Effect of polysaccharides with different ionic charge on the rheological, microstructural and textural properties of acid milk gels

Food Research International 72 (2015) 62–73

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Effect of polysaccharides with different ionic charge on the rheological, microstructural and textural properties of acid milk gels Zhihua Pang a,b, Hilton Deeth b, Nidhi Bansal b,⁎ a b

Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing 100048, China School of Agriculture and Food Sciences, The University of Queensland, Brisbane 4072, Australia

a r t i c l e

i n f o

Article history: Received 4 September 2014 Received in revised form 8 February 2015 Accepted 28 February 2015 Available online 9 March 2015 Keywords: Polysaccharides Ionic charge Acid milk gelation Rheology Microstructure

a b s t r a c t The effects of addition of polysaccharides with different ionic charge on rheology, microstructure, texture and water holding capacity (WHC) of acid milk gels were studied and compared to that of gelatin addition. Similar to gelatin, starch (neutral) and xanthan gum (anionic) did not prevent milk gelation in the first 30 min of the acidification stage, even at high concentrations, and the typical casein network in acid milk gels could still be seen from electron micrographs; gelling and melting of these hydrocolloids were observed during the cooling and heating stages at specific concentrations. On the other hand, two neutral polysaccharides, guar gum (≥0.05%) and locust bean gum [LBG] (≥0.1%) inhibited milk gelation from the beginning of the acidification stage; the microstructure of the gel was modified greatly and no gelling/melting was observed during the cooling or heating stages. Another anionic polysaccharide, carrageenan, induced earlier milk gelation at low concentration (≤ 0.05%), but inhibited gelation entirely at high concentration (0.2%); inflections at ~ 27 °C and 21 °C were also observed during the cooling and heating stages at 0.05% concentration. The gel microstructure was not changed greatly, but showed smaller particle size at a carrageenan concentration of 0.05% than control sample. None of the polysaccharides showed as much improvement in WHC of the milk gels as gelatin did. Hence, xanthan and starch were found to be closer to gelatin in their effect on acid milk gels compared to guar gum, LBG and carrageenan. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Stabilizers have been used in yogurt, especially stirred yogurt, to help maintain good textural properties and prevent syneresis. Among all the stabilizers used, gelatin has been preferred as it gives desirable appearance, texture and flavor to yogurt (Kumar & Mishra, 2004). It has the unique property of melting at body temperature, which provides fat-like sensory perception (Kalab, Emmons, & Sargant, 1975; Salvador & Fiszman, 1998), and is able to effectively reduce syneresis in yogurt without increasing the gel firmness excessively (Ares et al., 2007). However, religious beliefs and vegetarian lifestyle choices may prohibit certain consumer groups from consuming yogurt containing gelatin because of its mammalian source. The risk of potential contamination with viruses and prions is also a concern with gelatin use. Therefore, finding alternatives to gelatin has gained considerable interest in recent years, but an ideal alternative to gelatin has not been found so far (Karim & Bhat, 2008). One of the most common approaches to replacing gelatin in stirred yogurt is using alternative hydrocolloids. Non-ionic hydrocolloids (e.g. guar gum [guar] and locust bean gum [LBG]) stabilize the yogurt system ⁎ Corresponding author. Tel.: +61 7 33651673; fax: +61 7 33651177. E-mail address: [email protected] (N. Bansal).

http://dx.doi.org/10.1016/j.foodres.2015.02.009 0963-9969/© 2015 Elsevier Ltd. All rights reserved.

by increasing the viscosity of the continuous phase of yogurt (Everett & McLeod, 2005). Ionic hydrocolloids (e.g. pectin, carrageenan) strengthen the casein network and reduce syneresis by interacting with the positive charges on the surface of casein micelles. Gellan gum, modified starch, xanthan gum (xanthan), LBG and pectin have been suggested as potential alternatives to gelatin in stirred yogurt (Morrison, Clark, Chen, Talashek, & Sworn, 1999). In this study, two types of polysaccharides, non-ionic (guar, LBG and starch) and ionic (xanthan and carrageenan) were studied in milk gels to evaluate their potential for replacing gelatin. Both guar and LBG are galactomannans; only segregative interactions occur between these polysaccharides and milk proteins in yogurt (Corredig, Sharafbafi, & Kristo, 2011). Starch mainly contains two types of structurally distinct polysaccharides: amylose (a linear polymer) and amylopectin (a multi-branched polymer) (Morris, 1990), and it has been used widely in yogurt. Different from guar and LBG, both associative and segregative interactions between starch and milk proteins were reported (Allen, Carpenter, & Walsh, 2007; Corredig et al., 2011), which may indicate different mechanisms of stabilization in yogurt. Xanthan is a heteropolysaccharide with a primary structure of repeating pentasaccharide units. The existence of acetate and pyruvate in the molecules makes xanthan an anionic polysaccharide (Garcia-Ochoa, Santos, Casas, & Gomez, 2000). Carrageenans are highly sulphated polysaccharides and strong electrostatic interactions

