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Effects of addition of phenolic compounds on the acid gelation of milk
International Dairy Journal 21 (2011) 185e191
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International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Effects of addition of phenolic compounds on the acid gelation of milk Niamh Harbourne, Jean Christophe Jacquier, Dolores O’Riordan* School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 April 2010 Received in revised form 13 September 2010 Accepted 3 October 2010
Tannic acid (0.1e1%, w/w) and gallic acid (0.3e1%, w/w) were added to skim milk prior to acidification with GDL. The acid gelation of tannic and gallic acid fortified milk had a faster gelation time in comparison with the control gel without phenolic compounds. The addition of tannic acid and gallic acid (up to 0.8%) to the milk resulted in a higher storage modulus (G0 ), decrease in the water mobility (T2 time) and had no significant effect on the syneresis index (SI). However, the inclusion of 1% gallic acid resulted in a significant decrease in G0 , a significant increase in the SI and a wider T2 distribution. Lowering the temperature of the gels from 30 to 5 C caused the G0 for the gels with gallic and tannic acid to increase significantly in comparison with the control, possibly due to increased hydrogen bonding in the presence of phenolic compounds. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Phenolic compounds are secondary plant metabolites that have received much attention in the last few years due to their many potential health-benefits. They have antioxidant and anti-inflammatory properties and may protect against degenerative diseases such as cancer and cardiovascular disease (Bravo, 1998). There are numerous phenolic compounds ranging from the low molecular weight simple phenols to highly polymerised tannins. Phenolic compounds are ubiquitous in all plants, and recent studies have shown that medicinal plants contain a very high level of these polyphenols (Cai, Luo, Sun, & Corkes, 2004; Liu, Qiu, Ding, & Yao, 2008). In recent years, there has been an increase in the addition of phenolic compounds to dairy products to improve both their technological functionality and nutritional value (O’Connell & Fox, 2001). Some recent studies have looked at the effect of the addition of fruit extracts, rich in phenolic compounds, on the antioxidant (Wegrzyn et al., 2008) and sensory (Axten, Wohlers, & Wegrzyn, 2008) attributes of milk drinks. Phenolic compounds may affect the functional properties of dairy products as they readily interact with proteins; these interactions are well documented in the literature (Luck et al., 1994; Spencer et al., 1988). It is recognised that proline-rich proteins (e.g., casein) associate strongly to polyphenols and the major interactions that are thought to be involved in this association are hydrogen bonding and
* Corresponding author. Tel.: þ353 (1) 716 7016; fax: þ353 (1) 716 1149. E-mail address: [email protected] (D. O’Riordan). 0958-6946/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2010.10.003
hydrophobic interactions (Luck et al., 1994). These interactions are dependent on protein type and on the molecular size and flexibility of the polyphenol (Spencer et al., 1988). This proteinepolyphenol interaction is also dependent on pH and is at a maximum when proteins are at their isoelectric point (Spencer et al., 1988). The rheological properties of milk acidified using gluconodeltalactone (GDL) have been well characterised (Lucey & Singh, 1998; Van Vliet, Lakemond, & Visschers, 2004). More recently, the water mobility in acidified milk drinks has been studied using nuclear magnetic resonance (NMR) and it has been used to detect textural problems such as whey separation (Salomonsen, Sejersen, Viereck, Ipsen, & Engelsen, 2007). The rheological properties and water mobility of these gels may be modified by fortification with either dairy or non-dairy ingredients, such as starch (Oh, Anema, Wong, Pinder, & Hemar, 2007) or pectin (Matia-Merino & Singh, 2007; Salomonsen et al., 2007). Phenolic compounds or extracts rich in phenolic compounds may be ideal ingredients to add to milk gels as they could improve the nutritional value and may also enhance the rheological properties, but to date there has been little work published on the addition of phenolic compounds to acidified milk gels. Phenolic compounds are bitter and astringent in nature making them difficult to incorporate into food products; however, recent research has shown that milk may significantly decrease the bitterness and astringency of phenolic rich extracts (Ares, Barreiro, Deliza, & Gambaro, 2009). The objective of the present study was to determine the effect of simple phenols (gallic acid) and hydrolysable tannins (tannic acid) on the rheological properties, changes in water mobility and syneresis of acid milk gels.
N. Harbourne et al. / International Dairy Journal 21 (2011) 185e191
2. Materials and methods 2.1. Acid milk gel preparation Reconstituted skim milk was prepared by adding low-heat skim milk powder (SMP) (Arrabawn Co-op, Nenagh, Co. Tipperary, Ireland) to distilled water to give a final concentration of 12% (w/w) SMP. The solutions were stirred on a stirring plate for at least 2 h at room temperature and 0.01% (w/w) potassium sorbate (Hoechst A.G, Frankfurt am Main, Frankfurt, Germany) was added as a preservative. The reconstituted skim milk was heated in a water bath at 85 C for 30 min and after heating was rapidly cooled by immersion in ice-water. Tannic acid (Sigma Aldrich, St. Louis, MO, USA) (0.1, 0.5 and 1%, w/w) and gallic acid (Sigma Aldrich) (0.3, 0.5, 0.8 and 1%, w/w) were then added to the milk and stirred for 30 min using a stirring plate at room temperature. GDL (1.3%, w/w) (Sigma Aldrich) was then added and the sample was stirred for 2 min. The acidified milk was then incubated in a water bath at 30 C for 16 h. A control gel that contained no phenolic compounds was prepared in a similar fashion. The changes in pH during gelation after the addition of GDL were monitored continuously for 16 h by means of a pH meter fitted with a glass combination electrode. 2.2. Acid gel formation and rheological measurements A Bohlin Gemini HRnano rheometer (Malvern Instruments Ltd, Worcestershire, UK) was used for the rheological measurements using a cup (27 mm) and bob (25 mm) system (Bohlin C-25, Malvern Instruments Ltd), with a solvent trap to prevent evaporation of the sample. Samples were transferred to the rheometer after addition and mixing of GDL (as above). To monitor the gelation process an applied strain of 1%, frequency of 0.1 Hz and a constant temperature of 30 C were used. Measurements were taken every 10 min for 16 h. Gelation time was defined as the point when gels had a G0 1 Pa (Lucey, Teo, Munro, & Singh, 1997). A frequency sweep from 0.001 to 1 Hz was performed on the final gel. Finally, the gel was subjected to a temperature sweep. The temperature of the sample was dropped from 30 to 5 C at a rate of 1 C min1 at a frequency of 0.1 Hz and strain of 1%.
(Sarstedt, Numbrecht, Germany) of known weight. The samples were incubated at 30 C in a thermostatically controlled water bath. After 16 h the tubes were centrifuged (Beckman J2-HS, Beckman Instruments Inc., Palo Alto, CA, USA) at 300 g for 10 min at 4 C. The supernatant was then removed carefully and weighed. The syneresis index (SI) was expressed as percentage weight of supernatant over the initial weight of the gel. 2.5. Statistical analysis All samples were prepared in triplicate and results in the text are given as mean values and standard deviations. A one-way analysis of variance (ANOVA) and Tukey’s pairwise comparisons were used to determine significant differences between the various treatments. SAS 9.1.3 was used for all analyses (SAS Institute, Cary, NC, USA). Results with p 0.05 were considered significantly different. 3. Results and discussion 3.1. Effect of addition of phenolic compounds on pH The effect of phenolic compound addition on the evolution of pH during the acidification of milk is shown in Fig. 1. The inclusion of tannic acid to the skim milk did not have a significant effect on the change of pH with time after GDL addition (Fig. 1a). The final pH was 4.58 for the control gel (no added tannic or gallic acid) and pH 4.54 for those containing 1% tannic acid (Table 1). The addition of gallic acid at all concentrations (0.3e1%) resulted in a decrease in the initial pH of the skim milk (Table 1), caused the pH to decrease faster than the control and also caused a decrease in the final pH of the milk gel from pH 4.58 in the control to pH 4.14 in gels
a
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2.3. Nuclear magnetic resonance (NMR)
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All NMR experiments were performed using an Oxford Instruments Maran Ultra spectrometer (Oxford Instruments, Tubney Woods, Oxfordshire, UK). The resonance frequency was 23 MHz. About 7 mL of each sample was placed in NMR glass sample tubes (18 mm diameter). Measurements of the milk and polyphenol mixture were taken both before and after acidification with GDL. After GDL addition (Section 2.1), samples were incubated at 30 C in a water bath and measurements were determined at 30 min time intervals during gelation. All measurements were taken at 30 C. T2 relaxation times were obtained using the Carr, Purcell, Meiboom and Gill (CPMG) pulse sequence with a relaxation delay of 5000 ms and a 90e180 pulse gap of 1.0 ms. The 90 pulse was 7.7 ms and 180 pulse was 15.4 ms in length. A total of 256 points were acquired for each of the sixteen scans. T2 distributions were calculated as a continuous distribution of exponentials using WinDXP software (Version 1.8.1, Oxford Instruments).
