at
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25 1C (where i- and k-carrageenan are in the helix conformation), and depletion flocculation can take place at around 2 g L1 at 65 1C in the coil conformation (Langendorff et al., 1997). Other than these studies, there has been little reported on the stabilisation mechanisms of l-carrageenan acting upon casein micelles. Bridging of casein aggregates by l-carrageenan is likely to take place by electrostatic interaction under the conditions employed in this study (Dalgleish & Morris, 1988; Langendorff et al., 1999). 4.3. Adsorbing mechanisms For increasing concentration of adsorbing polymer, the stabilisation–destabilisation mechanisms undergo transitions from (1) bridging flocculation, to (2) steric stabilisation, to (3) depletion flocculation by unadsorbed polysaccharide in the serum phase, to (4) colloidal aggregates trapped in a viscous polymer network (Syrbe et al., 1998). Up to 5 g L1 LM-pectin the data are consistent with the first three mechanisms taking place. For l-carrageenan, mechanisms 2 and 3 are likely to occur, although mechanism 1 may also take place at lower concentrations. The complexity of this model is compounded by the nature of the colloidal material in this study. The casein aggregates are capable of interacting at pH values near the isoelectric point without the presence of a stabiliser. Thus, at high stabiliser concentrations the aggregates are compacted by both electrostatic attraction and stabiliser-induced depletion flocculation. Gelation of l-carrageenan does not take place. At a high concentration of LM-pectin a gel may form if sufficient Ca2+ is present. Van Marle, van den Ende, de Kruif, & Mellema (1999) reported that adsorbing exopolysaccharides increase the viscosity of the serum phase in stirred yoghurt. A mechanism of ‘‘brush friction’’ was invoked to explain this observation, whereby the adsorbed brush layer promotes increased interaction between protein aggregates. If this occurs in the present work, it is not possible to disentangle this mechanism from the other processes of bridging flocculation and steric stabilisation from the data presented. Indeed, the aggregate volume fraction is more likely to contribute to viscosity, and this will decrease as the extent of adsorbed polysaccharide increases. 4.4. Guar gum No electrostatic interaction between non-adsorbing neutral polysaccharides and casein aggregates takes place. As the concentration of guar increased to 1 g L1 the micellar aggregate structure became more compact due to depletion flocculation. The resultant lower effective volume fraction reduced apparent viscosity (Fig. 1C), and the yoghurt became more
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liquid like, as evident from the phase angle data (Fig. 3C). A more compact aggregate structure will have a smaller density of aggregate cross-links, and thus lower elasticity (van Vliet et al., 1991), and a higher level of syneresis. Guar may form a viscous continuous phase at 5 g L1 concentration containing trapped compact micellar aggregates. This would result in a higher viscosity and reduced water holding capacity. The viscous solution may also be inhibiting the breakage of interaggregate bonds under strain, making the yoghurt more solid-like as evident from the phase angle data (Fig. 3C). 4.5. Locust bean gum Similar behaviour occurs for LBG, although the increase in phase angle at 5 g L1 stabiliser concentration indicates that aggregates are not as efficiently trapped to inhibit the breaking of inter-aggregate bonds, as in the case of guar. Depletion flocculation may occur as LBG concentration increases. This mechanism has been observed for a suspension of 50 g L1 casein micelles at pH 6.7 with 0.5 g L1 LBG (Schorsch et al., 1999). 4.6. Xanthan gum Apparent viscosity and dynamic moduli of xanthan showed similar trends to the other two non-adsorbing polysaccharides, indicating that the proposed casein interactive mechanisms may also be applicable to this stabiliser. Depletion flocculation may also take place, as has been observed with reconstituted skim milk powder and milk protein concentrate with a xanthan concentration as low as 0.25 g L1 (Hemar et al., 2001). 4.7. Non-adsorbing mechanisms For increasing concentration of non-adsorbing polymer, the stabilisation–destabilisation mechanisms undergo a transition from depletion flocculation to colloidal particles trapped in a viscous polymer network (Syrbe et al., 1998). Depletion flocculation may occur up to 1 g L1 concentration for all three non-adsorbing polysaccharides. At higher concentrations, the casein aggregates may be trapped within the increasingly viscous polysaccharide solution, explaining the increase in apparent viscosity and dynamic moduli. 4.8. Relaxation between casein aggregate structural units The trends in peak values of phase angle as a function of frequency of oscillation warrants a more complex analysis. Despite being within what is known as the linear viscoelastic region, bonds between structural units will generally break and reform. At low frequencies, the bonds between structural flow units (casein aggregates) will stretch and relax with little breakage taking place,
and this is reflected in the low values for phase angle at low frequency where solid-like behaviour predominates. There is sufficient time during one period of oscillation at these low frequencies for bonds to break and form new linkages. As the frequency increases, some of the bonds between flow units will break and not have sufficient time to form new linkages before a second oscillation occurs, and the material will become increasingly more liquid-like, as reflected by higher values for phase angle. At the highest frequencies another mechanism takes place. The most tenacious bonds will not have time to break and re-establish during a rapid period of oscillation. This period will be shorter than the average characteristic bond relaxation time; therefore the phase angle will decrease. High-frequency behaviour is best explained by the Deborah number (Barnes et al., 1989), which quantifies the notion that all materials tend to exhibit increasingly solid-like characteristics over shorter experimental time scales (such as at higher frequencies). The peak values for phase angle thus correspond to an average bond relaxation time between structural flow units in the stabilised yoghurt, equal to the reciprocal of the frequency. The tangent of the phase angle has previously been argued to be an indicator of bond relaxation behaviour (van Vliet et al., 1991). It must be pointed out that the bonds between structural flow units will have a spectrum of relaxation times rather than a single characteristic relaxation time (Barnes et al., 1989) due to the inhomogeneous nature of the gel network. The relaxation times of guar-stabilised casein aggregates occur in the experimentally measurable range of 0.1–10 s, corresponding to a frequency range of 10–0.1 Hz. As the concentration of guar increases, casein aggregates are slowed down during bond stretching and relaxing due to the higher viscosity of the continuous phase; thus the characteristic relaxation time increases from around 0.22 s at 0.5 g L1 guar to 5.7 s at 5 g L1 guar. The relaxation time for unstabilised yoghurt may be outside this range. As yoghurt stabilised with 0.5 g L1 guar ages over a period of 42 days, proteolysis of caseins takes place (as it will for all other samples) and the structure changes from being a network of intact aggregated casein to one where the network is weakened. The result is a lengthening of the characteristic relaxation time from 0.22 s at day 1 to 0.46 s at day 42, as the number density of bonds between aggregates in yoghurt was reduced. This is supported by the lower dynamic moduli and apparent viscosity as the yoghurt ages (data not published).