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were reported between κ-carrageenan and milk proteins (κ-casein and whey proteins) (Mleko, Li-Chan, & Pikus, 1997; Snoeren, Payens, Jeunink, & Both, 1975). Both ι- and κ-carrageenan undergo a coil-tohelix transition during temperature decrease, resulting in thermoreversible gelation (Tye, 1988). This process also needs the presence of cations such as K+ and Ca2 + that are naturally present in milk (Drohan, Tziboula, McNulty, & Horne, 1997). The development of gelatin replacements should be product specific (Morrison et al., 1999). However, not many studies on gelatin replacement in yogurt could be found. Only a few studies comparing the effect of ingredients, including gelatin, on yogurt properties have been reported (Ares et al., 2007; Keogh & O'Kennedy, 1998; Modler, Larmond, Lin, Froehlich, & Emmons, 1983) and these have focused only on the texture and sensory properties of the final yogurt with gelatin or other stabilizers. The underlying mechanism of stabilization of the gel network and syneresis control using gelatin alternatives and microstructural characteristics of the resulting yogurts has been barely discussed. In our study, the manufacturing process of yogurt from fermentation to storage and consumption was followed by dynamic oscillatory rheological measurements and microstructural analysis of the product by scanning electronic microscopy. Instead of culture fermentation, the acidulantglucono-delta-lactone (GDL) was used to simulate yogurt acidification to reduce variation caused by differences in culture performance between trials. GDL has been extensively applied in milk gel studies (Hemar, Hall, Munro, & Singh, 2002; Lucey, Tamehana, Singh, & Munro, 1998; Sanchez, Zuniga-Lopez, Schmitt, Despond, & Hardy, 2000). The aims of this research were to study the effect of addition of polysaccharides with different ionic charge on the mechanism of their stabilization of milk protein gels and to evaluate the potential of these polysaccharides as gelatin replacements in yogurt. 2. Materials and methods 2.1. Materials The gelatin used in this study was supplied by Gelita (Beaudesert, Australia). It was a light colored edible beef skin (type B) gelatin powder with bloom 200, mesh 20 and isoelectric point of ~5.0, which is a commercial product commonly used in the food industry. Skim milk powder (SMP, protein 33%, moisture 3.6%, fat 0.9%, lactose 54.7% and ash 7.8%) was obtained from Murray Goulburn Co-Operative Ltd (Melbourne, Australia) and the acidulantglucono-delta-lactone (GDL) was purchased from Sigma Chemical Co. (St. Louis, USA). Xanthan (GRINDSTED 80 ANZ), guar (GRINDTED 250) and LBG (GRINDTED 246) were donated by Danisco, France. Carrageenan (GENULACTA type LRA-50, a 1:1 mixture of κ- and ι-carrageenan) was kindly provided by CP Kelco ApS, Denmark. Hydroxypropyl distarch phosphate modified tapioca starch (NATIONAL FRIGEX) was provided by National Starch, Singapore. 2.2. Methods 2.2.1. Measurement of zeta potential Five polysaccharides stock solutions (1% w/v) were prepared in MilliQ water by heating and stirring at 85 °C for 30 min followed by further stirring for 2 h at room temperature. The solutions were diluted with MilliQ water to 0.1% (w/v) and pH was adjusted by hydrochloric acid or sodium hydroxide to 4.6, 5.3 and 6.6. The zeta potential of all samples was measured using Malvern Zetasizer Nano-ZS (Malvern, UK). 2.2.2. Preparation of stirred acid milk gels The acid milk gels were prepared as described previously (Pang, Deeth, Sopade, Sharma, & Bansal, 2014). SMP was reconstituted in distilled water under continuous stirring for 30 min to obtain a milk protein concentration of 4.5% (w/w). Selected hydrocolloids were added from stock solutions at various concentrations. Gelatin was studied at

