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Proposed mechanism for the effect of polyphenols on the heat stability of milk
International Dairy Journal 9 (1999) 523}536
Proposed mechanism for the e!ect of polyphenols on the heat stability of milk J.E. O'Connell, P.F. Fox* Department of Food Chemistry, University College, Cork, Ireland Received 12 March 1999; accepted 29 June 1999
Abstract Ca!eic acid at 5.5 mmol l\ markedly enhanced the heat stability of milk at 1403C, the minimum was eliminated and stability increased as a function of pH; the apparent activation energy for coagulation was increased. The stabilizing e!ect of ca!eic acid was reduced or eliminated when milk was heated at 1503C or when the air in the headspace was replaced by N . The stabilising e!ect of ca!eic acid was reduced when stability was assessed as the temperature required to cause instantaneous coagulation, i.e., a maximum and minimum were evident. The thermal degradation/transformation of ca!eic acid in milk was a zero-order reaction with an activation energy of &40 kJ mol\ and Q value of &1.3 from 130}1403C; when the air in the headspace was replaced by N , thermal degradation/transformation of ca!eic acid was reduced. The addition of ca!eic acid resulted in a reduction of the reactive lysine and sulphydryl content and inhibited the dissociation of i-casein-rich protein from the casein micelles in milk on heating. It is postulated that on heating in milk, ca!eic acid is thermally oxidised to quinones which then interact with nucleophilic amino acid residues to inhibit i-casein dissociation from the casein micelle. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Milk; Heat stability; Polyphenols; Ca!eic acid
1. Introduction Polyphenols are common constituents in plants, in which they probably exist in conjugated and/or glycosylated forms. They are probably produced as secondary metabolites and act as natural detergents to grazing animals and as natural antimicrobial agents (Haslam & Lilley, 1988; Haslam, 1998). Recently, polyphenols have received attention for their ability to inhibit the oxidation of human low-density lipoproteins, to enhance the stability of b-lactoglobulin foams and to extend the shelf life of processed meat products by inhibiting oxidative rancidity (Sarker, Wilde & Clark, 1995; Landau & Yang, 1997; Chung, Wei & Johnson, 1998; Meyer, Heinoen & Frankel, 1998). The ability of polyphenol-rich extracts from a variety of plant sources, e.g., tea, co!ee, cocoa, wine, aloe vera and oak leaves and bark, and also individual polyphenols, e.g., ferulic acid, ca!eic acid, ellagic acid and epigallocatechingallate, to enhance the heat stability of skimmed milk
and concentrated milk has been reported (O'Connell, Fox, Tan-Kintia & Fox, 1998; O'Connell & Fox, 1999a, b). Ca!eic acid [3-(3,4-dihyroxyphenyl) 2-propenic acid, also referred to as 3,4-dihydroxycinnamic acid], the most e!ective polyphenol examined, markedly increased heat stability at 5.5 mmol l\. It was suggested (O'Connell et al., 1998; O'Connell & Fox, 1999a) that the stabilising e!ect of polyphenols on the heat stability of milk is due partially to calcium chelation. This communication reports on other possible mechanisms by which ca!eic acid may increase the heat stability of milk. 2. Materials and methods All chemicals used were reagent grade. The dialysis tubing used (code No. T88-T108) was obtained from Medicell International (London N1 1LX, UK). The whey protein isolate used was obtained from BIPRO (Davisco Foods Int. Inc, Le Sueur, MN 56058). 2.1. Milk supply
* Corresponding author. Tel.: #353-21-902362; fax: #353-21270001. E-mail address: [email protected] (P.F. Fox)
Raw milk, obtained from a local dairy, was defatted by centrifugation at 2000 g for 20 min at 203C, followed by
0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 1 2 4 - 7
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"ltration through glass wool to remove fat particles. The skimmed milk was stored at 43C until required (not more than 3 days).
2.2. Ewect of caweic acid on some physicochemical properties of milk The e!ect of ca!eic acid on the heat stability (as determined by the heat coagulation time-pH pro"le, HCTpH), alcohol stability, rennet coagulation and the calcium ion concentration of milk were determined by the methods described by O'Connell et al. (1998). The heat coagulation temperature-pH (HCTemp) pro"le of skimmed milk was determined by the method of Miller and Sommer (1940), with the temperature required to cause coagulation in 4 min (including a come-up time of 2 min) being recorded as the heat coagulation temperature (HCTemp) (Miller & Sommer, 1940).
2.5. Determination of total protein and N-acetyl neuraminic acid (NANA) The total protein and 12% trichloroacetic acid-insoluble NANA content of ultracentrifugal supernatants (90,000 g for 1 h at 203C) from milk at pH values in the range of 6.5}7.3 and heated at 903C for 10 min, with and without the addition of ca!eic acid to 5.5 mmol l\, were determined by the macro-Kjeldhal (AOAC, 1975) and thiobarbituric acid (Warren, 1959) methods, respectively. 2.6. Determination of zeta potential The zeta potential of control and experimental milks at pH 6.7, diluted 1 : 250 with SMUF (Jenness & Koops, 1962), was determined using a Malvern Zetamaster instrument (Malvern Instruments Ltd, Malvern, UK) at an applied voltage of 120 V and a modulation frequency of 250 Hz. The instrument was calibrated using a standard provided by Malvern Instruments.