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2.4. Syneresis
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Time (min)
Syneresis was determined using low-speed centrifugation (Keogh & O’Kennedy, 1998). After the addition of GDL w20 mL of each sample were poured into centrifuge tubes (25 90 mm)
Fig. 1. Change in pH with time after GDL addition to skim milk containing (a) 0 (C) and 1 (B) % (w/w) tannic acid, and (b) 0 (C), 0.3 (-), 0.5 (6), 0.8 (;), and 1 (B) % (w/w) gallic acid. Data presented are means of three replicates.
N. Harbourne et al. / International Dairy Journal 21 (2011) 185e191
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Table 1 Gelation properties of skim milk samples with added phenolic compounds acidified with GDL. Acid gel samplea Gelation time (min) Initial pH of skim milk Final pH of acid milk gels after 16 h Final G0 at 30 C (Pa)b Final G0 at 5 C (Pa)b Syneresis index (%)b Control 0.1% TA 0.5% TA 1% TA 0.3% GA 0.5% GA 0.8% GA 1% GA a
91 81 57 18 31 19 <10 <10
5 10 6 6 4 2
6.50 6.48 6.20 6.14 5.63 5.50 5.32 5.25
0.10 0.07 0.10 0.10 0.03 0.10 0.07 0.02
4.58 4.54 4.40 4.54 4.46 4.37 4.23 4.14
0.07 0.05 0.10 0.07 0.01 0.05 0.02 0.03
553 588 796 1929 609 633 624 56
68B 30B 34B 297A 48B 68B 57B 14C
1102 1756 4703 10411 1581 2124 2763 196
139D 25CD 562B 1429A 51CD 363CD 301C 48E
242A 19 2A 18 2A 19 2A 18 5A 21 7A 21 7A 58 4B
TA, tannic acid; GA, gallic acid. Data presented are means of three replicates the standard deviation. Means not sharing the same letter differ at p 0.05.
b
containing 1% gallic acid (Fig. 1b; Table 1). The difference between the effect of tannic and gallic acid on pH of the milk gels is possibly because gallic acid (pka 4.41; Wang, Chen, De Hu, & Ju, 2003) is a more acidic compound than tannic acid (pka 7e8; Cruz, DiazCruz, Arino, & Esteban, 2000). 3.2. Gelation kinetics The effect of phenolic compound addition on the storage modulus (G0 ) as a function of time after the addition of GDL is presented in Fig. 2. For all samples, the G0 increased rapidly after gelation and then reached a plateau during ageing of the gel, at a pH of w4.6. Similar results for the gelation of acid milk gels have been reported (Anema, Lauber, Lee, Henle, & Klostermeyer, 2005; Lucey et al., 1997; Van Vliet, Roefs, Zoon, & Walstra, 1989). The addition of tannic acid had a significant effect on the gelation kinetics of skim milk (Fig. 2a). Gelation time decreased with increasing tannic acid concentration (Table 1), the addition of 1% tannic acid reduced
a 1000
G' (Pa)
100
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1
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Time (min)
b
the gelation time dramatically from 91 5 to 18 6 min. The final G0 at 30 C also increased with increasing tannic acid concentration (Table 1). There was a 3.5-fold increase in the G0 at a tannic acid concentration of 1% in comparison with the control from 553 68 to 1929 297 Pa. This indicates that there may be an interaction between tannic acid and the protein in the gel resulting in faster gelation and a higher final G0 (Fig. 2a; Table 1). Several recent publications have studied the covalent (Chen & Hagerman, 2004) and non-covalent (Charlton et al., 2002; Simon et al., 2003) interactions between polyphenols and proteins. Gallic acid addition also had a significant effect on the gelation of skim milk (Fig. 2b), causing a decrease in the gelation time (Table 1) possibly due to the decrease in the initial pH of the milk and a faster decrease of pH with time (Fig. 1b; Table 1). Peng, Horne, and Lucey (2009) found that yogurts made from milk with a lower preacidification pH had lower G0 values. This was not the case with gallic acid as its addition up to 0.8% had no significant effect on the final G0 at 30 C (Fig. 1b; Table 1). However, the inclusion of 1% gallic acid caused a significant decrease in the final G0 (56 14 Pa) of the milk gel possibly due to the pH of the milk dropping too quickly and not allowing for proper formation of the gel network. It has been previously noted that the rapid acidification of milk to pH 4.6 can result in aggregation of casein and the formation of large dense aggregates that precipitate rather than form a gel (Fox, Cogan, Guinee, & Mc Sweeney, 2000). There have been few studies on the effect of acidification time or rate on the rheological properties of milk gels acidified using GDL, and the results are varied. Anema (2008) found a decrease in the final G0 of acid milk gels as acidification time was decreased, whereas Horne (2003) found an increase in G0 of the acid milk gels as acidification time was decreased. To examine the nature of the bonds within the gel network a frequency sweep test was completed at the end of gelation. 3.3. Frequency sweep
1000
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Time (min)
Fig. 2. Storage modulus, G0 , as a function of time for acid milk gels containing (a) 0 (C), 0.1(-), 0.5 (6), and 1 (B) % (w/w) tannic acid, and (b) 0 (C), 0.3 (-), 0.5 (6), 0.8 (;), and 1 (B) % (w/w) gallic acid. Data presented are means of three replicates.
Logarithm plots of frequency against G0 produced straight lines (R2 0.98) (results not shown), the control gel and those containing gallic acid had slopes of approximately 0.15, which is in agreement with previous studies (Anema et al., 2005; Bikker, Anema, Li, & Hill, 2000; Lucey et al., 1997; Oh et al., 2007). The slope of the gels containing 0.1, 0.5 and 1% tannic acid increased to 0.17, 0.23 and 0.26 respectively, indicating possible rearrangement of the bonds within the acid gel network. To further our understanding of the nature of the bonds within the gel network, tan d (¼G00 /G0 ) was plotted as a function of frequency. Tan d values for the control gel, the gels made from skim milk containing gallic acid (0.3e1%) and 0.1% tannic acid were hardly affected by frequency (Fig. 3a,b). The gels containing 0.5 and 1% tannic acid had a similar tan d at the high frequencies studied, but at low frequencies the tan d was much higher for these gels.
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N. Harbourne et al. / International Dairy Journal 21 (2011) 185e191
3.4. Temperature sweep
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3.5. Nuclear magnetic resonance (NMR)
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The acidified milk gels underwent a temperature sweep from 30 to 5 C (Fig. 4). As the temperature of the acid milk gels was decreased, the G0 of the control gel increased 2-fold, which is in agreement with previous reports (Anema et al., 2005; Bikker et al., 2000; Lucey et al., 1997; Oh et al., 2007). This increase in G0 with a decrease in temperature may be due to swelling of casein particles resulting in an increase in the contact area between them (Bikker et al., 2000; Lucey et al., 1997), or an increase in the rigidity of the gel network and viscosity of the continuous phase (Anema et al., 2005; Oh et al., 2007). Fortifying skim milk with tannic acid had a significant effect on the rheology of acid milk gels when the temperature was decreased in comparison with the control (Fig. 4a; Table 1). The addition of 0.1% tannic acid caused a 3-fold increase in the G0 , and there was w5-fold increase in gels containing 0.5% or 1% tannic acid. Furthermore, the final G0 at 5 C increased linearly with increasing tannic acid concentration (R2 ¼ 0.99). Similarly, 0.3% or 0.5% gallic acid caused w3-fold increase in the G0 of the gels as the temperature decreased from 30 to 5 C and gels containing 0.8 or 1% gallic acid increased w4 fold (Fig. 4b). These results suggest that this dramatic increase in G0 with decreasing temperature is probably due to increased hydrogen bonding in the presence of phenolic compounds, as the strength of hydrogen bonds increase with decreasing temperature (Bellissent-Funnel & Teixeira, 2004), whereas hydrophobic interactions tend to decrease.