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concentrations of 0.4 and 1% (w/w). A series of concentrations of guar and LBG (0.01, 0.05, 0.1 and 0.5%), xanthan (0.001, 0.005 and 0.01%), carrageenan (0.01, 0.05 and 0.1%) and starch (0.2, 1 and 2%) were studied. A sample with no stabilizer was studied as control. Mixtures of milk powder and gelatin were reconstituted and stored at 4 °C overnight before use and, where applicable, the polysaccharides were added the next day before heating. The samples were heated in a water bath at 95 °C for 10 min with continuous stirring at 300 rpm. Distilled water was added at the end of stirring in order to account for water evaporation. The samples were cooled to 45 °C immediately using cold water. For gel formation, 1.5% (w/w) GDL was added to the mixtures to decrease the pH to ~4.6 in 4 h at 45 °C. The samples were stirred using an overhead stirrer at 1200 rpm for 2 min. Samples were then distributed in cylindrical containers of diameter 11 cm and height 5 cm for texture analysis, and in 15 ml centrifuge tubes for measurement of water holding capacity. Samples were held at 10 °C for 48 h before testing. 2.2.3. Dynamic oscillatory rheological measurement The dynamic oscillatory rheological measurements were carried out on the samples on a stress-controlled rheometer (Model AR-G2, TA Instruments, USA) according to the method reported previously (Pang et al., 2014). Aliquots of mixture solutions were poured onto the bottom plate of the rheometer equipped with a 4 cm, 2° cone-plate measuring system immediately after GDL was added. The measurements were performed in a four-stage process: Acidification stage: measurement commenced at 45 °C and this temperature was maintained for 4 h, promoting formation of the milk protein gel; Cooling stage: the temperature was lowered from 45 to 10 °C at a constant rate of 1 °C/min to observe the gelling property of the hydrocolloids; Annealing stage: the oscillatory tests were performed at 10 °C for 2.5 h to observe the maturation of the gelling samples; Heating stage: the temperature was increased from 10 to 45 °C at 1 °C/min to observe melting property of the hydrocolloids. 2.2.4. Texture analysis Texture profile analysis (TPA) was performed using a TA-XT2 Texture Analyzer (Godalming, UK). The probe used was cylindrical with a flat base of 35 mm diameter. Two cycles were applied, at a constant speed of 1 mm/s, to a sample depth of 10 mm. From the resulting force–time curve, the values for texture attributes were obtained using the Exponent (version 5.0.8.0) equipment software. The following parameters, as defined by Bourne (2002), were quantified: firmness (N), adhesiveness (Nm), cohesiveness (ratio) and springiness (mm). 2.2.5. Water holding capacity The measurement was performed using the method reported previously (Pang et al., 2014) with some modifications. After 48 h storage at 10 °C, samples were centrifuged at 200 g for 10 min at 10 °C. The water holding capacity (WHC) was defined as follows: WHCð%Þ ¼ 100ðMG weight–SE weightÞ=MG weight: Where MG ¼ milk gel and SE ¼ serum expelled:

2.2.6. Microstructure The microstructure of the stirred acid milk gels was observed using scanning electron microscopy according to the method described by Pang, Deeth, Sharma, and Bansal (2014). Gels after 48 h storage at 10 °C were fixed with glutaraldehyde at room temperature, dehydrated with ethanol at room temperature and then dried with a CO2 critical point dryer (Tousimis Automatic). Dried samples were platinum-

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coated and observed with a scanning electron microscope (JEOL 6610) at an acceleration voltage of 10 kV. 2.2.7. Statistical analysis The trials were replicated twice and, unless otherwise stated, all analyses were carried on two independent replicates. Minitab ver. 16 software (Minitab Inc., USA) was used for analysis of variance (ANOVA) and test of significance (p b 0.05).

Table 1 Water holding capacity and textural characteristics of stirred acid milk gels with gelatin. Samples

Firmness (N)

Cohesiveness (ratio)

Adhesiveness (Nm)

WHC (%)

Control Gel with 0.4% gelatin Gel with 1% gelatin

0.40b 0.37b 2.4a

0.62a 0.64a 0.6a

0.15b 0.21b 0.45a

87.6b 99.7a 100a

Means (n = 3) within a column with different letters are significantly different (p b 0.05).