2.3. Preparation of whey protein-free milk Whey protein-free milk was prepared by dispersing casein micelles, obtained by ultracentrifugation (90,000 g for 1 h at 203C), in synthetic milk ultra"ltrate without lactose (SMUF; Jenness & Koops, 1962), followed by exhaustive dialysis against bulk milk (100 ml against &2;4 l of bulk milk) for 48 h at 43C to allow for the addition of lactose and urea to levels similar to that in milk.
2.7. Measurement of casein micelle size The size distribution of casein micelles in control and experimental milks, heated at pH 6.7, was determined by photon correlation spectroscopy using a Malvern Zetamaster instrument. Milks were diluted with SMUF (3.4 ll milk ml\), "ltered through Whatman No. 40 "lter paper (Whatman International, Maidstone, UK) and analysed in triplicate.
2.4. Determination of available lysine, sulphydryl groups and hydroxymethyl furfural (HMF) content
2.8. Measurement of crosslinking of milk proteins on heating
Available sulphydryl groups and HMF levels in control and experimental milks were determined by the methods of Patrick and Swaisgood (1976) and Fink and Kessler (1986), respectively. Reactive lysine was determined by the method of Yaylayan, Huyghues-Despointes and Polydorides (1992): 0.10 ml of milk were diluted with 4 ml of 0.2 M potassium tetraborate and 1 ml of 0.1 M EDTA. One ml of #uorescamine (10 mg ml\ dissolved in acetone) was added and rapidly mixed. Fluorescence was measured at an excitation wavelength of 390 nm and an emission wavelength of 475 nm and reactive lysine was determined from a standard curve of lysine versus #uorescence.
Na-caseinate solutions (3.0%, w/v, in SMUF) at pH 6.7, with or without ca!eic acid at 5.5 mmol l\, were heated at 1303C for up to 35 min. The solutions were cooled and diluted 30 : 1 with H O and the absorbance at 440 nm determined. 2.9. Determination of relative viscosity The relative viscosity of control and experimental skimmed milks was determined at room temperature using an Ostwald U-tube viscometer (size C BS/U, Technico, Gallenkamp, Leicestershire, LE11 OTR, England) and expressed as relative viscosity using the equation:
flow time of sample;specific gravity of milk;specific gravity of H O Relative viscosity" flow time of H O
J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536
2.10. Determination of caweic acid concentration in heated milk The concentration of ca!eic acid in heated milk was determined by RP-HPLC. Milks (containing 5.5 mmol l\ca!eic acid) were ultra"ltrated using Centriprep centrifugal concentrators with a nominal cut-o! value of 10 kDa (Amicon Inc, Beverly, MA, USA). The ultra"ltrate was diluted 1 : 5 with millipore water (Millipore Corp, Milford, USA) and 20 ll were injected onto a Nucleosil C RP-HPLC column (4.5;250 mm, 5 mm, 300 As pore size). RP-HPLC was performed using a Waters HPLC system (Waters, Millipore Corp, Milford, USA), consisting of a Waters 717 autosampler, a Waters 600 programmable pump and a Varian/Nainin UV detector (Varian Associates, Walnut Creek, CA, USA) set at 280 nm interfaced to a Dell Dimension PC running on Waters Millennium 2.10 software. The solvent used was methanol : H O : formic acid (20 : 79.7 : 0.3, v/v/v) at a #ow rate of 1 ml min\. 3. Results 3.1. Ewect of caweic acid on the heat stability of milk under diwerent experimental conditions As shown in Fig. 1a, ca!eic acid at 5.5 mmol l\ markedly enhanced the heat stability of milk. The apparent activation energy (E ) for the heat-induced coagulation of milk, with or without ca!eic acid at 5.5 mmol l\, was 174 and 142 at pH 6.7 and 198 and 140 kJ mol\ at pH 6.9, respectively (calculated from data in Table 1 as described by Parker & Dalgleish, 1977). The Q values for heat-induced coagulation was una!ected by the addition of ca!eic acid at all temperature ranges except for temperatures from 140}1503C, the signi"cance of which is discussed later. The stabilising e!ect of ca!eic acid was markedly reduced when the assay temperature was increased to 1503C and a minimum was reintroduced, i.e., a type A HCT-pH pro"le was evident (Fig. 1b). The presence of a shallow minimum in milk containing ca!eic acid was also apparent in the HCTemp-pH pro"le (Fig. 1c). Heat stability as determined by the HCTemp-pH pro"le is, essentially, a measure of the temperature required to cause instantaneous coagulation (Miller & Sommer, 1940). The method was developed to study coagulation under conditions where heat-induced changes, such as thermal oxidation of lactose, dephosphorylation and covalent polymerization, did not play a signi"cant role. The presence of a minimum in the HCTemp-pH pro"le of milk containing ca!eic acid indicates that the stabilizing mechanism of ca!eic acid involves some type of heat-induced reaction, and that at 1503C, coagulation occurs before ca!eic acid can undergo this reaction and exert its stabilizing e!ect.