5. Conclusions As the level of anionic LM-pectin and l-carrageenan increased, the mechanism of casein aggregate
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stabilisation was consistent with an adsorbed steric repulsion layer on the aggregate surface. However, this was countered by the electrostatic attraction of chargereduced aggregates and an increasing tendency towards depletion flocculation by the unadsorbed fraction of the stabiliser. The rheological data is consistent with bridging flocculation of aggregates by LM-pectin at low concentrations. Increasing levels of anionic stabiliser resulted in higher water holding capacity as the extent of casein aggregation was less able to trap serum phase within the protein matrix. This indicates that a steric stabilisation mechanism was becoming predominant to counter the aggregation process. For LBG, guar and xanthan, the lower apparent viscosity (compared to the unstabilised yoghurt) at low stabiliser concentrations, where the aggregate structure of caseins is becoming more compact, was consistent with a model of depletion flocculation of aggregates. Further increases in the stabiliser concentration will trap the compact casein aggregates in a viscous polysaccharide continuous phase. At higher concentrations of guar gum the average characteristic relaxation time between structural units (casein aggregates) undergoing shear strain increased. The proposed depletion flocculation mechanism evidently increased the time required for bonds between casein aggregates to relax upon the cessation of an applied strain. Ageing of yoghurt also increased the relaxation time at low guar concentration as the compact aggregate structure underwent rearrangement. The number density of bonds between aggregates in yoghurt would be reduced over time as the structure underwent proteolysis. Yoghurt samples tested using continuous shear viscometry underwent a well-defined shear treatment before subsequent measurements. The oscillatory rheological data must be interpreted with the knowledge that the pre-measurement shear treatment was not quantified in this case. References Afonso, I. M., & Joao, M. M. (1999). Rheological monitoring of structure evolution and development in stirred yoghurt. Journal of Food Engineering, 42, 183–190. Barnes, H. A., Hutton, J. F., & Walters, K. (1989). An introduction to rheology. Oxford, UK: Elsevier. Bourriot, S., Garnier, C., & Doublier, J.-L. (1999). Phase separation, rheology and microstructure of micellar casein–guar gum mixtures. Food Hydrocolloids, 13, 43–49. Dalgleish, D. G., & Morris, E. R. (1988). Interactions between carrageenans and casein micelles: electrophoretic and hydrodynamic properties of the particles. Food Hydrocolloids, 2, 311–320. Dickinson, E., Semenova, M. G., Antipova, A. S., & Pelan, E. G. (1998). Effect of high-methoxy pectin on properties of caseinstabilized emulsions. Food Hydrocolloids, 12(4), 425–432. Everett, D. W., & Jameson, G. W. (1993). Physicochemical aspects of cheddar cheese made from ultrafiltered milk. Australian Journal of Dairy Technology, 48(1), 20–29.
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Fiszman, S. M., Lluch, M. A., & Salvador, A. (1999). Effect of addition of gelatin on microstructure of acidic milk gels and yoghurt and on their rheological properties. International Dairy Journal, 9, 895–901. Hansen, P. M. T. (1993). Food hydrocolloids in the dairy industry. In K. Nishinari, & E. Doi (Eds.), Food hydrocolloids: structures, properties and functions (pp. 211–224). New York: Plenum Press. Hemar, Y., Tamehana, M., Munro, P. A., & Singh, H. (2001). Viscosity, microstructure and phase behavior of aqueous mixtures of commercial milk protein products and xanthan gum. Food Hydrocolloids, 15(4–6), 565–574. Keogh, M. K., & O’Kennedy, B. T. (1998). Rheology of stirred yogurt as affected by added milk fat, protein and hydrocolloids. Journal of Food Science, 63(1), 108–112. Kosikowski, F. V., & Mistry, V. V. (1999). Cheese and fermented milk foods: procedures and analysis. Great Falls, VA, USA: F. V. Kosikowski LLC. Langendorff, V., Cuvelier, G., Launay, B., Michon, C., Parker, A., & De Kruif, C. G. (1999). Casein micelle/iota carrageenan interactions in milk: influence of temperature. Food Hydrocolloids, 13(3), 211–218. Langendorff, V., Cuvelier, G., Launay, B., & Parker, A. (1997). Gelation and flocculation of casein micelle/carrageenan mixtures. Food Hydrocolloids, 11(1), 35–40. Lucey, J. A. (2002). Formation and physical properties of milk protein gels. Journal of Dairy Science, 85(2), 281–294. Lucey, J. A., & Singh, H. (1997). Formation