3. Results and discussion 3.1. Gelatin as a stabilizer in stirred acid milk gels The dynamic oscillatory rheological properties of milk gels with different levels of gelatin were reported previously (Pang et al., 2014). The microstructure, texture and WHC of stirred milk gels containing 0.4 or 1% gelatin are shown in this study. Micrographs of gels with no stabilizers showed a porous branched structure (Fig. 1A), which was similar to the structure of set milk gels reported by Pang et al. (2014). Similar microstructures of stirred and set yogurts were reported by Kalab et al. (1975). Compared to control sample, the gels with 0.4% gelatin had a more compact network with smaller pores (Fig. 1B), and no gelatin strands were observed. Addition of 1% gelatin induced formation of extensive gelatin strands while the primary size of the casein particles remained unmodified (Fig. 1C). The results of texture analysis and WHC of the stirred milk gels with gelatin are shown in Table 1. Springiness of milk gels was not significantly affected by any stabilizer studied; hence the results were not included. Kumar and Mishra (2004) have also reported that the springiness of a mango-soy-fortified milk gel was not influenced by the addition of stabilizers. The gel with 1% gelatin had about 6 times higher firmness than control sample and showed 100% WHC. Milk gels with 0.4% gelatin did not show significant differences in firmness, adhesiveness and cohesiveness from the control sample, but had significantly higher WHC. Similar results with set milk gels have been reported by Pang et al. (2014). The ability of gelatin to increase WHC without increasing gel firmness at low concentration deserves more attention. Syneresis (which is strongly related to WHC) is one of the major problems in yogurt; however, the approaches that are taken to improve WHC must maintain the moderate gel firmness that is characteristic of yogurt gels (Damin, Alcantara, Nunes, & Oliveira, 2009). This is a major reason why gelatin is the preferred stabilizer in yogurt, besides its “melt-in-mouth” property (Karim & Bhat, 2008). Gelatin gelation was observed in milk gels, according to the rheological results in a previous study (Pang et al., 2014). The basic mechanism of gelation of gelatin is a coil-to-helix transition during which the helices that are created are similar to the collagen structure (Djabourov,

Lechaire, & Gaill, 1993). This transition occurs at temperatures below 30 °C, even at low gelatin concentrations (Djabourov, 1988). It was reported that at low gelatin concentration, the formation of crosslinks is very likely to occur between two segments of the same molecule or molecules already joined (Ferry & Eldridge, 1949). During this transition, some water molecules are incorporated into the triple helices, called ‘structural water’ (Maquet, Theveneau, Djabourov, Leblond, & Papon, 1986), inducing immobilization and orientation of the water molecules in the triple helix (Badii & Howell, 2006). This could be the reason why even low concentrations of gelatin increased the WHC. On the other hand, the intramolecular crosslinks do not contribute to the rigidity of a gel, which is more related to the concentration of intermolecular crosslinks (Ferry & Eldridge, 1949; Joly-Duhamel, Hellio, Ajdari, & Djabourov, 2002). Therefore, at low concentration (0.4%), gelatin did not increase the firmness of milk gels. Moreover, the interaction between stabilizers and milk components should be considered to produce the desired properties. No strong interactions between gelatin and milk proteins occur during the acidification stage (Pang et al., 2014), which could also be important to maintain the typical milk gel structure. 3.2. Polysaccharides as stabilizers in milk gels The zeta potential of the five polysaccharides was measured as a function of pH (4.6, 5.3 and 6.6) (results not shown). At all these pH levels, guar, LBG and starch showed zeta potential around zero or slightly negative, with values above −10 mV, while xanthan and carrageenan showed zeta potential between −60 and −70 mV. Therefore, the results suggested that the polysaccharides used in this study could be classified as non-ionic (guar, LBG and starch) or ionic (xanthan and carrageenan). 3.2.1. Rheology Rheology during four stages (acidification, cooling, annealing and heating) was studied. Since the G′ did not change much during the annealing stage for all the samples, the results for the annealing stage are not shown.

Fig. 1. SEM micrographs of stirred acid milk gel with no stabilizer (A), with 0.4% gelatin (B) and 1% gelatin (C).

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A

B

C

Fig. 2. Changes in G′ of samples without ( ), with 0.01 (…), 0.05 ( ), 0.1 (—··—) and 0.5% ( acidification stage; B. the cooling stage from 45 to 10 °C; C. the heating stage from 10 to 45 °C.

3.2.1.1. Guar and LBG. Fig. 2 shows the rheology results of samples with guar. The gelation time was considered as the time of an obvious increase in the G′ from the baseline (Pang et al., 2014). During the acidification stage, 0.01% guar had little effect on the gelation of milk; the G′ was very close to that of control sample. A sharp increase in G′ was seen after 10 min, which indicated casein micelle fusion, and casein dissociation and rearrangement, as discussed by Pang et al. (2014). At 0.05% addition, guar inhibited the gelation considerably. However, earlier gelation was observed and the G′ reached ~ 55 Pa within 25 min (Fig. 2A inset), which might indicate the formation of loosely entangled casein aggregates as reported by McMahon, Du, McManus, and Larsen (2009). The earlier gelation is likely due to phase separation, which forced the casein micelles closer together causing aggregation at higher pH (Girard & Schaffer-Lequart, 2007; Perrechil, Braga, & Cunha, 2009). However, no further increase of G′ was seen; instead, G′ declined immediately after that peak. Addition of 0.1% guar inhibited the gelation completely and the G′ was very close to zero. The sample containing 0.5% guar showed obvious gelation during the acidification stage, although it had a much lower G′ than control sample. The G′ increased from the beginning of acidification stage, at a much lower increase rate than for control sample. However, the increase in G′ was possibly mainly due to the high viscosity of guar solution caused by the high