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The e!ect of ca!eic acid was eliminated if air in the headspace of the assay tubes was replaced by N (Fig. 1d). When an aqueous solution of ca!eic acid (5.5 mmol l\) was heated at 1403C for 15 min, lyophilized and the product added to milk at a weight basis equivalent to 5.5 mmol l\ ca!eic acid, it increased the heat stability of milk, regardless of the composition of the headspace (Fig. 1e). To investigate whether ca!eic acid interacts with milk proteins (either whey proteins or casein), 100 ml of milk containing 5.5 mmol l\ ca!eic acid were dialysed against 2;4 l of bulk milk for 48 h at 43C and the HCT-pH pro"le determined (Fig. 2a). The stabilising e!ect of ca!eic was lost on dialysis, presumably due to the di!usion of ca!eic acid into the bulk milk. When skimmed milk (50 ml) was dialysed against 500 ml milk containing 5.5 mmol l\ ca!eic acid, it assumed a HCTpH pro"le similar to that of milk containing ca!eic acid (Fig. 2b). These results suggest that most of the ca!eic acid in the experimental milk was dialysable and presumably had not interacted with the milk proteins at 43C. It should be noted that hydrophobic bonding, which has been proposed to be the principal binding force between proteins and polyphenols, is very weak at the temperature at which dialysis was carried out (&43C). The concentration of ca!eic acid in heated milk (1403C for 0}25 min) was determined in order to ascertain whether it interacted with some milk constituent or was degraded when heated at the assay temperature. The concentration of ca!eic acid in ultra"ltrate from milk containing 5.5 mmol l\ decreased from 5.36 to 2.31 mmol l\ on heating for 25 min (Table 2). Degradation/transformation of ca!eic acid in milk was a zero order reaction with respect to time and had an activation energy of&40 kJ mol\ and a Q value of 1.3 from 130 to 1403C. Degradation/transformation of ca!eic acid was reduced if air in the headspace was replaced by N (Table 2). When considered in conjunction with the data in Fig. 1d, it would appear that the heat-induced degradation/transformation of ca!eic acid is a prerequisite for its stabilising e!ect and that oxygen is required for the degradation/transformation of ca!eic acid. The low heat dependence for the degradation/transformation of ca!eic acid (E &40 KJ mol\ and Q value of&1.3) compared to heat-induced coagulation (E &140 KJ mol\ and Q value of &2}3) may explain why the e!ect of ca!eic acid on the heat stability of milk is less marked at 150 than at 1403C, i.e., at 1503C coagulation occurs before su$ciently enough ca!eic acid has been degraded/transformed to the active stabilizing agent. 3.2. Ewect of caweic acid on the calcium ion concentration of milk Polyphenols are capable of chelating metals (Radhakrishnan & Sivaprasad, 1980; McDonald, Mila &
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J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536
Fig. 1. E!ect of ca!eic acid at 5.5 mmol l\ or heat-trated ca!eic acid at 0.1%, w/v, on the HCT-pH pro"le of milk; control (air headspace) (}䢇}), control (N headspace) (}*}), with ca!eic acid (air headspace) (}䊏}), with ca!eic acid (N headspace) (}䊐}), heat-treated ca!eic acid (air headspace) (}䉱}), heat-treated ca!eic acid (N headspace) (}䉭}).
Scalbert, 1996). Ca!eic acid at 5.5 mmol l\ reduced the concentration of Ca> in unheated milk from 3.25 to 2.55 mM as determined using a calcium selective electrode (Table 3). Readjustment of the calcium ion concentration to the value of the control by adding 1.0 M CaCl
reduced, but did not eliminate, the stabilising e!ect of ca!eic acid (Fig. 3), suggesting that chelation of calcium is only partially responsible for the stabilising e!ect of ca!eic acid on the heat stability of milk. This conclusion is supported by the fact that addition of 5.5 mmol l\
J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536
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Table 1 E!ect of ca!eic acid at 5.5 mmol l\ on the heat coagulation times and Q values of milk at di!erent temperatures System
Q for coagulation at temperature
Heat coagulation time, min at 1253C
1303C
Control, 60.0$3.5 39.1$4.5 pH 6.7 Control, 21.4$5.4 12.1$1.2 pH 6.9 With ca!eic 112.8$3.0 69.1$1.6 acid, pH 6.7 With ca!eic 127$6.5 77.1$2.1 acid, pH 6.9
1353C
1403C
1453C
1503C
125}1353C
130}1403C
135}1453C
140}1503C
25.2$4.2
15.6$2.2
10.2$1.1
5.7$0.9
2.38
2.51
2.47
2.74
7.3$2.3
3.7$1.1
2.5$0.8
1.9$0.2
2.93
3.27
2.92
1.95
47.2$0.7
31.0$1.5
17.6$2.3
3.4$0.5
2.39
2.23
2.68
9.12
60.1$3.2
35.4$2.6
14.8$1.2
2.5$0.7
2.12
2.18
4.06
14.16
Table 2 Concentration of ca!eic acid in ultra"ltration permeates from milk containing 5.5 mmol l\ ca!eic acid and heated at 1403C with air or nitrogen in the headspace (mean$SD, n"3) Heating, min at 1403C
Concentration (mM)
0 5 10 15 20 25
Air
Nitrogen
5.36$0.11 4.58$0.60 4.22$0.34 3.81$0.41 3.08$0.19 2.31$0.54
5.36$0.11 5.24$0.06 5.17$0.09 4.93$0.24 N.D. N.D.