and physical properties of acid milk gels. Food Research International, 30(7), 529–542. Lucey, J. A., Teo, C. T., Munro, P. A., & Singh, H. (1997). Rheological properties at small (dynamic) and large (yield) deformations of acid gels made from heated milk. Journal of Dairy Research, 64, 591–600. Maroziene, A., & de Kruif, C. G. (2000). Interaction of pectin and casein micelles. Food Hydrocolloids, 14, 391–394. Ramirez-Figueroa, E., Salgado-Cervantes, M. A., Rodriguez, G. C., & Garcia, H. S. (2002). Addition of hydrocolloids to improve the functionality of spray-dried yoghurt. Milchwissenschaft, 57(2), 87–89. Schorsch, C., Jones, M. G., & Norton, I. T. (1999). Thermodynamic incompatibility and microstructure of milk protein/locust bean gum/sucrose systems. Food Hydrocolloids, 13(2), 89–99. Snoeren, T. H. M., Payens, T. A. J., Jeurnink, J., & Both, P. (1975). Electrostatic interaction between kappa-carrageenan and kappacasein. Milchwissenschaft, 30, 393–395. Syrbe, A., Bauer, W. J., & Klostermeyer, H. (1998). Polymer science concepts in dairy systems—an overview of milk protein and food hydrocolloid interaction. International Dairy Journal, 8(3), 179–193. Tamime, A. Y., & Robinson, R. K. (1985). Yoghurt: science and technology. Oxford, UK: Pergamon Press. Tuinier, R., Rolin, C., & de Kruif, C. G. (2002). Electrosorption of pectin onto casein micelles. Biomacromolecules, 3(3), 632–638. van Marle, M. E., van den Ende, D., de Kruif, C. G., & Mellema, J. (1999). Steady-shear viscosity of stirred yoghurts with varying ropiness. Journal of Rheology, 43, 1643–1662. van Vliet, T., Lucey, J. A., Grolle, K., & Walstra, P. (1997). Rearrangements in acid-induced casein gels during and after gel formation. In E. Dickinson, & B. Bergensta˚hl (Eds.), Food colloids: proteins, lipids and polysaccharides (pp. 335–345). Cambridge, UK: Royal Society of Chemistry. van Vliet, T., van Dijk, H. J. M., Zoon, P., & Walstra, P. (1991). Relation between syneresis and rheological properties of particle gels. Colloid and Polymer Science, 269(6), 620–627. Velez-Ruiz, J. F., & Barbosa Canovas, G. V. (1997). Rheological properties of selected dairy products. Critical Reviews in Food Science and Nutrition, 37(4), 311–359.
International Dairy Journal 15 (2005) 1175–1183 www.elsevier.com/locate/idairyj
Interactions of polysaccharide stabilisers with casein aggregates in stirred skim-milk yoghurt David W. Everett, Rosalind E. McLeod Department of Food Science, University of Otago, Dunedin 9001 New Zealand Received 18 February 2004; accepted 8 December 2004
Abstract The stabilisation mechanisms of low-methoxy pectin, l-carrageenan, guar gum, locust bean gum and xanthan in stirred yoghurt were investigated using dynamic oscillatory rheometry and water holding capacity measurements. As the level of low-methoxy pectin and l-carrageenan increased, the stabilisation mechanism was consistent with the model of casein aggregates passing through a region of bridging flocculation, followed by partial steric stabilisation. For guar gum, locust bean gum and xanthan, the results were consistent with depletion flocculation at a low concentration of stabiliser, and trapped aggregated casein within a viscous polysaccharide solution at higher concentrations. A peak was observed for the phase angle of guar-stabilised yoghurt as a function of frequency, from 4.5 to 0.2 Hz as guar increased from 0.5 to 5 g L1 for 1-day yoghurt, and from 4.5 to 2.2 Hz as the yoghurt containing 0.5 g L1 guar gum was aged from 1 to 42 days. Results are interpreted with an understanding that the rheological behaviour is affected by the pre-sharing conditions of the stirred yogurt. r 2005 Elsevier Ltd. All rights reserved. Keywords: Yoghurt; Stabilisers; Rheology; Syneresis
1. Introduction Yoghurt contains a network of three-dimensional casein strands aggregated through iso-electric precipitation brought about by the action of lactic acid bacteria (Tamime & Robinson, 1985). The casein strands can be broken apart by shearing, and the size of the aggregates decreases as the rate of shearing increases (Afonso & Joao, 1999). Rearrangement and syneresis of the acidinduced casein network in yoghurt occurs during storage (van Vliet, Lucey, Grolle, & Walstra, 1997), although this process is far from being completely understood. Syneresis can be reduced by increasing the casein content of the milk (Fiszman, Lluch, & Salvador, 1999), by reducing the incubation temperature and rate of acidification (Lucey, 2002), or by adding stabilisers that interact with the casein network.
Corresponding author. Tel.: +64 3 479 7545; fax: +64 3 479 7567.