0.01
0.1
1
Frequency (Hz)
Fig. 3. Tan d as a function of frequency of acid milk gels containing (a) 0 (C),0.1(-), 0.5 (6) and 1 (B) % (w/w) tannic acid, and (b) 0 (C), 0.3 (-), 0.5 (6), 0.8 (;), and 1 (B) % (w/w) gallic acid. Data presented are means of three replicates.
To further investigate the interaction of the phenolic compounds within the acidified milk gel matrix, the mobility of the water was studied using NMR. During acidification of the skim milk
a
12000 10000
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This indicates that at lower frequencies the gels made with tannic acid become more viscous and liquid in nature, possibly due to tannic acid addition resulting in the formation of large aggregates within the network which may be connected by a greater number of weak interactions that can withstand low frequencies. Similarly, the addition of oxidized tannic acid to a surimi gel resulted in a gel with a compact structure which contained large aggregates in the matrix (Balange & Benjakul, 2009). In addition, previous studies have reported similar results for tan d in rennet casein gels and proposed that at longer time scales (low frequencies) the proteineprotein bonds had a more dynamic character, possibly due to differences in the type and form of the major interaction forces in and between the casein molecules (Van Vliet et al., 1989). As mentioned above, the ability of phenolic compounds to interact with proteins (especially proline-rich proteins including casein) is well established in the literature (Luck et al., 1994) and both hydrophobic interactions and hydrogen bonding appear to be the major driving forces between the polyphenoleprotein interaction. Furthermore, the final pH of the milk gels was close to the isoelectric point (pI) of the protein. It has been established that at the pI the proteinephenolic interaction is at a maximum (Siebert, 1999; Spencer et al., 1988). Therefore, the addition of tannic acid (0.5e1%) may cause an increase in either hydrophobic interactions or hydrogen bonding in the acid milk gels. So, to further understand the interactions involved between phenolic compounds and proteins in the acidified milk gel matrix, a temperature sweep was performed.
G' (Pa)
8000
1500 1000 500 0
5
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20
Temperature (oC) Fig. 4. Storage modulus, G0 , as a function of temperature for acid milk gels containing (a) 0 (C), 0.1(-), 0.5 (6), and 1 (B) % (w/w) tannic acid, and (b) 0 (C), 0.3 (-), 0.5 (6), 0.8 (;), and 1 (B) % (w/w) gallic acid. Data presented are means of three replicates.
N. Harbourne et al. / International Dairy Journal 21 (2011) 185e191 5000
Magnitude
4000
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0 1
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T2 (ms) Fig. 5. Distribution of T2 times in acidified milk gels after 5 h gelation: e e e, control; $$$$$$, 1% (w/w) gallic acid.
a
189
two mobile water phases T21 (0.1 mse100 ms) and T22 (100 mse1000 ms) were detectable by means of T2 relaxation times (Fig. 5), in agreement with Mok, Qi, Chen, and Ruan (2008) and Salomonsen et al. (2007). In these studies the T21 phase appeared immediately after GDL addition; however in the present study T21 appeared after w1 h of gelation. The T22 relaxation time of the control skim milk increased from 137 ms to 299 ms as the incubation time increased from 5 to 300 min, indicating the water became more mobile as the gel network developed. Similarly, Mok et al. (2008) found an increase in T2 times as yogurt gel network formed and Hinrichs, Bulca, and Kulozik (2007) reported an increase in the relaxation time of the mobile phase during the acid coagulation of casein solutions due to the evolving microstructure of these gels. The T22 times obtained for the control gel were similar to previous studies although the milk was not acidified in the same way; milks acidified with HCl were reported to have a T2 value of w250 ms (Roefs, van As, & van Vilet, 1989), a lower T2 value of w194 ms was reported for milk drinks acidified with bacteria (Salomonsen et al., 2007) and a T2 value of w250 ms was found for set yogurts (Mok et al., 2008).
350
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T22 (ms)
250
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50 0
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260 240 220 200 180 160 140 120 0
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Gelation time (min) Fig. 6. (a) The T22 times of acid milk gels containing (a) 0 (C), 0.1(-), 0.5 (6) and 1 (B) % tannic acid, and (b) 0 (C), 0.3 (-), 0.5 (6), 0.8 (;), and 1 (B) % (w/w) gallic acid during incubation at 30 C for 300 min. Data presented are means of three replicates.
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The addition of tannic acid (1%) to the skim milk before GDL addition resulted in an immediate decrease in the T22 time from 137.2 ms in reconstituted skim milk powder to 62.9 ms, indicating that the addition of tannic acid immediately caused the water within the skim milk to become less mobile. The T2 time of water in tannic acid (1%) solution was also determined to see if this decrease in water mobility was solely due to tannic acid binding water; however there was no significant difference between the T2 time of water and water in tannic acid solution. In comparison with the control gel the T22 times decreased with an increase in tannic acid concentration indicating that the mobility of the water in the gel matrix decreased with increasing tannic acid (Fig. 6a). This suggests that the water molecules became more tightly bound within the gel network with increasing tannic acid concentration. This increased water binding with an increase in tannic acid concentration is correlated with the increase in gel strength, indicated by an increase in G0 (Section 3.2). The addition of gallic acid resulted in higher T22 times after 5 min gelation in comparison with the control (Fig. 6b) indicating an increase in the water mobility, probably due to gallic acid causing a decrease in the gelation time of acid milk gels (Table 1). However, after 3 h gelation there was a decrease in the water mobility of these gels in comparison with the control. The addition of 1% gallic acid caused the biggest decrease in the water mobility; however it resulted in samples with a much broader distribution in comparison with the control (Fig. 5). Previous studies have suggested that a broader distribution of T2 corresponds to more inhomogeneous samples (Salomonsen et al., 2007). This was in agreement with the rheological properties of the gel containing 1% gallic acid, as the final G0 at 30 C was very low (56 14 Pa) indicating manufacture of a very weak gel.
Overall, the addition of tannic and gallic acid (0.8%) decreased the gelation time of acidified milk gels and caused an increase in the final G0 at 5 C. Also, the addition of these phenolic compounds had no adverse effects on the syneresis index of acidified milk gels. 4. Conclusions The addition of both simple phenols and hydrolysable tannins influenced the rheological properties and water mobility of acidified milk gels. Tannic acid caused an increase in the final G0 at 30 C, an increase in tan d at low frequencies, a w5-fold increase in G0 when the temperature dropped from 30 to 5 C and an increase of water binding within the gel matrix. This indicates that the addition of tannic acid to the gel probably resulted in the formation of stronger gels, with large aggregates within the network connected by a greater number of weak interactions (probably hydrogen bonding). The addition of gallic acid to acidified milk gels resulted in a drop in the initial pH of the milk prior to acidification, an increase in the rate of acidification and a decrease in the gelation pH. Despite the very fast acidification time the addition of gallic acid up to 0.8% had no significant effect on the final G0 at 30 C. Overall, the addition of both phenolic compounds resulted in faster gelation times, an increase in G0 with a decrease in temperature (probably due to increased hydrogen bonding in the presence of phenols) and an increase in water binding within the gel network. Acknowledgements This research was funded by a grant under the Food Institutional Research Measure, which is administered by the Department of Agriculture, Fisheries and Food, Ireland.