N.D.: not determined.
ca!eic acid had little e!ect on the ethanol stability or rennet coagulation of milk, both of which are strongly in#uenced by [Ca>] (Table 3).
& Jenness, 1984; van Boekel, Nieuwenhuijse & Walstra, 1989), is partially responsible for the heat-induced coagulation of milk at pH values outside the minimum, due to covalent polymerization. Ca!eic acid reduced the concentration of HMF, which is an index of Maillard reaction, formed in milk on heating, but only after 12 min of heating (Fig. 4). Interaction of ca!eic acid with lysine residues may also increase heat stability in a manner analogous to formaldehyde, i.e., by preventing the dissociation of i-caseinrich protein from the casein micelles by cross-linking proteins (Holt, Muir & Sweetsur, 1978; Singh & Fox, 1985). The ability of ca!eic acid to crosslink milk proteins on heating was determined by measuring the turbidity of diluted Na caseinate solutions at 440 nm; turbidity increased (Fig. 5a) markedly in the presence of ca!eic acid, suggesting that the ability of ca!eic acid to increase heat stability may be due to crosslinking of proteins. Ca!eic acid also increased casein micelle size upon heating (Fig. 5b), which may be due to crosslinking.
3.3. Ewect of caweic acid on the reactive lysine residues content of milk
3.4. Ewect of caweic acid on the sulphydryl content of milk
Ca!eic acid reduced the lysine content of heated milk by&10%; again, replacement of air in the headspace by N eliminated this e!ect (Table 4). Blocking of e-amino groups of lysine may be important in inhibiting the Maillard reaction, which, it has been suggested (Walstra
Addition of ca!eic acid (5.5 mmol l\) reduced the sulphydryl content of a heated milk (953C for 10 min), Table 4 [note: milk was heated at 953C for 10 min and not at assay temperature because at the latter temperature the decrease of cysteine is so rapid that it is not
Table 3 E!ect of ca!eic acid (5.5 mmol l\) on some properties of skimmed milk at 213C (with the exception of rennet coagulation time (RCT) which was determined at 313C). (mean$SD, n"3) System
Control With ca!eic acid
[Ca>], mM
3.25$0.22 2.55$0.13
RCT, min
6.98$0.92 6.75$0.25
Milk was heated at 1303C for 10 min.
Ethanol stability, pH
Relative viscosity
Zeta potential, mV
6.5
7.0
Unheated
Heated
Unheated
Heated
65.0$0 67.5$0
99.18$1.44 98.33$1.44
1.60 1.69
1.70 1.73
!15.6$0.5 !15.2$0.5
!18.7$1.2 !18.7$0.7
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J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536
Fig. 2. E!ect of dialysis of (a) skimmed milk containing ca!eic acid at 5.5 mmol l\ against bulk on its HCT-pH pro"le; control (}䢇}), dialysed control (}*}), with ca!eic acid (}䊏}), with ca!eic acid and dialysed (}䊐}); (b) of skimmed milk agianst an excess milk containing ca!eic acid at 5.5 mmol l\, control (}䉱}), with ca!eic acid (}䉬}), control milk dialysed against milk containing 5.5 mmol l\ ca!eic acid (}䉭}).
possible to analyse]. Loss of cysteine residues was not a!ected by the composition of the headspace (Table 4). A decrease of cysteine content of milk proteins, by chemical oxidation, markedly enhanced the heat stability of milk (Singh & Fox, 1987). Decreasing of the sulphydryl content inhibits or disrupts the formation of whey protein-i-casein complexes and consequently the dissociation of i-casein-rich protein from the casein micelles at pH values'pH 6.8 is inhibited (Singh & Fox, 1987). The
ability of ca!eic acid to inhibit heat-induced dissociation of protein and i-casein was shown by measuring the protein content and N-acetyl neuraminic acid content of ultracentrifugal supernatants of heated milk with and without the addition of ca!eic acid (90,000 g for 1 h at 203C) (Fig. 6). The possibility that ca!eic acid eliminates the minimum in the HCT-pH pro"le of milk by interacting with the cysteine group of b-lactoglobulin and other whey
J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536
529
Fig. 3. E!ect of ca!eic acid at 5.5 mmol l\ on the HCT-pH pro"le of skimmed milk, with and without supplementation with CaCl ; control (}䢇}), with ca!eic acid (}䊏}), with ca!eic acid and calcium activity readjusted to level of control (}䊐}).