E-mail address: [email protected] (D.W. Everett). 0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.12.004
The firmness of yoghurt is affected by homogenisation, pH, processing parameters (stirred or set yoghurt), and heat treatment of milk that denatures b-lactoglobulin and that subsequently may bridge adjacent casein micelles (Lucey & Singh, 1997; Lucey, Teo, Munro, & Singh, 1997). Yoghurt is usually prepared from homogenised milk to improve stability. This process coats the increased surface of fat globules with casein, enabling the fat globules to participate as a copolymer with casein to strengthen the gel network and reduce syneresis (Keogh & O’Kennedy, 1998). Stabilisers are used in yoghurt to improve consistency (increase viscosity) and reduce syneresis (Lucey, 2002). These may include gelatin, starch, pectin, alginate, carrageenan, derivatives of methylcellulose, gum arabic, tragacanth, karaya, locust bean gum (LBG) or guar (Tamime & Robinson, 1985). Xanthan, k-carrageenan and LBG have been used to improve the consistency and dispersion properties of spray-dried yoghurt powder in water (Ramirez-Figueroa, Salgado-Cervantes, Rodriguez, & Garcia, 2002). A primary stabiliser such as
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carboxymethyl cellulose (CMC), LBG, alginate, or guar gum can be used as a thickener in conjunction with a secondary stabiliser such as carrageenan to reduce syneresis (Hansen, 1993). Anionic hydrocolloids (e.g. CMC, pectin, l-carrageenan) interact with the positive charges on the surface of casein micelles to strengthen the casein network and reduce syneresis, and will be classified as adsorbing polysaccharides for the purposes of this study. Neutral hydrocolloids (e.g. xanthan, guar, LBG) do so through a different mechanism by increasing the viscosity of the continuous phase (Hansen, 1993), and will be classified as non-adsorbing polysaccharides. At low concentration of some types of stabilisers, destabilisation can occur by the bridging of casein micelles (Langendorff et al., 1999; Langendorff, Cuvelier, Launay, & Parker, 1997; Maroziene & de Kruif, 2000; Syrbe, Bauer, & Klostermeyer, 1998). Non-adsorbing or an excess of adsorbing polysaccharides can cause depletion flocculation of suspended colloids (Bourriot, Garnier, & Doublier, 1999; Dickinson, Semenova, Antipova, & Pelan, 1998; Hemar, Tamehana, Munro, & Singh, 2001; Langendorff et al., 1997, 1999; Maroziene & de Kruif, 2000; Schorsch, Jones, & Norton, 1999), and also increase the viscosity of the continuous phase and form an entangled polymeric network (Syrbe et al., 1998). The quality of the solvent will affect the ability of adsorbed polymers to stabilise casein micelles. In good solvents such as water, the polymer will dissolve in the water phase and casein micelles will be prevented from aggregating. In poor solvents such as ethanol, the adsorbed polymer will not dissolve in the solvent phase and will lie compact on the surface of the micelle. In this case, the steric repulsive mechanism is minimised (Syrbe et al., 1998). To examine the effect of stabilisers on yoghurt viscosity, elasticity and water holding capacity, five polysaccharides were added to cultured yoghurt at concentrations ranging from 0.5 to 5 g L1. Two adsorbing polysaccharides, low-methoxy (LM) pectin and l-carrageenan, and three non-adsorbing polysaccharides, guar gum, LBG and xanthan were selected and the mechanisms of stability examined as the concentration of stabiliser increased. This study provides evidence for structural models of casein aggregates as the concentrations of adsorbing and non-adsorbing polysaccharides increase.
2. Materials and methods LM-pectin, l-carrageenan, LBG (Sigma Chemical Company, St. Louis, Missouri, USA), guar gum (Tic Gums, Belcamp, Maryland, USA) and xanthan (Jungbunzlauer, Vienna, Austria) were purchased and used without further purification. LM-pectin, with a 9.4%
methoxy and an 83% galacturonic acid content, was derived from citrus peel. This particular pectin preparation comprised 0.91% sodium and 0.57% calcium. No low molecular weight polysaccharides were present. Non-gelling l-carrageenan was derived from Gigartina aciculaire and G. pistillata seaweed and comprised 0.65% calcium, 4.72% potassium and 4.71% sodium, with less than 40% ester sulphate content. LBG, a straight chain polymer of mannose with one galactose branch on every fourth mannose and a molecular weight of approximately 310,000 was derived from Ceratonia siliqua seeds. Guar gum, consisting mainly of non-ionic galactomannan and with a molecular weight of approximately 250,000 was derived from the seeds of Cyamopsis tetragonolobus. Unpasteurised skim milk was obtained fresh (Meadowfresh Dairies, Dunedin, New Zealand) and used to make yoghurt within 4 days. 2.1. Yoghurt manufacture The casein contents of skim milk and skim milk powder were measured using a modified micro-Kjeldahl technique. Casein was first precipitated at the isoelectric point and filtered to remove undenatured whey proteins prior to nitrogen determination (Everett & Jameson, 1993). Skim milk powder was mixed with the skim milk to an initial casein content of 50 g L1 and standardised to a final casein content of 45 g L1 1 day after manufacture. The milk was heated at 85 1C for 20 min, dry powdered stabiliser added, and the mixture heated for a further 10 min at 85 1C. The types and amounts of stabilisers (by volume) were as follows: LM-pectin (0.5, 1, 2 3, 4 and 5 g L1), l-carrageenan (0.5, 1, 3 and 5 g L1), guar gum (0.5, 1 and 5 g L1), LBG (0.5, 1 and 5 g L1) and xanthan (0.5, 1, 3 and 5 g L1). Yoghurt manufacture was adapted from a standard technique (Kosikowski & Mistry, 1999). The mixture of milk and added stabiliser was cooled to 30 1C and a Streptococcus thermophilus and Lactobacillus bulgaricus direct vat set culture (YC-380, Chr. Hansen, Hamilton, New Zealand) was mixed into the milk and allowed to ferment for 16 h at 30 1C in autoclaved glass jars. Yoghurt samples were stored at 4 1C for 1, 21 and 42 days until required for analysis. The pH of the yoghurt was adjusted to 4.25 on day 1 after manufacture using 1M-NaOH or 1M-HCl as appropriate, and the volume of added acid or base solution kept constant (100 mL L1 of yoghurt volume) by further addition of deionised water such that the final casein content of the milk was 45 g L1. All utensils and equipment were washed with 200 mg L1 sodium hypochlorite solution before use. 2.2. Water holding capacity Water holding capacity measurements of yoghurt were made in triplicate (Keogh & O’Kennedy, 1998).
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Samples (30 g) were centrifuged at 200 g for 10 min at 4 1C and the extent of trapped serum phase quantified and expressed as a percentage of yoghurt volume by measuring the volume of supernatant. Water holding capacity measurements were made in triplicate and the average and absolute error range calculated.