) guar gum. A. The acidification stage at 45 °C, inset: the first 60 min during

concentration of guar, rather than milk protein gelation. The viscous solution induced by high concentration of guar may inhibit the breakage of inter-aggregate bonds under strain, making the system behave more solid-like (Everett & McLeod, 2005); and the reduced molecular mobility of casein micelles could prevent phase separation (Perrechil et al., 2009). Similar results were reported by Kontogiorgos, Ritzoulis, Biliaderis, and Kasapis (2006) for the mixed gels of sodium caseinate and β-glucan, in which the gel properties were dominated by the protein component at low concentrations of β-glucan, but governed by β-glucan at high concentrations. During the later stages of cooling, annealing and heating, similar trends to that of control sample were observed in all guar-containing samples except that the G′ of the sample with 0.1% guar remained close to zero. No inflection was observed in the profiles during the cooling or heating stages. Similar effects of guar and LBG on yogurt were reported by Everett and McLeod (2005) and β-glucan in acid milk gels (Lazaridou, Vaikousi, & Biliaderis, 2008). Fig. 3 shows the rheological results of samples with LBG. Effects similar to guar were observed for addition of LBG on milk gelation except at a concentration of 0.05%. During acidification, earlier gelation occurred with addition of 0.05% LBG but, unlike the 0.05% guar sample, the G′ of the sample with 0.05% LBG did not decrease throughout the acidification stage and remained much higher than that of control sample. This

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A

B

C

Fig. 3. Changes in G′ of samples without ( ), with 0.01 (…), 0.05 ( ), 0.1 (—··—) and 0.5% ( stage; B. the cooling stage from 45 to 10 °C; C. the heating stage from 10 to 45 °C.

difference is likely due to differences in degree of branching of LBG and guar; ~50% for guar and ~25% for LBG (Doublier & Launay, 1981; Everett & McLeod, 2005). 3.2.1.2. Starch. The rheology results of samples with starch are shown in Fig. 4. The starch used in this study was hydroxypropyl distarch phosphate modified starch, which is superior to native starch as a thickener and stabilizer. The cross-link modification improves the acid, heat and shear stability of the starch, which is crucial in application of yogurt, due to the heat treatment, acidification and stirring steps used in manufacturing stirred yogurt. The etherification provides the starch with much greater stability which leads to an improvement in WHC (BeMiller & Whistler, 2009; Tharanathan, 2005). Samples with 0.2, 1 and 2% starch showed lower final G′ than that of control sample during the acidification stage, with the 1% sample having the lowest G′ of only ~70 Pa. This could be due to phase separation or steric interference caused by the pasted starch (Zuo, Hemar, Hewitt, & Saunders, 2008), although the pasting characteristics of starch were governed by the level of cross-linking (Wongsagonsup et al., 2014). Different from guar and LBG, in the first 25 min, all samples containing starch showed an increase of G′ similar to that of control sample, even

) LBG. A. The acidification stage at 45 °C, inset: the first 60 min during acidification

at high concentration, indicating formation of a casein network. It has been reported that both associative and segregative interactions exist between starch and milk proteins (Allen et al., 2007; Corredig et al., 2011), but only segregative interaction between milk protein and guar or LBG (Corredig et al., 2011; de Jong, Klok, & van de Velde, 2009), which could make starch more compatible with milk protein than the other two gums. In addition, both 1 and 2% starch induced earlier gelation and the sample containing 2% starch showing a higher G′ than control sample in this short period, which could be related to the uptake of water by the starch granules during swelling and the consequent increase in protein concentration (Oh, Anema, Wong, Pinder, & Hemar, 2007). During the cooling and heating stages (Fig. 4B, C), inflections were observed at ~28 °C and ~22 °C, respectively, for the sample with 2% starch, which are related to the gelling and melting temperature of starch. It was reported that upon cooling, starch tends to form a gel or paste-like mass and the gelation was partly reversible upon heating (Kim & Yoo, 2010; Lund, 1984). It was also reported that the gelation only happens when the concentration is above the critical gelling concentration of the starch (Morris, 1990). A study on pure cross-linked tapioca starch showed that this starch had gelling property and 1% concentration was high enough to produce an elastic network-like

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A

B

Fig. 4. Changes in G′ of samples without ( ), with 0.2 (…), 1 ( ) and 2% ( cooling stage from 45 to 10 °C; C. the heating stage from 10 to 45 °C.