Fig. 4. E!ect of ca!eic acid at 5.5 mmol l\ on the concentration of hydroxymethylfurfural in heated milk; control (}䢇}), with ca!eic acid (}䊏}).
proteins is supported by the fact that addition of whey protein isolate (WPI) to ca!eic acid-containing milk (5.5 mmol l\) reintroduced the minimum in the HCTpH pro"le (Fig. 7a). However, it should be noted that preheat treatment at 903C for 10 min prior to addition of ca!eic acid did not eliminate the stabilising in#uence of ca!eic acid (Fig. 8). Pre-heating under such conditions
should result in complex formation between b-lactoglobulin and i-casein and consequently if the exclusive stabilising mechanism of ca!eic acid was by inhibition of complex formation via reduction of suphydryl groups it would be expected to have little e!ect on preheated milk (Fig. 8). Also, the addition of ca!eic acid to whey protein-free milk enhanced stability (Fig. 7b).
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Fig. 5. E!ect of ca!eic acid 0 (}䢇}), 5.5 (}*}), 11.0 (}䊏}) or 22.0 (}䊐}) mmol l\ on (a) turbidity of Na-caseinate solution and (b) casein micelle size in milk.
3.5. Ewect of caweic acid derivatives and related compounds on the heat stability of milk The molecular reactivity of polyphenols is generally attributed to the presence of hydroxyl groups (Spencer et al., 1988; Haslam & Lilley, 1988). Indeed, replacement of the hydroxyl groups of ca!eic acid by methoxy groups, i.e., 3,4-dimethoxycinnamic acid, eliminated the stabilizing e!ect of ca!eic acid (Fig. 9). In an attempt to determine which speci"c functional group(s) may be responsible for the ability of ca!eic acid to enhance the heat
stability of milk, the e!ects of derivatives of ca!eic acid and related compounds on the heat stability of milk were examined (Fig. 9). Benzene had no e!ect on the heat stability. Phenol (monohydroxybenzene) had no e!ect on the heat stability of milk, nor did the monohydroxyl derivative of ca!eic acid, coumaric acid, regardless of the isomer. Introduction of a second hydroxyl group on the benzene ring a!ected the HCT-pH pro"le of skimmed milk but this e!ect was dependent on the position of the second hydroxyl group. Catechol (1,2-benzenediol) and
J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536 Table 4 E!ect of ca!eic acid at 5.5 mmol l\ on the free cysteine and lysine content of milk with and without heating (953C for 10 min at pH 6.7, with air or nitrogen in the headspace). (mean$SD, n"3) System
Cysteine (mM)
Lysine (mM)
Control, unheated Control, heated (air) Control, heated (N ) With ca!eic acid, unheated With ca!eic acid, heated (air) With ca!eic acid, heated (N )
0.012$0.003 0.032$0.004 0.032$0.003 0.008$0.002 0.012$0.001 0.013$0.002
26.52$0.66 26.13$0.80 25.48$0.78 25.27$0.26 23.62$0.63 26.12$1.26
hydroquinone (1,4-benzenediol) converted a type A milk to a type B milk, while resorcinol (1,3-benzenediol) had little e!ect on the heat stability of milk, suggesting that positioning of the hydroxyl groups at the ortho or para position is necessary for the stabilizing e!ect of polyphenols. Supporting this assertion, 1,2-naphthalene enhanced heat stability while 1,3-naphthalene had little e!ect. Saturation of the aromatic ring eliminated the stabilizing e!ect, e.g., the saturated derivative of catechol, 1,2-cyclohexanediol, did not a!ect the heat stability of milk. However, ca!eic acid had a much greater e!ect on the heat stability of milk than catechol, which destabilized milk in the region of the maximum. The stabilizing e!ect of ca!eic acid at the maximum in the HCT-pH pro"le may be attributed to the chain on C-1. This would appear
531
to be supported by the fact that ferulic acid, which contains the same C-1 moiety as ca!eic acid, stabilized milk in the region of the maximum but had a destabilizing e!ect at the pH of the minimum and on the alkaline side of the minimum, presumably because it does not possess a second hydroxyl group. This e!ect is analogous to other mono phenolic compounds containing a carboxylic acid moiety, e.g., vanillic acid (O'Connell & Fox, 1999b). The e!ect of these monophenolic compounds (ferulic acid and vanillic acid) was similar to that of other acids, e.g., tartaric acid and citric acid (Mohammad & Fox, 1983). Perhaps it is the carboxylic acid moiety of monophenols that stabilises milk in the region of the maximum by chelation of calcium. Modi"cation of the carboxylic acid moiety at C-1 of ca!eic acid had a profound e!ect on the ability of diphenols to increase the heat stability of milk. 3,4-Dihydroxypropiophenone or 3,4-dihydroxyphenyl-acetic acid did not eliminate the minimum in the HCT-pH pro"le. The double bond in the C-1 chain of ca!eic acid did not appear to be important, as hydroca!eic acid also stabilised milk. 3,4-Dihydroxybenzaldehyde, which differs from ca!eic acid in that it has an aldehyde group, rather than propenic acid, at C-1, markedly increased the heat stability of milk. However, the signi"cance of this result is unclear considering that aldehyde groups alone also increase the heat stability of milk (Holt et al., 1978; Singh & Fox, 1985). However, vanillin (4-hydroxy 3methoxy benzaldehyde) had no e!ect on the heat stability of milk.