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3. Results Increasing the concentration of the two adsorbing stabilisers (LM-pectin and l-carrageenan) up to 1 g L1 resulted in no change in apparent viscosity, and a decrease in apparent viscosity at higher concentrations (Fig. 1A, B). For two of the non-adsorbing stabilisers
0.0 3.5
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3.0 2.5 2.0 1.5 1.0 0.5 Apparent viscosity (Pa s)
Apparent viscosity and elasticity of yoghurt samples were measured 1, 21 and 42 days after manufacture at 10 1C using a cup and spindle configuration (RheoStress 1, Gebru¨der Haake GmbH, Karlsruhe, Germany) surrounded by a circulating water bath. For apparent viscosity measurements, samples were pre-sheared at 1000 s1 until the shear stress reached a constant value over a 20 s period (with a change of less than 0.05 Pa), typically taking less than 150 s to achieve. Shear stress was then measured as a function of decreasing shear rate from 1000 to 0.03 s1, allowing sufficient time at each shear rate step for the shear stress to attain a new equilibrium value. Elastic parameters were measured using dynamic oscillatory rheometry (Velez-Ruiz & Barbosa Canovas, 1997). The pre-measurement shear history of each sample was minimised by allowing time for the structure to be regained after carefully pouring the sample into the rheometer cup. To determine the region of linear viscoelasticity, samples were sheared under oscillatory conditions at 1 Hz and at an increasing strain (from 0.001 to 1 dimensionless units), and the storage modulus, G0 (in Pa, defined as the ratio of in-phase stress to strain) and the loss modulus, G00 (defined as the ratio of out-of-phase stress to strain) measured as a function of strain (Barnes, Hutton, & Walters, 1989). A subsequent sample of yoghurt was then sheared under oscillatory conditions at a strain of 0.003 units, determined to be within the linear viscoelastic region, and G0 and G00 measured as a function of frequency from 0.1 to 10 Hz. The ratio of G00 to G0 (equal to the tangent of the phase angle, d; between the shear stress and strain) was also calculated. A characteristic relaxation time was calculated by taking the inverse of the frequency of oscillation at the point where the dynamic moduli (storage and loss) showed peak values. Rheological measurements were made in triplicate and the average and absolute error range calculated.
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Stabiliser (g L−1) Fig. 1. Apparent viscosity of yoghurt at different concentrations of two adsorbing stabilisers (A) low-methoxy pectin and (B) l-carrageenan, and three non-adsorbing stabilisers (C) guar gum, (D) locust bean gum and (E) xanthan (pH 4.25). Measurements were made 1 day after manufacture at a shear rate of 10 s1 and a temperature of 10 1C (n ¼ 3; error bars are absolute range).
(LBG and guar gum), the apparent viscosity decreased up to 1 g L1 stabiliser concentration, then increased at
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5 g L1 (Fig. 1C, D). For the other non-adsorbing stabiliser (xanthan), the apparent viscosity remained unchanged up to 3 g L1, followed by an increase at higher concentrations (Fig. 1E), consistent with previously reported results for xanthan mixed with reconstituted skim milk powder or milk protein concentrate (Hemar et al., 2001). Two types of behaviour were evident for G0 and G00 at 1 Hz as a function of stabiliser concentration. For the two non-adsorbing stabilisers (Fig. 2A, B), a decrease in the two moduli occurred as the concentration of stabiliser increased above 1 g L1 for LM-pectin and between 0 and 1 g L1 for l-carrageenan. For the three non-adsorbing stabilisers, LBG, guar gum and xanthan, a decrease occurred up to 1 g L1 followed by an increase at 5 g L1 stabiliser, indicating a transition between two stabilising mechanisms at around 1 g L1 (Fig. 2C, D, E). The pH of 1-day yoghurt was adjusted to 4.25; however, the pH values after 21 and 42 days were not standardised. These were recorded above the appropriate set of data for 21-and 42-day-old yoghurt (Figs. 3 and 4). In most cases the change in pH over the 42-day storage period decreased by less than 0.2 pH units. The phase angle of the yoghurt samples at 1 Hz was calculated from G0 and G00 as a function of stabiliser concentration after 1, 21 and 42 days storage (Fig. 3). For the adsorbing stabilisers LM-pectin and l-carrageenan, the phase angle was constant up to 1 g L1 stabiliser. Phase angle increased above 2 g L1 LMpectin but remained constant up to 5 g L1 l-carrageenan. For two of the non-adsorbing stabilisers, LBG and guar gum, d increased up to 1 g L1, then remained unchanged at 5 g L1. The phase angle of the other nonadsorbing stabiliser, xanthan, remained constant over the concentration range examined (0.5 to 5 g L1). In most cases, for all five stabilisers, phase angle was not affected by the length of storage time of the yoghurt, although at higher concentrations of adsorbing stabiliser (X3 g L1), the 42-day yoghurt was more liquid as shown by the higher value for d (Fig. 3A, B). The water holding capacity decreased up to 1 g L1 then increased up to 5 g L1 for LM-pectin to an amount more than for the unstabilised yoghurt (Fig. 4A). This occurred for both 1- and 42-day yoghurt, and also for 21-day yoghurt with the exception of 5 g L1 LM-pectin. For the other adsorbing stabiliser, the water holding capacity increased up to 3 g L1 l-carrageenan, then decreased at 5 g L1 (Fig. 4B). A similar trend occurred for LBG where a decrease in water holding capacity took place between 1 and 5 g L1 stabiliser (Fig. 4C). There was no consistent trend for guar gum (Fig. 4D). Water holding capacity of xanthan yoghurt remained relatively unchanged compared to the other four stabilisers (Fig. 4E).
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Stabiliser (g L−1) Fig. 2. Dynamic moduli (storage, G0 , and loss, G00 ) of yoghurt (pH 4.25) at different concentrations of two adsorbing stabilisers (A) lowmethoxy pectin and (B) l-carrageenan, and three non-adsorbing stabilisers (C) guar gum, (D) locust bean gum and (E) xanthan. Measurements were made 1 day after manufacture at a strain of 0.003, frequency of 1 Hz and a temperature of 10 1C (n ¼ 3; error bars are absolute range). (’) G0 , (m) G00 .