3.6. Syneresis References In this study low-speed centrifugation was used to assess the level of syneresis in acidified milk gels and the results are presented in Table 1. The addition of tannic acid (up to 1%) had no significant effect on the syneresis index of acidified milk gels (Table 1). As previously mentioned syneresis may be linked to large scale rearrangement of the gel; a high tan d indicates a likely increase in syneresis (Walstra, 1993). However, in the present study this does not seem to be the case. The addition of tannic acid to acidified milk gels resulted in rearrangement of the gel network (Fig. 3a) but did not result in an increase in syneresis. Balange and Benjakul (2009) reported that the addition of oxidized phenolic compounds (including tannic acid) to surimi gels resulted in enhanced crosslinking of proteins and the formation of gels with greater water holding capacity. Unlike this study, the addition of tannic acid in milk gels did not cause a significant decrease in SI; however, neither did it cause an increase even though it caused an increase in tan d that is linked to an increase in syneresis. Similarly, the addition of up to 0.8% gallic acid did not have a significant effect on the syneresis index of acidified milk gels (Table 1). However, an increase in gallic acid to 1% caused a significant increase in the syneresis index of the acidified milk gel from 24 2 in the control gel to 58 5%. It has been previously reported that the rapid acidification of milk may cause an increase in whey separation of acid milk gels (Lucey, Munro, & Singh, 1998). Therefore, the increase in the syneresis index and decrease in G0 of the gel containing 1% gallic acid may be due to the rapid decrease in pH. This is in agreement with the NMR and rheological results, as samples containing 1% gallic acid exhibited a much wider T2 distribution (Fig. 5) than the other samples indicating inhomogeneous milk gels and had a much lower G0 indicating a weaker gel.
Anema, S. G. (2008). Effect of temperature and rate of acidification on the rheological properties of acid skim milk gels. Journal of Food Processing and Preservation, 32, 1016e1033. Anema, S. G., Lauber, S., Lee, S. K., Henle, T., & Klostermeyer, H. (2005). Rheological properties of acid gels prepared from pressure and transglutaminase-treated skim milk. Food Hydrocolloids, 19, 879e887. Ares, G., Barreiro, C., Deliza, R., & Gambaro, A. (2009). Alternatives to reduce the bitterness, astringency and characteristic flavour of antioxidant extracts. Food Research International, 42, 871e878. Axten, L. G., Wohlers, M. W., & Wegrzyn, T. (2008). Using phytochemicals to enhance health benefits of milk: impact of polyphenols on flavour profile. Journal of Food Science, 73, H122eH126. Balange, A., & Benjakul, S. (2009). Enhancement of gel strength of bigeye snapper (Priacanthus tayenus) surimi using oxidized phenolic compounds. Food Chemistry, 113, 61e70. Bellissent-Funnel, M. C., & Teixeira, J. (2004). Structural and dynamic properties of bulk and confined water. In L. Rey, & J. C. May (Eds.), Freeze-drying/lyophilization of pharmaceutical and biological products (2nd ed, pp. 33e62). New York, NY, USA: Informa Health Care. Bikker, J. F., Anema, S. G., Li, Y., & Hill, P. J. (2000). Rheological properties of acid gels prepared from heated milk fortified with whey protein mixtures containing the A, B and C variants of b-lactoglobulin. International Dairy Journal, 10, 723e732. Bravo, L. (1998). Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutrition Reviews, 56, 317e333. Cai, Y., Luo, Q., Sun, M., & Corkes, H. (2004). Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sciences, 74, 2157e2184. Charlton, A., Baxter, N. J., Lokman Khan, M., Moir, A. J. G., Haslam, E., Davies, A. P., et al. (2002). Polyphenol/peptide binding and precipitation. Journal of Agriculture and Food Chemistry, 50, 1593e1601. Chen, Y. M., & Hagerman, A. E. (2004). Characterization of soluble non-covalent complexes between bovine serum albumin and beta-1,2,3,4,6-penta-o-galloyld-glucopyranose by MALDI-TOF MS. Journal of Agriculture and Food Chemistry, 52, 4008e4011. Cruz, B. H., Diaz-Cruz, J. M., Arino, C., & Esteban, M. (2000). Heavy metal binding by tannic acid: a voltammetric study. Electroanalysis, 12, 1130e1137. Fox, P. F., Cogan, T. M., Guinee, T. P., & Mc Sweeney, P. L. H. (2000). Principles of acid gel formation. In P. F. Fox, P. L. H. Mc Sweeney, T. M. Cogan, & T. P. Guinee (Eds.),
N. Harbourne et al. / International Dairy Journal 21 (2011) 185e191 Fundamentals of cheese science (pp. 364e368). Maryland, MO, USA: Aspen Publishers. Hinrichs, R., Bulca, S., & Kulozik, U. (2007). Water mobility during renneting and acid coagulation of casein solutions: a differentiated low-resolution nuclear magnetic resonance analysis. International Journal of Dairy Technology, 60, 37e43. Horne, D. S. (2003). Casein micelles as hard spheres: limitations of the model in acidified gel formation. Colloids and Surfaces A, 213, 255e263. 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, 108e112. Liu, H., Qiu, N., Ding, H., & Yao, R. (2008). Polyphenols contents and antioxidant capacity of 68 Chinese herbals suitable for medical or food uses. Food Research International, 41, 363e370. Lucey, J. A., Munro, P. A., & Singh, H. (1998). Whey separation in acid skim milk gels made with glucono-d-lactone: effects of heat treatment and gelation temperature. Journal of Texture Studies, 29, 413e426. Lucey, J. A., & Singh, H. (1998). Formation and physical properties of acid milk gels: a review. Food Research International, 30, 529e542. 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, 591e600. Luck, G., Liao, H., Murray, N. J., Grimmer, H. R., Warminski, E. E., Williamson, M. P., et al. (1994). Polyphenols, astringency and proline-rich proteins. Phytochemistry, 37, 357e371. Matia-Merino, L., & Singh, H. (2007). Acid-induced gelation of milk protein concentrates with added pectin: effect of casein micelle dissociation. Food Hydrocolloids, 21, 765e775. Mok, C., Qi, J., Chen, P., & Ruan, R. (2008). NMR relaxometry of water in set yogurt during fermentation. Food Science and Biotechnology, 17, 895e898. O’Connell, J. E., & Fox, P. F. (2001). Significance and applications of phenolic compounds in the production and quality of milk and dairy products: a review. International Dairy Journal, 11, 103e120.
191
Oh, H. E., Anema, S. G., Wong, M., Pinder, D. N., & Hemar, Y. (2007). Effect of potato starch addition on the acid gelation of milk. International Dairy Journal, 17, 808e815. Peng, Y., Horne, D. S., & Lucey, J. A. (2009). Impact of preacidification of milk and fermentation time on the properties of yogurt. Journal of Dairy Science, 92, 2977e2990. Roefs, S. P. F. M., van As, H., & van Vliet, T. (1989). Pulse NMR of casein dispersions. Journal of Food Science, 54, 704e708. Salomonsen, T., Sejersen, M. T., Viereck, N., Ipsen, R., & Engelsen, S. B. (2007). Water mobility in acidified milk drinks studied by low-field 1H NMR. International Dairy Journal, 17, 294e301. Siebert, K. J. (1999). Effects of proteinepolyphenol interactions on beverage haze, stabilization, and analysis. Journal of Agriculture and Food Chemistry, 47, 353e362. Simon, C., Barathieu, K., Laguerre, M., Schmitter, J. M., Fouquet, E., Pianet, I., et al. (2003). Three-dimensional structure and dynamics of wine tanninesaliva protein complexes. A multitechnique approach. Biochemistry, 42, 10385e10395. Spencer, C. M., Cai, Y., Martin, R., Gaffney, S. H., Goulding, P. N., Magnolato, D., et al. (1988). Polyphenol complexation: some thoughts and observations. Phytochemistry, 27, 2397e2409. Van Vliet, T., Lakemond, C. M. M., & Visschers, R. W. (2004). Rheology and structure of milk protein gels. Current Opinion in Colloid and Interface Science, 9, 298e304. Van Vliet, T., Roefs, S. P. F. M., Zoon, P., & Walstra, P. (1989). Rheological properties of casein gels. Journal of Dairy Research, 56, 529e534. Walstra, P. (1993). The syneresis of curd. In P. F. Fox, P. L. H. Mc Sweeney, T. M. Cogan, & T. P. Guinee (Eds.), Cheese: chemistry, physics, and microbiology (2nd ed, pp. 141e191). London, UK: Chapman and Hall. Wang, S. F., Chen, X. G., De Hu, Z., & Ju, J. (2003). Analysis of three effective components in Fructus corni and its preparations by micellar electrokinetic capillary chromatography. Biomedical Chromatography, 17, 306e311. Wegrzyn, T. F., Farr, J. M., Hunter, D. C., Au, J., Wohlers, M. W., Skinner, M. A., et al. (2008). Stability of antioxidants in an apple polyphenolemilk model system. Food Chemistry, 109, 310e318.