Fig. 6. E!ect of pH on the level of protein and 12% TCA-insoluble NANA in the ultracentrifugal supernatant (90,000 g for 1 h at 203C) of heated milk (903C for 10 min) with and without 5.5 mmol l\ ca!eic acid; control: protein (}䊏}), experimental protein (2䊐2), control, NANA (}䢇}), experimental NANA (2*2).
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Fig. 7. E!ect of ca!eic acid at 5.5 mmol l\ on the HCT-pH pro"le of (a) skimmed milk with and without WPI supplementation at 1.0%, w/v; control (}䢇}), control with WPI (}*}), ca!eic acid (}䊏}), with ca!eic acid and WPI (}䊐}); (b) of whey-protein free milk; control (}䢇}), with ca!eic acid (}䉱}).
Quinic acid (1,3,4,5-tetrahydroxycyclohexanecarboxylic acid) at 0.1}0.4%, w/v, markedly destabilised milk throughout the pH range 6.3}7.3. The e!ect of tri or tetra hydroxycinnamic acids (i.e., ca!eic acid with additional hydroxyl groups) on the heat stability of milk would be of interest. Unfortunately, these compounds are not available. The e!ect of derivatives of ca!eic acid and related compounds on the heat stability of milk suggests that the presence of two hydroxyl groups at ortho or para positions and the aromatic ring are prerequisites. The pres-
ence of a carboxylic group in the C-1 also appears to be important. 3.6. Ewect of quinones on the heat stability of milk 1,4-Benzoquinone, the quinone derivative of hydroquinone (1,4-benzenediol) which converted a type A milk to a type B HCT-pH pro"le, stabilized skimmed milk (Fig. 9) regardless of the headspace in the test tube (air or N ). 1,2-Naphthoquinone, the quinone derivative of 1,2-naphthalene, also stabilized milk
J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536
533
Fig. 8. E!ect of preheating (903C for 10 min) on the heat coagulation time-pH pro"le of skimmed milk with and without ca!eic acid at 5.5 mmol l\; control (}䢇}), control (preheated) (}*}), with ca!eic acid (}䊏}), with ca!eic acid (preheated) (}䊐}).
(Fig. 9) regardless of the composition of the head space.
4. Discussion From the data presented, it appears that the e!ect of ca!eic acid on the HCT-pH pro"le of skimmed milk is not due exclusively to one mechanism; rather, it appears to be a multifactorial e!ect. Chelation of calcium appears to be involved. Two possible binding mechanisms of calcium by ca!eic acid are presented in Fig. 10 (reactions 1 and 2). Interaction of ca!eic acid with proteins also appears to be important in the stabilizing e!ect of ca!eic acid. The ability of polyphenols to interact with proteins is established (Asquith, Uhlig, Mehansho, Putman, Carlson & Butler, 1987; Haslam & Lilley, 1988). Most of the literature suggests that hydrophobic and hydrogen bonding are responsible for polyphenol}protein interactions (Spencer et al., 1988; Haslam & Lilley, 1988). However, the heat stability assay is carried out at 1403C, at which hydrophobic and hydrogen bonding are negligible. The ability of polyphenols to interact covalently with proteins has also been suggested (Meek & Weiss, 1979; Beart, Lilley & Haslam, 1985). Beart et al. (1985) proposed that under certain conditions, e.g., enzymatic oxidation, polyphenols are converted to quinones which may then react with nucleophilic amino acids, e.g., sulphydryl or amino
groups. Horigome and Kandatsu (1968) reported a 3}11% reduction in the lysine content of casein when incubated with ca!eic acid and polyphenol oxidase. It is conceivable that heating at high temperatures may cause thermal oxidation of ca!eic acid to quinones (Fig. 10, reaction 3). Pyrolysis (2283C for 15 min) of ca!eic acid resulted in the production of strong reducing compounds (Guillot, Malmoe & Stadler, 1996). Possible binding modes of quinones with amino groups are shown in Fig. 10 (reactions 4 and 5); the possible implications of such interactions on the heat stability of milk has been discussed. Loss of cysteine residues may be due to interaction between products of ca!eic acid oxidation and cysteine (Fig. 10, reactions 5 and 6) or reduction of quinones to ca!eic acid resulting in a conversion of cysteine to cystine (Fig. 10, reaction 7). Alternatively, loss of cysteine may be due to direct complexation with ca!eic acid, forming cysteinyl-ca!eic acid (Fig. 10, reaction 8; Cillers & Singleton, 1990). The signi"cance of the interaction of ca!eic acid with sulphydryl residues in relation to heat stability has already been discussed. The likelihood that thermal oxidation of ca!eic acid to quinones is responsible for the ability of ca!eic acid to increase the heat stability of milk is supported by the fact that replacement of air in the headspace by N , i.e., removal of oxygen, inhibited thermal oxidation of ca!eic acid (Table 2) and consequently eliminated the stabilising e!ect of ca!eic acid (Fig. 1d). It is also noteworthy that heat-treated ca!eic acid (Fig. 1e) and quinones stabilised
534
J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536
Fig. 9. E!ect of ca!eic acid and related compounds on the HCT-pH pro"le of skimmed milk (graphs are representative, i.e., not exact) (*) control, (- - -) with added phenolic compound.