The phase angle of 1-day yoghurt containing guar gum showed peak values that differed over the range in frequency of 0.1–10 Hz according to the amount of
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Stabiliser (g L−1) Fig. 3. Phase angle (d) of yoghurt at different concentrations of two adsorbing stabilisers (A) low-methoxy pectin and (B) l-carrageenan, and three non-adsorbing stabilisers (C) guar gum, (D) locust bean gum and (E) xanthan. Measurements were made at 1 (’), 21 (K) and 42 (m) days after manufacture at a strain of 0.003, frequency of 1 Hz and at a temperature of 10 1C (n ¼ 3; error bars are absolute range). The pH at day 1 was 4.25. Value for pH at days 21 (upper figure) and 42 (lower figure) are shown above each set of data. With no added stabiliser, the pH is 4.13 at day 21 and 4.14 at day 42 for all samples.
Fig. 4. Water holding capacity as a percentage of yoghurt volume at different concentrations of two adsorbing stabilisers (A) low-methoxy pectin and (B) l-carrageenan, and three non-adsorbing stabilisers (C) guar gum, (D) locust bean gum and (E) xanthan. Measurements were made at 1 (’), 21 (K) and 42 (m) days after manufacture at a temperature of 4 1C (n ¼ 3; error bars are absolute range). The pH at day 1 was 4.25. Value for pH at days 21 (upper figure) and 42 (lower figure) are shown above each set of data. With no added stabiliser, the pH is 4.13 at day 21 and 4.14 at day 42 for all samples.
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90 0.5 g L-1 guar
80
day 1 80
1 g L-1 guar 5 g L-1 guar
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day 42 70
unstabilised
60
Phase angle (˚)
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Phase angle (˚)
day 21
50 40
50 40
30
30
20
20
10
10
0 0.1
1
10
Frequency (Hz)
0 0.1
1
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Frequency (Hz)
Fig. 5. Phase angle (d) of yoghurt (pH 4.25) at different concentrations of guar gum as a function of frequency of oscillation. Measurements were made 1 day after manufacture at a strain of 0.003 and a temperature of 10 1C (n ¼ 3; error bars are absolute range). Arrows indicate peaks.
Fig. 6. Phase angle (d) of yoghurt containing 0.5 g L1 guar gum as a function of frequency of oscillation. Measurements were made at 1 (pH 4.25), 21 (pH 3.96) and 42 days (pH 3.89) after manufacture at a strain of 0.003 and a temperature of 10 1C (n ¼ 3; error bars are absolute range). Arrows indicate peaks.
stabiliser (Fig. 5). Unstabilised yoghurt did not show a peak value. For increasing amounts of guar gum (0.5 and 1 g L1) the peak shifted to lower values, from 4.5 to 2.2 Hz, respectively. There was no clear peak for the 5 g L1 guar sample. The inverse of the frequency peak yielded characteristic relaxation times for each stabiliser concentration (0.22 and 0.46 s) for increasing concentration of guar. For the other stabilisers, peaks were not always evident (data not shown), possibly because the putative peak occurred outside the frequency range measured. Frequency dependency of phase angle has also been observed for acidified reconstituted skim milk (Lucey et al., 1997), where the phase angle decreased as frequency increased. A shift in the frequency peak occurred for yoghurt stabilised with 0.5 g L1 guar gum after different storage periods (Fig. 6). The peaks occurred at 4.5 Hz at 1 day, 2.5 Hz at 21 days, and 2.2 Hz at 42 days. The corresponding characteristic relaxation times were 0.22, 0.39 and 0.46 s for 0.5 g L1 guar gum sample aged for 1, 21 and 42 days, respectively.
not trap serum phase, or in other words, the volume fraction is not changed. Increasing the LM-pectin concentration above 1 g L1 resulted in a decrease in apparent viscosity and dynamic moduli and an increase in water holding capacity. At the higher levels of LMpectin the flow units are increasingly covered by this polysaccharide and the aggregates are partially sterically stabilised. This will reduce the effective volume fraction, reducing viscosity, and increasing water holding capacity as the casein network begins to lose structural integrity and expel serum phase. Adsorption and bridging of casein micelles by LM-pectin at pH 5.3 occurs at low concentrations of pectin, leading to steric stabilisation at higher concentrations (Maroziene & de Kruif, 2000), although it must be pointed out that the micelles are non-interacting at this pH, unlike the aggregates in this present study. Pectin with 61% esterification adsorbs onto the surface of casein micelles at pH values of 5.0 and below, where the net charge on the aggregating micelles becomes positive (Tuinier, Rolin, & de Kruif, 2002). Bridging takes place at low pectin concentrations, with stabilisation at higher concentrations. These authors conclude that pectin electrostatically adsorbed to the micellar surface and formed multilayers at pH values at least between 3.5 and 4.8, possibly though a combination of electrostatic and hydrophobic interaction. The yoghurt cannot become completely dispersed at high stabiliser concentrations as micellar aggregates would not then form and syneresis could not occur. There must be some residual degree of micellar
4. Discussion 4.1. Low-methoxy pectin Bridging of casein aggregates by up to 1 g L1 LMpectin may strengthen the casein network, thus reducing water holding capacity (Fig. 4A), but not affect viscosity to a large extent (Fig. 1A) if the protein flow units do
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aggregation induced by either depletion flocculation by unadsorbed LM-pectin, or electrostatic interaction of micellar aggregates. Higher values of phase angle provide evidence that the network is becoming more liquid like at greater than 2 g L1 LM-pectin (Fig. 3A). An increasing tendency towards liquid-like behaviour, with concomitant increase in the phase angle, will make syneresis more likely (van Vliet, van Dijk, Zoon, & Walstra, 1991). An LM-pectin gel will eventually form in the continuous phase at concentrations above 6 g L1 providing there is enough Ca2+ present (Maroziene & de Kruif, 2000). This would increase the elasticity of yoghurt; however, the effect was not examined in this present study. 