C

) starch. A. The acidification stage at 45 °C, inset: the first 60 min during acidification stage; B. the

structure (Wongsagonsup et al., 2014). Gelation occurs during cooling through cross-linking of the chains of amylose by hydrogen bonds (BeMiller & Whistler, 2009; Zhang et al., 2015). As starch existed in a mixed system with milk protein gel in our study, a higher gelling concentration than in a pure starch system could be expected. This may explain our results that low concentrations of starch, 0.2 and 1%, did not show any inflection during cooling, but 2% did. 3.2.1.3. Xanthan. The rheology results of samples with xanthan are shown in Fig. 5. During the acidification stage, the low concentration (0.001%) of xanthan did not affect the G′; the medium concentration (0.005%) increased the G′ of the control sample slightly; while high concentration (0.01%) interfered with the gelation severely and the G′ was very close to zero at the end of acidification, which could be attributed to the depletion destabilization (Syrbe, Bauer, & Klostermeyer, 1998). However, all the samples showed an increase in G′ in the first 60 min, even with 0.01% xanthan, indicating the formation of casein network, which was different from the effect of guar or LBG at concentration 0.1%. The results are attributable to attractive interactions between positively charged patches on κ-casein and negatively charged xanthan, which makes xanthan more compatible with milk proteins and lead to bridging flocculation (Braga & Cunha, 2004; Syrbe et al., 1998). Unlike

guar and LBG containing samples, for the sample containing 0.005% xanthan, inflections were observed at ~ 24 °C during the cooling (Fig. 5B) and ~19 °C during heating stages (Fig. 5C). These results should be related to the conformational change in the xanthan molecules. Xanthan molecules undergo conformation shifts from disordered state to ordered state through coil-helix transition when temperature is lowered. This conformational change is reversible and the transition temperature is concentration dependent (Braga & Cunha, 2004; Garcia-Ochoa et al., 2000). 3.2.1.4. Carrageenan. The rheology results of samples with carrageenan are shown in Fig. 6. During the acidification stage, low concentration (0.02%) of carrageenan did not change the rheology profiles much, and addition of 0.05% carrageenan decreased the G′ of the control sample; however, earlier gelation than the control sample was also observed at both concentrations (see inset in Fig. 6A). Higher concentrations (0.1 and 0.2%) of carrageenan prevented gelation of the milk, although the sample with 0.1% carrageenan showed weak gelation with a final G′ of 33 Pa and G″ of 8 Pa at the end of this stage. Carrageenans are highly sulphated polysaccharides. Hence, interaction between κ/ι-carrageenan and milk proteins could occur over the entire pH range, especially the interaction between κ-carrageenan and

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A

B

Fig. 5. Changes in G′ of samples without ( ), with 0.001 (…), 0.005 ( ) and 0.01% ( B, the cooling stage from 45 to 10 °C; C, the heating stage from 10 to 45 °C.

κ-casein (Drohan et al., 1997; Mleko et al., 1997), thereby affecting the milk protein–protein interactions. It has also been proven by microstructural study that κ-carrageenan can adsorb to the surface of the casein micelles (Spagnuolo, Dalgleish, Goff, & Morris, 2005). At low concentration of carrageenan, the strong interaction between carrageenan and milk proteins leads to the extensive exposure of protein hydrophobic groups and formation of large aggregates (Mleko et al., 1997), which could induce earlier gelation; at high concentration of carrageenan, the interaction could severely interfere with milk protein interactions and fusion of casein micelles, inducing low gel strength. During the later stages, similar to xanthan and starch, inflections at ~ 27–23 °C (Fig. 6B) during cooling and at ~ 21–28 °C during heating stages (Fig. 6C) were observed for the sample containing 0.05% carrageenan, which could be related to the gelling property of carrageenan (Tziboula & Horne, 1999). Therefore, comparing the effect of gelatin and the effects of these five polysaccharides on acid milk gels, some similarities and differences were observed. Gelatin decreased the G′ of the milk protein gels with increasing gelatin concentration during acidification. However, there was no change in the gelation time with any change in gelatin concentrations (Pang et al., 2014), which was different from the effects of the