milk, regardless of the composition of the headspace. The proposed mechanism, i.e., that heating converts ca!eic acid to quinone, may also explain why ca!eic acid stabilises milk while closely related compounds do not.
The fact that ca!eic acid, catechol, hydroquinone and 1,2-naphthalene exerted an e!ect on the heat stability of milk while resorcinol, 1,2-cyclohexanediol and 1,3naphthalene had little e!ect is likely to be due to the
J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536
535
Fig. 10. Possible molecular interactions of ca!eic acid and quinones formed upon oxidation with nucleophilic amino acids, lysine and cysteine.
inability of the latter group of compounds to oxidise to quinones. The e!ect of ca!eic acid on the heat stability of skimmed milk is of interest in that its addition markedly increases heat stability at relatively low concentrations, 5.5}20 mmol l\, without a!ecting other properties such as rennet coagulation time, alcohol stability, viscosity or zeta potential (Table 2). Secondly, since ca!eic acid is a naturally occurring constituent of plants and can be easily chemically synthesised (Horigome & Kandatzu, 1968), it may be considered as a &natural functional ingredient' in foods. Polyphenol-rich extracts of tea are currently being incorporated into sweet paste biscuits, milk drinks and chewing gum for nutritional reasons due to their attributed bene"cial health e!ects, e.g., anticarcinogenic, antimutagenic and antioxidative properties (Landau & Yang, 1997; Anon, 1997; Chung et al., 1998; Hennessy, 1998). The addition of phenolic compounds to meat products as antioxidants (Pratt & Hudson, 1990), to b-lactoglobulin foams as stabilizers (Sarker et al., 1995), to milk as antifungal agents (Rosenthal, Rosen & Bernstein, 1997) and to foods as inhibitors of browning reactions (Cillers & Singleton, 1990) has been advocated.
Acknowledgements This research was partly funded by grant aid under the Food Sub Programme for Industrial Development which is administered by the Department of Agriculture, Food and Forestry and supported by National and EU funds. References Anonymous (1997). The bene"ts of tea. Foods Ingredients and Analysis International, 16}17. AOAC (1975). O$cial methods of analysis (12th ed., p. 248). Washington DC: Association of O$cial Analytical Chemists. Asquith, T. N., Uhlig, T., Mehansho, H., Putman, L., Carlson, D. M., & Butler, L. (1987). Binding of condensed tannins to silivary proline-rich glycoproteins: the role of carbohydrate. Journal of Agricultural and Food Chemistry, 35, 331}334. Beart, J. E., Lilley, T. H., & Haslam, E. (1985). Polyphenol interactions. Part 2. Covalent binding of procyanidins to proteins during acidcatalysed decomposition; observations of some polymeric procyanidins. Journal of the Chemical Society, Perkin Transactions, 2, 1439}1443. Cillers, J. J. L., & Singleton, V. L. (1990). Ca!eic acid autoxidation and the e!ects of thiols. Journal of Agricultural and Food Chemistry, 38, 1789}1796.