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Effects of addition of phenolic compounds on the acid gelation of milk Niamh Harbourne, Jean Christophe Jacquier, Dolores O’Riordan* School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 April 2010 Received in revised form 13 September 2010 Accepted 3 October 2010
Tannic acid (0.1e1%, w/w) and gallic acid (0.3e1%, w/w) were added to skim milk prior to acidification with GDL. The acid gelation of tannic and gallic acid fortified milk had a faster gelation time in comparison with the control gel without phenolic compounds. The addition of tannic acid and gallic acid (up to 0.8%) to the milk resulted in a higher storage modulus (G0 ), decrease in the water mobility (T2 time) and had no significant effect on the syneresis index (SI). However, the inclusion of 1% gallic acid resulted in a significant decrease in G0 , a significant increase in the SI and a wider T2 distribution. Lowering the temperature of the gels from 30 to 5 C caused the G0 for the gels with gallic and tannic acid to increase significantly in comparison with the control, possibly due to increased hydrogen bonding in the presence of phenolic compounds. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Phenolic compounds are secondary plant metabolites that have received much attention in the last few years due to their many potential health-benefits. They have antioxidant and anti-inflammatory properties and may protect against degenerative diseases such as cancer and cardiovascular disease (Bravo, 1998). There are numerous phenolic compounds ranging from the low molecular weight simple phenols to highly polymerised tannins. Phenolic compounds are ubiquitous in all plants, and recent studies have shown that medicinal plants contain a very high level of these polyphenols (Cai, Luo, Sun, & Corkes, 2004; Liu, Qiu, Ding, & Yao, 2008). In recent years, there has been an increase in the addition of phenolic compounds to dairy products to improve both their technological functionality and nutritional value (O’Connell & Fox, 2001). Some recent studies have looked at the effect of the addition of fruit extracts, rich in phenolic compounds, on the antioxidant (Wegrzyn et al., 2008) and sensory (Axten, Wohlers, & Wegrzyn, 2008) attributes of milk drinks. Phenolic compounds may affect the functional properties of dairy products as they readily interact with proteins; these interactions are well documented in the literature (Luck et al., 1994; Spencer et al., 1988). It is recognised that proline-rich proteins (e.g., casein) associate strongly to polyphenols and the major interactions that are thought to be involved in this association are hydrogen bonding and
* Corresponding author. Tel.: þ353 (1) 716 7016; fax: þ353 (1) 716 1149. E-mail address: [email protected] (D. O’Riordan). 0958-6946/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2010.10.003
hydrophobic interactions (Luck et al., 1994). These interactions are dependent on protein type and on the molecular size and flexibility of the polyphenol (Spencer et al., 1988). This proteinepolyphenol interaction is also dependent on pH and is at a maximum when proteins are at their isoelectric point (Spencer et al., 1988). The rheological properties of milk acidified using gluconodeltalactone (GDL) have been well characterised (Lucey & Singh, 1998; Van Vliet, Lakemond, & Visschers, 2004). More recently, the water mobility in acidified milk drinks has been studied using nuclear magnetic resonance (NMR) and it has been used to detect textural problems such as whey separation (Salomonsen, Sejersen, Viereck, Ipsen, & Engelsen, 2007). The rheological properties and water mobility of these gels may be modified by fortification with either dairy or non-dairy ingredients, such as starch (Oh, Anema, Wong, Pinder, & Hemar, 2007) or pectin (Matia-Merino & Singh, 2007; Salomonsen et al., 2007). Phenolic compounds or extracts rich in phenolic compounds may be ideal ingredients to add to milk gels as they could improve the nutritional value and may also enhance the rheological properties, but to date there has been little work published on the addition of phenolic compounds to acidified milk gels. Phenolic compounds are bitter and astringent in nature making them difficult to incorporate into food products; however, recent research has shown that milk may significantly decrease the bitterness and astringency of phenolic rich extracts (Ares, Barreiro, Deliza, & Gambaro, 2009). The objective of the present study was to determine the effect of simple phenols (gallic acid) and hydrolysable tannins (tannic acid) on the rheological properties, changes in water mobility and syneresis of acid milk gels.
N. Harbourne et al. / International Dairy Journal 21 (2011) 185e191
2. Materials and methods 2.1. Acid milk gel preparation Reconstituted skim milk was prepared by adding low-heat skim milk powder (SMP) (Arrabawn Co-op, Nenagh, Co. Tipperary, Ireland) to distilled water to give a final concentration of 12% (w/w) SMP. The solutions were stirred on a stirring plate for at least 2 h at room temperature and 0.01% (w/w) potassium sorbate (Hoechst A.G, Frankfurt am Main, Frankfurt, Germany) was added as a preservative. The reconstituted skim milk was heated in a water bath at 85 C for 30 min and after heating was rapidly cooled by immersion in ice-water. Tannic acid (Sigma Aldrich, St. Louis, MO, USA) (0.1, 0.5 and 1%, w/w) and gallic acid (Sigma Aldrich) (0.3, 0.5, 0.8 and 1%, w/w) were then added to the milk and stirred for 30 min using a stirring plate at room temperature. GDL (1.3%, w/w) (Sigma Aldrich) was then added and the sample was stirred for 2 min. The acidified milk was then incubated in a water bath at 30 C for 16 h. A control gel that contained no phenolic compounds was prepared in a similar fashion. The changes in pH during gelation after the addition of GDL were monitored continuously for 16 h by means of a pH meter fitted with a glass combination electrode. 2.2. Acid gel formation and rheological measurements A Bohlin Gemini HRnano rheometer (Malvern Instruments Ltd, Worcestershire, UK) was used for the rheological measurements using a cup (27 mm) and bob (25 mm) system (Bohlin C-25, Malvern Instruments Ltd), with a solvent trap to prevent evaporation of the sample. Samples were transferred to the rheometer after addition and mixing of GDL (as above). To monitor the gelation process an applied strain of 1%, frequency of 0.1 Hz and a constant temperature of 30 C were used. Measurements were taken every 10 min for 16 h. Gelation time was defined as the point when gels had a G0 1 Pa (Lucey, Teo, Munro, & Singh, 1997). A frequency sweep from 0.001 to 1 Hz was performed on the final gel. Finally, the gel was subjected to a temperature sweep. The temperature of the sample was dropped from 30 to 5 C at a rate of 1 C min1 at a frequency of 0.1 Hz and strain of 1%.
(Sarstedt, Numbrecht, Germany) of known weight. The samples were incubated at 30 C in a thermostatically controlled water bath. After 16 h the tubes were centrifuged (Beckman J2-HS, Beckman Instruments Inc., Palo Alto, CA, USA) at 300 g for 10 min at 4 C. The supernatant was then removed carefully and weighed. The syneresis index (SI) was expressed as percentage weight of supernatant over the initial weight of the gel. 2.5. Statistical analysis All samples were prepared in triplicate and results in the text are given as mean values and standard deviations. A one-way analysis of variance (ANOVA) and Tukey’s pairwise comparisons were used to determine significant differences between the various treatments. SAS 9.1.3 was used for all analyses (SAS Institute, Cary, NC, USA). Results with p 0.05 were considered significantly different. 3. Results and discussion 3.1. Effect of addition of phenolic compounds on pH The effect of phenolic compound addition on the evolution of pH during the acidification of milk is shown in Fig. 1. The inclusion of tannic acid to the skim milk did not have a significant effect on the change of pH with time after GDL addition (Fig. 1a). The final pH was 4.58 for the control gel (no added tannic or gallic acid) and pH 4.54 for those containing 1% tannic acid (Table 1). The addition of gallic acid at all concentrations (0.3e1%) resulted in a decrease in the initial pH of the skim milk (Table 1), caused the pH to decrease faster than the control and also caused a decrease in the final pH of the milk gel from pH 4.58 in the control to pH 4.14 in gels
a
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6.5
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2.3. Nuclear magnetic resonance (NMR)
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All NMR experiments were performed using an Oxford Instruments Maran Ultra spectrometer (Oxford Instruments, Tubney Woods, Oxfordshire, UK). The resonance frequency was 23 MHz. About 7 mL of each sample was placed in NMR glass sample tubes (18 mm diameter). Measurements of the milk and polyphenol mixture were taken both before and after acidification with GDL. After GDL addition (Section 2.1), samples were incubated at 30 C in a water bath and measurements were determined at 30 min time intervals during gelation. All measurements were taken at 30 C. T2 relaxation times were obtained using the Carr, Purcell, Meiboom and Gill (CPMG) pulse sequence with a relaxation delay of 5000 ms and a 90e180 pulse gap of 1.0 ms. The 90 pulse was 7.7 ms and 180 pulse was 15.4 ms in length. A total of 256 points were acquired for each of the sixteen scans. T2 distributions were calculated as a continuous distribution of exponentials using WinDXP software (Version 1.8.1, Oxford Instruments).