4.2. Carrageenan Similar behaviour for the other adsorbing polysaccharide, l-carrageenan, was found up to 3 g L1 stabiliser. This polysaccharide adopts a coil conformation under the ionic conditions and temperatures used in this present study (Dalgleish & Morris, 1988). It may adsorb to the surface of casein micelles (Syrbe et al., 1998); however, it does not gel unlike k- and i- variants. The reduction in apparent viscosity and dynamic moduli and the increase in water holding capacity up to 3 g L1 l-carrageenan may have occurred by a partial steric stabilisation mechanism with residual aggregation. In this case, the aggregate structure partially unfolds to reveal aggregated casein strands with a lower effective volume fraction of aggregated flow units, and a lower viscosity. The aggregates may be bridged by l-carrageenan at low concentrations, although this is not conclusive from the water holding capacity data, which did not decrease up to 1 g L1 l-carrageenan. Depletion flocculation of unadsorbed l-carrageenan above 3 g L1 may explain the higher dynamic moduli compared to LM-pectin. Adsorption of negatively charged l-carrageenan onto the casein micelle surface may take place by electrostatic interaction with the net positively charged moiety (f20–115) of k-casein (Snoeren, Payens, Jeurnink, & Both, 1975). It must be pointed out, however, that the positively charged part of the k-casein molecule is adsorbed close to the micelle surface and interaction with l-carrageenan may be hindered, and also that Snoeren et al. (1975) examined non-micellar k-casein. Tuinier et al. (2002) found that the adsorption of highmethoxy pectin does not occur above pH 5.0 in either the presence or absence of the k-casein brush layer, indicating an exclusively electrostatic interaction. Saturation coverage of casein micelles occurs at about 1 g L1 in equivalent unconcentrated skim milk (Dalgleish & Morris, 1988), indicating that depletion flocculation may take place above this concentration. Bridging of casein micelles by all three types of carrageenan is possible at concentrations less than around 1 g L1 at
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25 1C (where i- and k-carrageenan are in the helix conformation), and depletion flocculation can take place at around 2 g L1 at 65 1C in the coil conformation (Langendorff et al., 1997). Other than these studies, there has been little reported on the stabilisation mechanisms of l-carrageenan acting upon casein micelles. Bridging of casein aggregates by l-carrageenan is likely to take place by electrostatic interaction under the conditions employed in this study (Dalgleish & Morris, 1988; Langendorff et al., 1999). 4.3. Adsorbing mechanisms For increasing concentration of adsorbing polymer, the stabilisation–destabilisation mechanisms undergo transitions from (1) bridging flocculation, to (2) steric stabilisation, to (3) depletion flocculation by unadsorbed polysaccharide in the serum phase, to (4) colloidal aggregates trapped in a viscous polymer network (Syrbe et al., 1998). Up to 5 g L1 LM-pectin the data are consistent with the first three mechanisms taking place. For l-carrageenan, mechanisms 2 and 3 are likely to occur, although mechanism 1 may also take place at lower concentrations. The complexity of this model is compounded by the nature of the colloidal material in this study. The casein aggregates are capable of interacting at pH values near the isoelectric point without the presence of a stabiliser. Thus, at high stabiliser concentrations the aggregates are compacted by both electrostatic attraction and stabiliser-induced depletion flocculation. Gelation of l-carrageenan does not take place. At a high concentration of LM-pectin a gel may form if sufficient Ca2+ is present. Van Marle, van den Ende, de Kruif, & Mellema (1999) reported that adsorbing exopolysaccharides increase the viscosity of the serum phase in stirred yoghurt. A mechanism of ‘‘brush friction’’ was invoked to explain this observation, whereby the adsorbed brush layer promotes increased interaction between protein aggregates. If this occurs in the present work, it is not possible to disentangle this mechanism from the other processes of bridging flocculation and steric stabilisation from the data presented. Indeed, the aggregate volume fraction is more likely to contribute to viscosity, and this will decrease as the extent of adsorbed polysaccharide increases. 4.4. Guar gum No electrostatic interaction between non-adsorbing neutral polysaccharides and casein aggregates takes place. As the concentration of guar increased to 1 g L1 the micellar aggregate structure became more compact due to depletion flocculation. The resultant lower effective volume fraction reduced apparent viscosity (Fig. 1C), and the yoghurt became more
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liquid like, as evident from the phase angle data (Fig. 3C). A more compact aggregate structure will have a smaller density of aggregate cross-links, and thus lower elasticity (van Vliet et al., 1991), and a higher level of syneresis. Guar may form a viscous continuous phase at 5 g L1 concentration containing trapped compact micellar aggregates. This would result in a higher viscosity and reduced water holding capacity. The viscous solution may also be inhibiting the breakage of interaggregate bonds under strain, making the yoghurt more solid-like as evident from the phase angle data (Fig. 3C). 4.5. Locust bean gum Similar behaviour occurs for LBG, although the increase in phase angle at 5 g L1 stabiliser concentration indicates that aggregates are not as efficiently trapped to inhibit the breaking of inter-aggregate bonds, as in the case of guar. Depletion flocculation may occur as LBG concentration increases. This mechanism has been observed for a suspension of 50 g L1 casein micelles at pH 6.7 with 0.5 g L1 LBG (Schorsch et al., 1999). 4.6. Xanthan gum Apparent viscosity and dynamic moduli of xanthan showed similar trends to the other two non-adsorbing polysaccharides, indicating that the proposed casein interactive mechanisms may also be applicable to this stabiliser. Depletion flocculation may also take place, as has been observed with reconstituted skim milk powder and milk protein concentrate with a xanthan concentration as low as 0.25 g L1 (Hemar et al., 2001). 4.7. Non-adsorbing mechanisms For increasing concentration of non-adsorbing polymer, the stabilisation–destabilisation mechanisms undergo a transition from depletion flocculation to colloidal particles trapped in a viscous polymer network (Syrbe et al., 1998). Depletion flocculation may occur up to 1 g L1 concentration for all three non-adsorbing polysaccharides. At higher concentrations, the casein aggregates may be trapped within the increasingly viscous polysaccharide solution, explaining the increase in apparent viscosity and dynamic moduli. 4.8. Relaxation between casein aggregate structural units The trends in peak values of phase angle as a function of frequency of oscillation warrants a more complex analysis. Despite being within what is known as the linear viscoelastic region, bonds between structural units will generally break and reform. At low frequencies, the bonds between structural flow units (casein aggregates) will stretch and relax with little breakage taking place,