C

) xanthan. A. The acidification stage at 45 °C, inset: the first 60 min during acidification stage;

polysaccharides studied. Gelatin did not inhibit milk gelation from the beginning of acidification at any concentration (Pang et al., 2014); similar behaviors were observed with xanthan and starch, but not with guar, LBG and carrageenan, which may indicate that gelatin, xanthan and starch were more compatible with milk proteins than the other gums in our study. Moreover, gelling and melting were observed during the cooling and heating stages for gelatin at sufficient concentration (Pang et al., 2014); this was also observed with samples containing xanthan and starch. 3.2.2. Microstructure The micrographs of the samples were taken after 48 h storage at 10 °C. Fig. 7 shows the microstructure of stirred milk gels containing different gums at various concentrations. In some samples, those containing 0.05 and 0.1% guar, 0.1% LBG, 0.01% xanthan and 0.1% carrageenan, varying amounts of serum were observed on the top of the samples. The samples for SEM were thus taken from the bottom phase. Although the rheological analysis indicated no gelation, especially for samples with 0.1% guar or LBG, the bottom phase of the samples after 48 h storage looked like a ‘gel’, but could be called ‘sediment’. Samples with 0.5% guar and LBG and 0.2% carrageenan did not show any characteristics of

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A

B

C

Fig. 6. Changes in G′ of samples without ( ), with 0.02 (…), 0.05 ( ), 0.1% (—··—) and 0.2% carrageenan ( acidification stage; B. the cooling stage from 45 to 10 °C; C. the heating stage from 10 to 45 °C.

milk gels after storage for 48 h at 10 °C. Instead, the samples with 0.5% guar and LBG became very viscous even before acidification and the sample with 0.2% carrageenan became very liquid. Therefore, these concentrations were considered inappropriate for yogurt and the corresponding samples were not studied further. Addition of 0.01% guar did not change the microstructure of the gel much (Fig. 7A), which is in agreement with the rheological results. Addition of 0.05 and 0.1% guar resulted in a radically different structure. The gel network could not be seen at the magnification used for other samples (× 13000); only a few large clusters or pores were observed (Fig. 7M, N). Hence, the samples were viewed under lower magnification (×1000) (Fig. 7B, C). It could be clearly seen for the sample containing 0.05% guar that the apparent diameter of pores was greater than 10 μm and much thicker chains of the casein network were formed. Milk gels with 0.1% guar showed an even more compact structure (Fig. 7C), with the apparent diameter of the casein strands being N10 μm. Similar results were obtained for milk gels with LBG at 0.01 and 0.1% concentrations (Fig. 7D–F). Addition of 0.05% LBG did not dramatically change the structure of the milk gel and the size of the primary particles was the same as in control sample, although large pores with diameter ~ 5 μm were observed. This is consistent with the rheology results which showed that addition of 0.05% LBG increased the G′. Similar results were reported previously (Cavallieri & Cunha, 2009; Sanchez et al., 2000). Such a marked effect of addition of higher concentrations of

). A. The acidification stage at 45 °C, inset: the first 60 min during

guar and LBG may be responsible for their negative effect on the rheological properties of the samples as discussed in Section 3.2.1.1. Addition of non-ionic polysaccharide, starch, at concentrations from 0.2 to 2% did not affect the typical casein network (Fig. 7G–I). Comparable microstructures were observed for samples with or without starch. Similar results were reported by Tamime, Barrantes, and Sword (1996), who found very subtle differences between samples with and without a starch-based fat substitute by both SEM and transmission electron microscopy; detection of the fat substitute was very difficult, even at high concentration. Fig. 7J–L show micrographs of stirred milk gels with different concentrations of xanthan. At 0.001%, xanthan induced a more dense structure with fewer and smaller pores than in control sample. A looser structure with larger pores was observed with 0.005% xanthan while with 0.01% xanthan, the milk gel showed a highly compact structure with very large pores. However, unlike in samples with high concentrations of guar and LBG, the casein network could still be seen at relatively high magnification, with unchanged primary particle size. This is consistent with the rheology results which showed an increase of G′ at the beginning of the acidification stage. Sanchez et al. (2000) reported that although a highly open network was formed with a high concentration of xanthan (0.1%), the network was more organized than with a high concentration of LBG. Fig. 7M–O shows micrographs of stirred milk gels with ionic polysaccharide, carrageenan. Addition of 0.02%

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Fig. 7. SEM micrographs of acid stirred milk gels after 48 h storage at 10 °C, with 0.01-0.1% guar gum (A–C), 0.01–0.1% LBG (D–F), 0.2–1% starch (G–I), 0.001–0.01% xanthan gum (J–L), 0.02–0.1% carrageenan (M–O), and images (P–R) were micrographs of samples with 0.05 and 0.1% guar gum and 0.1% LBG, taken at same magnification as other samples. Scale bars in the images are 1 μm, except in images B, C and F (scale bars are 10 μm).