4.5
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2.4. Syneresis
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Syneresis was determined using low-speed centrifugation (Keogh & O’Kennedy, 1998). After the addition of GDL w20 mL of each sample were poured into centrifuge tubes (25 90 mm)
Fig. 1. Change in pH with time after GDL addition to skim milk containing (a) 0 (C) and 1 (B) % (w/w) tannic acid, and (b) 0 (C), 0.3 (-), 0.5 (6), 0.8 (;), and 1 (B) % (w/w) gallic acid. Data presented are means of three replicates.
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Table 1 Gelation properties of skim milk samples with added phenolic compounds acidified with GDL. Acid gel samplea Gelation time (min) Initial pH of skim milk Final pH of acid milk gels after 16 h Final G0 at 30 C (Pa)b Final G0 at 5 C (Pa)b Syneresis index (%)b Control 0.1% TA 0.5% TA 1% TA 0.3% GA 0.5% GA 0.8% GA 1% GA a
91 81 57 18 31 19 <10 <10
5 10 6 6 4 2
6.50 6.48 6.20 6.14 5.63 5.50 5.32 5.25
0.10 0.07 0.10 0.10 0.03 0.10 0.07 0.02
4.58 4.54 4.40 4.54 4.46 4.37 4.23 4.14
0.07 0.05 0.10 0.07 0.01 0.05 0.02 0.03
553 588 796 1929 609 633 624 56
68B 30B 34B 297A 48B 68B 57B 14C
1102 1756 4703 10411 1581 2124 2763 196
139D 25CD 562B 1429A 51CD 363CD 301C 48E
242A 19 2A 18 2A 19 2A 18 5A 21 7A 21 7A 58 4B
TA, tannic acid; GA, gallic acid. Data presented are means of three replicates the standard deviation. Means not sharing the same letter differ at p 0.05.
b
containing 1% gallic acid (Fig. 1b; Table 1). The difference between the effect of tannic and gallic acid on pH of the milk gels is possibly because gallic acid (pka 4.41; Wang, Chen, De Hu, & Ju, 2003) is a more acidic compound than tannic acid (pka 7e8; Cruz, DiazCruz, Arino, & Esteban, 2000). 3.2. Gelation kinetics The effect of phenolic compound addition on the storage modulus (G0 ) as a function of time after the addition of GDL is presented in Fig. 2. For all samples, the G0 increased rapidly after gelation and then reached a plateau during ageing of the gel, at a pH of w4.6. Similar results for the gelation of acid milk gels have been reported (Anema, Lauber, Lee, Henle, & Klostermeyer, 2005; Lucey et al., 1997; Van Vliet, Roefs, Zoon, & Walstra, 1989). The addition of tannic acid had a significant effect on the gelation kinetics of skim milk (Fig. 2a). Gelation time decreased with increasing tannic acid concentration (Table 1), the addition of 1% tannic acid reduced
a 1000
G' (Pa)
100
10
1
0.1
0.01
0.001 0
200
400
600
800
Time (min)
b
the gelation time dramatically from 91 5 to 18 6 min. The final G0 at 30 C also increased with increasing tannic acid concentration (Table 1). There was a 3.5-fold increase in the G0 at a tannic acid concentration of 1% in comparison with the control from 553 68 to 1929 297 Pa. This indicates that there may be an interaction between tannic acid and the protein in the gel resulting in faster gelation and a higher final G0 (Fig. 2a; Table 1). Several recent publications have studied the covalent (Chen & Hagerman, 2004) and non-covalent (Charlton et al., 2002; Simon et al., 2003) interactions between polyphenols and proteins. Gallic acid addition also had a significant effect on the gelation of skim milk (Fig. 2b), causing a decrease in the gelation time (Table 1) possibly due to the decrease in the initial pH of the milk and a faster decrease of pH with time (Fig. 1b; Table 1). Peng, Horne, and Lucey (2009) found that yogurts made from milk with a lower preacidification pH had lower G0 values. This was not the case with gallic acid as its addition up to 0.8% had no significant effect on the final G0 at 30 C (Fig. 1b; Table 1). However, the inclusion of 1% gallic acid caused a significant decrease in the final G0 (56 14 Pa) of the milk gel possibly due to the pH of the milk dropping too quickly and not allowing for proper formation of the gel network. It has been previously noted that the rapid acidification of milk to pH 4.6 can result in aggregation of casein and the formation of large dense aggregates that precipitate rather than form a gel (Fox, Cogan, Guinee, & Mc Sweeney, 2000). There have been few studies on the effect of acidification time or rate on the rheological properties of milk gels acidified using GDL, and the results are varied. Anema (2008) found a decrease in the final G0 of acid milk gels as acidification time was decreased, whereas Horne (2003) found an increase in G0 of the acid milk gels as acidification time was decreased. To examine the nature of the bonds within the gel network a frequency sweep test was completed at the end of gelation. 3.3. Frequency sweep
1000
G' (Pa)
100
10
1
0.1
0.01
0.001 0
200
400
600
800
Time (min)
Fig. 2. Storage modulus, G0 , as a function of time for acid milk gels containing (a) 0 (C), 0.1(-), 0.5 (6), and 1 (B) % (w/w) tannic acid, and (b) 0 (C), 0.3 (-), 0.5 (6), 0.8 (;), and 1 (B) % (w/w) gallic acid. Data presented are means of three replicates.
Logarithm plots of frequency against G0 produced straight lines (R2 0.98) (results not shown), the control gel and those containing gallic acid had slopes of approximately 0.15, which is in agreement with previous studies (Anema et al., 2005; Bikker, Anema, Li, & Hill, 2000; Lucey et al., 1997; Oh et al., 2007). The slope of the gels containing 0.1, 0.5 and 1% tannic acid increased to 0.17, 0.23 and 0.26 respectively, indicating possible rearrangement of the bonds within the acid gel network. To further our understanding of the nature of the bonds within the gel network, tan d (¼G00 /G0 ) was plotted as a function of frequency. Tan d values for the control gel, the gels made from skim milk containing gallic acid (0.3e1%) and 0.1% tannic acid were hardly affected by frequency (Fig. 3a,b). The gels containing 0.5 and 1% tannic acid had a similar tan d at the high frequencies studied, but at low frequencies the tan d was much higher for these gels.
188
a
N. Harbourne et al. / International Dairy Journal 21 (2011) 185e191
3.4. Temperature sweep
0.8
0.7
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3.5. Nuclear magnetic resonance (NMR)
0.15
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The acidified milk gels underwent a temperature sweep from 30 to 5 C (Fig. 4). As the temperature of the acid milk gels was decreased, the G0 of the control gel increased 2-fold, which is in agreement with previous reports (Anema et al., 2005; Bikker et al., 2000; Lucey et al., 1997; Oh et al., 2007). This increase in G0 with a decrease in temperature may be due to swelling of casein particles resulting in an increase in the contact area between them (Bikker et al., 2000; Lucey et al., 1997), or an increase in the rigidity of the gel network and viscosity of the continuous phase (Anema et al., 2005; Oh et al., 2007). Fortifying skim milk with tannic acid had a significant effect on the rheology of acid milk gels when the temperature was decreased in comparison with the control (Fig. 4a; Table 1). The addition of 0.1% tannic acid caused a 3-fold increase in the G0 , and there was w5-fold increase in gels containing 0.5% or 1% tannic acid. Furthermore, the final G0 at 5 C increased linearly with increasing tannic acid concentration (R2 ¼ 0.99). Similarly, 0.3% or 0.5% gallic acid caused w3-fold increase in the G0 of the gels as the temperature decreased from 30 to 5 C and gels containing 0.8 or 1% gallic acid increased w4 fold (Fig. 4b). These results suggest that this dramatic increase in G0 with decreasing temperature is probably due to increased hydrogen bonding in the presence of phenolic compounds, as the strength of hydrogen bonds increase with decreasing temperature (Bellissent-Funnel & Teixeira, 2004), whereas hydrophobic interactions tend to decrease.