and this is reflected in the low values for phase angle at low frequency where solid-like behaviour predominates. There is sufficient time during one period of oscillation at these low frequencies for bonds to break and form new linkages. As the frequency increases, some of the bonds between flow units will break and not have sufficient time to form new linkages before a second oscillation occurs, and the material will become increasingly more liquid-like, as reflected by higher values for phase angle. At the highest frequencies another mechanism takes place. The most tenacious bonds will not have time to break and re-establish during a rapid period of oscillation. This period will be shorter than the average characteristic bond relaxation time; therefore the phase angle will decrease. High-frequency behaviour is best explained by the Deborah number (Barnes et al., 1989), which quantifies the notion that all materials tend to exhibit increasingly solid-like characteristics over shorter experimental time scales (such as at higher frequencies). The peak values for phase angle thus correspond to an average bond relaxation time between structural flow units in the stabilised yoghurt, equal to the reciprocal of the frequency. The tangent of the phase angle has previously been argued to be an indicator of bond relaxation behaviour (van Vliet et al., 1991). It must be pointed out that the bonds between structural flow units will have a spectrum of relaxation times rather than a single characteristic relaxation time (Barnes et al., 1989) due to the inhomogeneous nature of the gel network. The relaxation times of guar-stabilised casein aggregates occur in the experimentally measurable range of 0.1–10 s, corresponding to a frequency range of 10–0.1 Hz. As the concentration of guar increases, casein aggregates are slowed down during bond stretching and relaxing due to the higher viscosity of the continuous phase; thus the characteristic relaxation time increases from around 0.22 s at 0.5 g L1 guar to 5.7 s at 5 g L1 guar. The relaxation time for unstabilised yoghurt may be outside this range. As yoghurt stabilised with 0.5 g L1 guar ages over a period of 42 days, proteolysis of caseins takes place (as it will for all other samples) and the structure changes from being a network of intact aggregated casein to one where the network is weakened. The result is a lengthening of the characteristic relaxation time from 0.22 s at day 1 to 0.46 s at day 42, as the number density of bonds between aggregates in yoghurt was reduced. This is supported by the lower dynamic moduli and apparent viscosity as the yoghurt ages (data not published).
5. Conclusions As the level of anionic LM-pectin and l-carrageenan increased, the mechanism of casein aggregate
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stabilisation was consistent with an adsorbed steric repulsion layer on the aggregate surface. However, this was countered by the electrostatic attraction of chargereduced aggregates and an increasing tendency towards depletion flocculation by the unadsorbed fraction of the stabiliser. The rheological data is consistent with bridging flocculation of aggregates by LM-pectin at low concentrations. Increasing levels of anionic stabiliser resulted in higher water holding capacity as the extent of casein aggregation was less able to trap serum phase within the protein matrix. This indicates that a steric stabilisation mechanism was becoming predominant to counter the aggregation process. For LBG, guar and xanthan, the lower apparent viscosity (compared to the unstabilised yoghurt) at low stabiliser concentrations, where the aggregate structure of caseins is becoming more compact, was consistent with a model of depletion flocculation of aggregates. Further increases in the stabiliser concentration will trap the compact casein aggregates in a viscous polysaccharide continuous phase. At higher concentrations of guar gum the average characteristic relaxation time between structural units (casein aggregates) undergoing shear strain increased. The proposed depletion flocculation mechanism evidently increased the time required for bonds between casein aggregates to relax upon the cessation of an applied strain. Ageing of yoghurt also increased the relaxation time at low guar concentration as the compact aggregate structure underwent rearrangement. The number density of bonds between aggregates in yoghurt would be reduced over time as the structure underwent proteolysis. Yoghurt samples tested using continuous shear viscometry underwent a well-defined shear treatment before subsequent measurements. The oscillatory rheological data must be interpreted with the knowledge that the pre-measurement shear treatment was not quantified in this case. References Afonso, I. M., & Joao, M. M. (1999). Rheological monitoring of structure evolution and development in stirred yoghurt. Journal of Food Engineering, 42, 183–190. Barnes, H. A., Hutton, J. F., & Walters, K. (1989). An introduction to rheology. Oxford, UK: Elsevier. Bourriot, S., Garnier, C., & Doublier, J.-L. (1999). Phase separation, rheology and microstructure of micellar casein–guar gum mixtures. Food Hydrocolloids, 13, 43–49. Dalgleish, D. G., & Morris, E. R. (1988). Interactions between carrageenans and casein micelles: electrophoretic and hydrodynamic properties of the particles. Food Hydrocolloids, 2, 311–320. Dickinson, E., Semenova, M. G., Antipova, A. S., & Pelan, E. G. (1998). Effect of high-methoxy pectin on properties of caseinstabilized emulsions. Food Hydrocolloids, 12(4), 425–432. Everett, D. W., & Jameson, G. W. (1993). Physicochemical aspects of cheddar cheese made from ultrafiltered milk. Australian Journal of Dairy Technology, 48(1), 20–29.
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