carrageenan did not change the microstructure dramatically. With the addition of 0.05% carrageenan, smaller casein particles and larger and denser clusters with large voids were formed (Fig. 2C). The micrograph of the sample containing 0.1% carrageenan was similar to that of the control sample, with a well-organized casein network. It seemed that guar and LBG, tended to induce highly compact casein clusters and phase separation with increasing concentration with a loss

of the typical casein network, while xanthan, carrageenan and starch had less effect on the milk gel structure, which was closer to the effect of gelatin on milk gels. 3.2.3. Texture and water holding capacity (WHC) Fig. 8 shows the texture and WHC results of stirred milk gels with different treatments. Addition of 0.01% guar did not change the texture

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Fig. 7 (continued).

properties (firmness, cohesiveness and adhesiveness) or WHC of milk gels compared to the control sample. Addition of 0.05% guar led to greater gel firmness and adhesiveness while addition of 0.1% guar resulted in lower gel firmness and adhesiveness. Surprisingly, the highest gel cohesiveness was observed for the sample containing 0.1% guar. High concentrations of guar (0.05 and 0.1%) induced lower WHC also, especially with 0.1% guar when a WHC of only 57% was obtained; this could be attributed to depletion flocculation. A similar effect on texture properties and WHC to that of guar was found with LBG, except that addition of 0.05% LBG had no significant effect on gel firmness and WHC. Similar with guar and LBG, addition of low concentration (0.2%) of starch did not introduce any difference for both texture properties and WHC of the samples. However, firmness and adhesiveness were significantly increased by addition of higher concentrations of starch (1 and 2%). Similar results have been reported with modified tapioca starch (Sandoval-Castilla, Lobato-Calleros, Aguirre-Mandujano, & VernonCarter, 2004). They attributed this to the increased viscosity of the sample by starch molecules binding and orienting water, which consequently dampened the effect of applied stress and the starch molecules functioned as “fillers”. WHC was not increased by starch even at 2% concentration, which was probably because the water binding effect was counteracted by its interference with milk protein gelation. Xanthan did not affect any texture parameters of milk gels at any concentration. WHC was decreased by addition of 0.005 and 0.01% xanthan, while no effect was observed at 0.001%. Different effects on WHC of xanthan-containing samples have been reported. Everett and McLeod (2005) showed that xanthan did not affect the WHC of yogurt at concentrations between 0.05 and 0.5% while El-Sayed, Abd ElGawad, Murad, and Salah (2002) reported that xanthan (0.005%) increased the gel firmness and decreased syneresis of yogurt due to the interaction between the gum and milk proteins. Carrageenan did not induce any significant difference to texture parameters or WHC at low concentration (0.02%), while the sample with 0.05% carrageenan

showed higher gel firmness, but lower WHC than control sample; lower gel firmness and WHC were observed for the sample containing 0.1% carrageenan. Improvement of the WHC of yogurt by carrageenan has been reported at the very low concentration of 0.01% (Hematyar, Samarin, Poorazarang, & Elhamirad, 2012); however, the type of carrageenan was not indicated. In summary, within the range studied, the gel firmness and adhesiveness were not altered at low concentrations of the polysaccharides, and increased at higher concentrations (except xanthan, which did not show any influence on these parameters at any concentration); with further increase in concentration, gel firmness and adhesiveness were decreased for LBG, guar and carrageenan. Cohesiveness was not affected by any of the polysaccharides, except guar and LBG at a high concentration (0.1%). None of the polysaccharides increased the WHC of the milk gels; however, even a low concentration of gelatin (0.4%) enhanced the WHC of milk gel. 4. Conclusions Polysaccharides behaved very differently in stirred acid milk gels, due to their different stabilizing mechanisms. The non-ionic polysaccharides induced a change in the system in a series of no influencedepletion flocculation-polymeric stabilization steps with concentration increase and only segregative interaction with milk proteins occurred (except starch, which has associative interaction with milk proteins). The ionic polysaccharide, led the system through a series of no influence-bridging-depletion destabilization steps with concentration increase, and both associative and segregative interactions with milk proteins occurred. These different mechanisms resulted in very different milk gel properties, especially in microstructure and rheology. Xanthan and starch did not prevent milk gelation from the beginning of acidification or cause severe aggregation of milk proteins as guar and LBG did, even at high concentration, and the typical casein network was still obtained, which was similar to the effect of adding gelatin.

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A

B

C

D

Fig. 8. Textural characteristics of milk gels with polysaccharides at different concentrations. A. Firmness; B. adhesiveness; C. cohesiveness; D. water holding capacity.

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