0.01
0.1
1
Frequency (Hz)
Fig. 3. Tan d as a function of frequency of acid milk gels containing (a) 0 (C),0.1(-), 0.5 (6) and 1 (B) % (w/w) tannic acid, and (b) 0 (C), 0.3 (-), 0.5 (6), 0.8 (;), and 1 (B) % (w/w) gallic acid. Data presented are means of three replicates.
To further investigate the interaction of the phenolic compounds within the acidified milk gel matrix, the mobility of the water was studied using NMR. During acidification of the skim milk
a
12000 10000
6000 4000 2000 0 5
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This indicates that at lower frequencies the gels made with tannic acid become more viscous and liquid in nature, possibly due to tannic acid addition resulting in the formation of large aggregates within the network which may be connected by a greater number of weak interactions that can withstand low frequencies. Similarly, the addition of oxidized tannic acid to a surimi gel resulted in a gel with a compact structure which contained large aggregates in the matrix (Balange & Benjakul, 2009). In addition, previous studies have reported similar results for tan d in rennet casein gels and proposed that at longer time scales (low frequencies) the proteineprotein bonds had a more dynamic character, possibly due to differences in the type and form of the major interaction forces in and between the casein molecules (Van Vliet et al., 1989). As mentioned above, the ability of phenolic compounds to interact with proteins (especially proline-rich proteins including casein) is well established in the literature (Luck et al., 1994) and both hydrophobic interactions and hydrogen bonding appear to be the major driving forces between the polyphenoleprotein interaction. Furthermore, the final pH of the milk gels was close to the isoelectric point (pI) of the protein. It has been established that at the pI the proteinephenolic interaction is at a maximum (Siebert, 1999; Spencer et al., 1988). Therefore, the addition of tannic acid (0.5e1%) may cause an increase in either hydrophobic interactions or hydrogen bonding in the acid milk gels. So, to further understand the interactions involved between phenolic compounds and proteins in the acidified milk gel matrix, a temperature sweep was performed.
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Temperature (oC) Fig. 4. Storage modulus, G0 , as a function of temperature for acid milk gels containing (a) 0 (C), 0.1(-), 0.5 (6), and 1 (B) % (w/w) tannic acid, and (b) 0 (C), 0.3 (-), 0.5 (6), 0.8 (;), and 1 (B) % (w/w) gallic acid. Data presented are means of three replicates.
N. Harbourne et al. / International Dairy Journal 21 (2011) 185e191 5000
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T2 (ms) Fig. 5. Distribution of T2 times in acidified milk gels after 5 h gelation: e e e, control; $$$$$$, 1% (w/w) gallic acid.
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two mobile water phases T21 (0.1 mse100 ms) and T22 (100 mse1000 ms) were detectable by means of T2 relaxation times (Fig. 5), in agreement with Mok, Qi, Chen, and Ruan (2008) and Salomonsen et al. (2007). In these studies the T21 phase appeared immediately after GDL addition; however in the present study T21 appeared after w1 h of gelation. The T22 relaxation time of the control skim milk increased from 137 ms to 299 ms as the incubation time increased from 5 to 300 min, indicating the water became more mobile as the gel network developed. Similarly, Mok et al. (2008) found an increase in T2 times as yogurt gel network formed and Hinrichs, Bulca, and Kulozik (2007) reported an increase in the relaxation time of the mobile phase during the acid coagulation of casein solutions due to the evolving microstructure of these gels. The T22 times obtained for the control gel were similar to previous studies although the milk was not acidified in the same way; milks acidified with HCl were reported to have a T2 value of w250 ms (Roefs, van As, & van Vilet, 1989), a lower T2 value of w194 ms was reported for milk drinks acidified with bacteria (Salomonsen et al., 2007) and a T2 value of w250 ms was found for set yogurts (Mok et al., 2008).
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Gelation time (min) Fig. 6. (a) The T22 times of acid milk gels containing (a) 0 (C), 0.1(-), 0.5 (6) and 1 (B) % tannic acid, and (b) 0 (C), 0.3 (-), 0.5 (6), 0.8 (;), and 1 (B) % (w/w) gallic acid during incubation at 30 C for 300 min. Data presented are means of three replicates.
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The addition of tannic acid (1%) to the skim milk before GDL addition resulted in an immediate decrease in the T22 time from 137.2 ms in reconstituted skim milk powder to 62.9 ms, indicating that the addition of tannic acid immediately caused the water within the skim milk to become less mobile. The T2 time of water in tannic acid (1%) solution was also determined to see if this decrease in water mobility was solely due to tannic acid binding water; however there was no significant difference between the T2 time of water and water in tannic acid solution. In comparison with the control gel the T22 times decreased with an increase in tannic acid concentration indicating that the mobility of the water in the gel matrix decreased with increasing tannic acid (Fig. 6a). This suggests that the water molecules became more tightly bound within the gel network with increasing tannic acid concentration. This increased water binding with an increase in tannic acid concentration is correlated with the increase in gel strength, indicated by an increase in G0 (Section 3.2). The addition of gallic acid resulted in higher T22 times after 5 min gelation in comparison with the control (Fig. 6b) indicating an increase in the water mobility, probably due to gallic acid causing a decrease in the gelation time of acid milk gels (Table 1). However, after 3 h gelation there was a decrease in the water mobility of these gels in comparison with the control. The addition of 1% gallic acid caused the biggest decrease in the water mobility; however it resulted in samples with a much broader distribution in comparison with the control (Fig. 5). Previous studies have suggested that a broader distribution of T2 corresponds to more inhomogeneous samples (Salomonsen et al., 2007). This was in agreement with the rheological properties of the gel containing 1% gallic acid, as the final G0 at 30 C was very low (56 14 Pa) indicating manufacture of a very weak gel.
Overall, the addition of tannic and gallic acid (0.8%) decreased the gelation time of acidified milk gels and caused an increase in the final G0 at 5 C. Also, the addition of these phenolic compounds had no adverse effects on the syneresis index of acidified milk gels. 4. Conclusions The addition of both simple phenols and hydrolysable tannins influenced the rheological properties and water mobility of acidified milk gels. Tannic acid caused an increase in the final G0 at 30 C, an increase in tan d at low frequencies, a w5-fold increase in G0 when the temperature dropped from 30 to 5 C and an increase of water binding within the gel matrix. This indicates that the addition of tannic acid to the gel probably resulted in the formation of stronger gels, with large aggregates within the network connected by a greater number of weak interactions (probably hydrogen bonding). The addition of gallic acid to acidified milk gels resulted in a drop in the initial pH of the milk prior to acidification, an increase in the rate of acidification and a decrease in the gelation pH. Despite the very fast acidification time the addition of gallic acid up to 0.8% had no significant effect on the final G0 at 30 C. Overall, the addition of both phenolic compounds resulted in faster gelation times, an increase in G0 with a decrease in temperature (probably due to increased hydrogen bonding in the presence of phenols) and an increase in water binding within the gel network. Acknowledgements This research was funded by a grant under the Food Institutional Research Measure, which is administered by the Department of Agriculture, Fisheries and Food, Ireland.
3.6. Syneresis References In this study low-speed centrifugation was used to assess the level of syneresis in acidified milk gels and the results are presented in Table 1. The addition of tannic acid (up to 1%) had no significant effect on the syneresis index of acidified milk gels (Table 1). As previously mentioned syneresis may be linked to large scale rearrangement of the gel; a high tan d indicates a likely increase in syneresis (Walstra, 1993). However, in the present study this does not seem to be the case. The addition of tannic acid to acidified milk gels resulted in rearrangement of the gel network (Fig. 3a) but did not result in an increase in syneresis. Balange and Benjakul (2009) reported that the addition of oxidized phenolic compounds (including tannic acid) to surimi gels resulted in enhanced crosslinking of proteins and the formation of gels with greater water holding capacity. Unlike this study, the addition of tannic acid in milk gels did not cause a significant decrease in SI; however, neither did it cause an increase even though it caused an increase in tan d that is linked to an increase in syneresis. Similarly, the addition of up to 0.8% gallic acid did not have a significant effect on the syneresis index of acidified milk gels (Table 1). However, an increase in gallic acid to 1% caused a significant increase in the syneresis index of the acidified milk gel from 24 2 in the control gel to 58 5%. It has been previously reported that the rapid acidification of milk may cause an increase in whey separation of acid milk gels (Lucey, Munro, & Singh, 1998). Therefore, the increase in the syneresis index and decrease in G0 of the gel containing 1% gallic acid may be due to the rapid decrease in pH. This is in agreement with the NMR and rheological results, as samples containing 1% gallic acid exhibited a much wider T2 distribution (Fig. 5) than the other samples indicating inhomogeneous milk gels and had a much lower G0 indicating a weaker gel.
Anema, S. G. (2008). Effect of temperature and rate of acidification on the rheological properties of acid skim milk gels. Journal of Food Processing and Preservation, 32, 1016e1033. Anema, S. G., Lauber, S., Lee, S. K., Henle, T., & Klostermeyer, H. (2005). Rheological properties of acid gels prepared from pressure and transglutaminase-treated skim milk. Food Hydrocolloids, 19, 879e887. Ares, G., Barreiro, C., Deliza, R., & Gambaro, A. (2009). Alternatives to reduce the bitterness, astringency and characteristic flavour of antioxidant extracts. Food Research International, 42, 871e878. Axten, L. G., Wohlers, M. W., & Wegrzyn, T. (2008). Using phytochemicals to enhance health benefits of milk: impact of polyphenols on flavour profile. Journal of Food Science, 73, H122eH126. Balange, A., & Benjakul, S. (2009). Enhancement of gel strength of bigeye snapper (Priacanthus tayenus) surimi using oxidized phenolic compounds. Food Chemistry, 113, 61e70. Bellissent-Funnel, M. C., & Teixeira, J. (2004). Structural and dynamic properties of bulk and confined water. In L. Rey, & J. C. May (Eds.), Freeze-drying/lyophilization of pharmaceutical and biological products (2nd ed, pp. 33e62). New York, NY, USA: Informa Health Care. Bikker, J. F., Anema, S. G., Li, Y., & Hill, P. J. (2000). Rheological properties of acid gels prepared from heated milk fortified with whey protein mixtures containing the A, B and C variants of b-lactoglobulin. International Dairy Journal, 10, 723e732. Bravo, L. (1998). Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutrition Reviews, 56, 317e333. Cai, Y., Luo, Q., Sun, M., & Corkes, H. (2004). Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sciences, 74, 2157e2184. Charlton, A., Baxter, N. J., Lokman Khan, M., Moir, A. J. G., Haslam, E., Davies, A. P., et al. (2002). Polyphenol/peptide binding and precipitation. Journal of Agriculture and Food Chemistry, 50, 1593e1601. Chen, Y. M., & Hagerman, A. E. (2004). Characterization of soluble non-covalent complexes between bovine serum albumin and beta-1,2,3,4,6-penta-o-galloyld-glucopyranose by MALDI-TOF MS. Journal of Agriculture and Food Chemistry, 52, 4008e4011. Cruz, B. H., Diaz-Cruz, J. M., Arino, C., & Esteban, M. (2000). Heavy metal binding by tannic acid: a voltammetric study. Electroanalysis, 12, 1130e1137. Fox, P. F., Cogan, T. M., Guinee, T. P., & Mc Sweeney, P. L. H. (2000). Principles of acid gel formation. In P. F. Fox, P. L. H. Mc Sweeney, T. M. Cogan, & T. P. Guinee (Eds.),
N. Harbourne et al. / International Dairy Journal 21 (2011) 185e191 Fundamentals of cheese science (pp. 364e368). Maryland, MO, USA: Aspen Publishers. Hinrichs, R., Bulca, S., & Kulozik, U. (2007). Water mobility during renneting and acid coagulation of casein solutions: a differentiated low-resolution nuclear magnetic resonance analysis. International Journal of Dairy Technology, 60, 37e43. Horne, D. S. (2003). Casein micelles as hard spheres: limitations of the model in acidified gel formation. Colloids and Surfaces A, 213, 255e263. 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, 108e112. Liu, H., Qiu, N., Ding, H., & Yao, R. (2008). Polyphenols contents and antioxidant capacity of 68 Chinese herbals suitable for medical or food uses. Food Research International, 41, 363e370. Lucey, J. A., Munro, P. A., & Singh, H. (1998). Whey separation in acid skim milk gels made with glucono-d-lactone: effects of heat treatment and gelation temperature. Journal of Texture Studies, 29, 413e426. Lucey, J. A., & Singh, H. (1998). Formation and physical properties of acid milk gels: a review. Food Research International, 30, 529e542. 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, 591e600. Luck, G., Liao, H., Murray, N. J., Grimmer, H. R., Warminski, E. E., Williamson, M. P., et al. (1994). Polyphenols, astringency and proline-rich proteins. Phytochemistry, 37, 357e371. Matia-Merino, L., & Singh, H. (2007). Acid-induced gelation of milk protein concentrates with added pectin: effect of casein micelle dissociation. Food Hydrocolloids, 21, 765e775. Mok, C., Qi, J., Chen, P., & Ruan, R. (2008). NMR relaxometry of water in set yogurt during fermentation. Food Science and Biotechnology, 17, 895e898. O’Connell, J. E., & Fox, P. F. (2001). Significance and applications of phenolic compounds in the production and quality of milk and dairy products: a review. International Dairy Journal, 11, 103e120.
191
Oh, H. E., Anema, S. G., Wong, M., Pinder, D. N., & Hemar, Y. (2007). Effect of potato starch addition on the acid gelation of milk. International Dairy Journal, 17, 808e815. Peng, Y., Horne, D. S., & Lucey, J. A. (2009). Impact of preacidification of milk and fermentation time on the properties of yogurt. Journal of Dairy Science, 92, 2977e2990. Roefs, S. P. F. M., van As, H., & van Vliet, T. (1989). Pulse NMR of casein dispersions. Journal of Food Science, 54, 704e708. Salomonsen, T., Sejersen, M. T., Viereck, N., Ipsen, R., & Engelsen, S. B. (2007). Water mobility in acidified milk drinks studied by low-field 1H NMR. International Dairy Journal, 17, 294e301. Siebert, K. J. (1999). Effects of proteinepolyphenol interactions on beverage haze, stabilization, and analysis. Journal of Agriculture and Food Chemistry, 47, 353e362. Simon, C., Barathieu, K., Laguerre, M., Schmitter, J. M., Fouquet, E., Pianet, I., et al. (2003). Three-dimensional structure and dynamics of wine tanninesaliva protein complexes. A multitechnique approach. Biochemistry, 42, 10385e10395. Spencer, C. M., Cai, Y., Martin, R., Gaffney, S. H., Goulding, P. N., Magnolato, D., et al. (1988). Polyphenol complexation: some thoughts and observations. Phytochemistry, 27, 2397e2409. Van Vliet, T., Lakemond, C. M. M., & Visschers, R. W. (2004). Rheology and structure of milk protein gels. Current Opinion in Colloid and Interface Science, 9, 298e304. Van Vliet, T., Roefs, S. P. F. M., Zoon, P., & Walstra, P. (1989). Rheological properties of casein gels. Journal of Dairy Research, 56, 529e534. Walstra, P. (1993). The syneresis of curd. In P. F. Fox, P. L. H. Mc Sweeney, T. M. Cogan, & T. P. Guinee (Eds.), Cheese: chemistry, physics, and microbiology (2nd ed, pp. 141e191). London, UK: Chapman and Hall. Wang, S. F., Chen, X. G., De Hu, Z., & Ju, J. (2003). Analysis of three effective components in Fructus corni and its preparations by micellar electrokinetic capillary chromatography. Biomedical Chromatography, 17, 306e311. Wegrzyn, T. F., Farr, J. M., Hunter, D. C., Au, J., Wohlers, M. W., Skinner, M. A., et al. (2008). Stability of antioxidants in an apple polyphenolemilk model system. Food Chemistry, 109, 310e318.