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Chung, K. T., Wei, C. I., & Johnson, M. G. (1998). Are tannins a double edged sword in health and biology? Trends in Food Science and Technology, 9, 168}175. Fink, R., & Kessler, H. G. (1986). HMF levels in heat treated and stored milk. Milchwissenschaft, 41, 638}641. Guillot, F. L., Malmoe, A., & Stadler, R. H. (1996). Antioxidant properties of novel tetraoxygenated phenylinyan isomers formed during thermal decomposition of ca!eic acid. Journal of Agricultural and Food Chemisty, 44, 2503}2510. Haslam, E. (1998). In E. Haslam, Practical polyphenols: from structure to molecular recognition and physiological action. Cambridge: Cambridge University Press. Haslam, E., & Lilley, T. H. (1988). Natural astringency in foodstu!s*a molecular interpretation. CRC Critical Reviews in Food Science and Nutrition, 27, 1}40. Hennessey, A. J. (1998). Anyone for tea? Chemistry and Industry, 518}519. Holt, D., Muir, D. D., & Sweetsur, A. W. M. (1978). The heat stability of milk and concentrated milk containing added sugars and aldehydes. Journal of Dairy Research, 45, 47}52. Horigome, T., & Kandatsu, M. (1968). Biological value of proteins allowed to interact with phenolic compounds in the presence of o-diphenol oxidase. Agricultural and Biological Chemistry, 32, 1093}1102. Jenness, R., & Koops, J. (1962). Preparation and properties of salt solution which simulates milk ultra"ltrate. Netherlands Milk and Dairy Journal, 16, 154}164. Landau, J. M., & Yang C. S. (1997). The e!ect of tea on health. Chemistry and Industry, 903}906. McDonald, M., Mila, I., & Scalbert, A. (1996). Precipitation of metal ions by polyphenols: optimal conditions and origin of precipitation. Journal of Agricultural and Food Chemistry, 44, 599}606. Meek, K. M., & Weiss, J. B. (1979). Di!erential "xation of poly(Larginine) and poly(L-lysine) by tannic acid and its application to the "xation of collagen in electron microscopy. Biochimica and Biophysica Acta, 587, 112}120. Meyer, A. S., Heinoen, M., & Frankel, E. N. (1998). Antioxidant properties of catechin, cyanidin, ca!eic acid, quercetin and ellagic acid on human LDL oxidation. Food Chemistry, 61, 71}75. Miller, P. G., & Sommer, H. H. (1940). The coagulation temperature of milk as a!ected by pH, salts, evaporation and previous heat treatments. Journal of Dairy Science, 23, 405}421. Mohammad, K. S., & Fox, P. F. (1983). In#uence of some polyvalent acids and salts on the colloidal stability of milk. Journal of the Society of Dairy Technology, 36, 112}118. O'Connell, J. E., Fox, P. D., Tan-Kintia, R., & Fox, P. F. (1998). E!ect of tea, co!ee and cocoa extracts on the colloidal stability of milk and concentrated milk. International Dairy Journal, 8, 689}693.
O'Connell, J. E., & Fox, P. F. (1999a). E!ects of extracts of oak (Quercus petraea) bark, oak leaves, aloe vera (Curacao aloe), coconut shell and wine on the colloidal stability of milk and concentrated milk. Food Chemistry, 66, 93}96. O'Connell, J. E., & Fox. P. F. (1999b). E!ect of phenolic compounds on the heat stability of milk and concentrated milk. Journal of Dairy Research, in press. Parker, T. G., & Dalgleish, D. G. (1977). The theory of branching processes applied to milk proteins. I. Heat-induced coagulation of whole milk. Journal of Dairy Research, 44, 85}92. Patrick, P. S., & Swaisgood, H. E. (1976). Sulphydryl and disulphide groups of skim milk as a!ected by direct ultra-high-temperature. Journal of Dairy Science, 59, 594}600. Pratt, D. E., & Hudson, B. J. F. (1990). Natural antioxidants not exploited commercially. In B. J. F. Hudson, Food antioxidants (p. 171). London: Elsevier. Radhakrishnan, M. R., & Sivaprasad, J. (1980). Tannin content of sorgum varieties and their role in iron bioavailability. Journal of Agricultural and Food Chemistry, 28, 55}57. Rosenthal, I., Rosen, B., & Bernstein, S. (1997). Phenols in milk. Evaluation of ferulic acid and other phenols as antifungal agents. Milchwissenschaft, 52, 134}138. Sarker, D. K., Wilde, P. J., & Clark, D. C. (1995). Control of surfactantinduced destabilization of foams through polyphenol-mediated protein}protein interactions. Journal of Agricultural and Food Chemistry, 43, 295}300. Singh, H., & Fox, P. F. (1985). Heat stability of milk: the mechanism of stabilization of formaldehyde. Journal of Dairy Research, 52, 65}76. Singh, H., & Fox, P. F. (1987). Heat stability of milk: in#uence of modifying sulphydryl}disulphide interactions on the HCT-pH pro"le. Journal of Dairy Research, 54, 347}359. Spencer, C. M., Cai, Y., Martin, R., Ga!ney, S. H., Goulding, P. N., Magnolato, D., Lilley, T. H., & Haslam, E. (1988). Polyphenol complexation: some thoughts and observations. Phytochemistry, 27, 2397}2409. Van Boekel, M. A. J. S., Nieuwenhuijse, J. A., & Walstra, P. (1989). The coagulation of milk 1. Mechanisms. Netherlands Milk and Dairy Journal, 43, 97}127. Walstra, P., & Jenness, R. (1984). Dairy chemistry and physics (pp. 162}185). New York: Wiley Interscience (Chapter 10). Warren, L. (1959). The thiobarbituric acid assay of sialic acid. Journal of Biological Chemistry, 234, 1971}1975. Yaylayan, V. A., Huyghues-Despointes, A., & Polydorides, A. (1992). A #uorescamine-based assay for the degree of glycation in bovine serum albumin. Food Research International, 25, 269}275.
Proposed mechanism for the e!ect of polyphenols on the heat stability of milk J.E. O'Connell, P.F. Fox* Department of Food Chemistry, University College, Cork, Ireland Received 12 March 1999; accepted 29 June 1999
Abstract Ca!eic acid at 5.5 mmol l\ markedly enhanced the heat stability of milk at 1403C, the minimum was eliminated and stability increased as a function of pH; the apparent activation energy for coagulation was increased. The stabilizing e!ect of ca!eic acid was reduced or eliminated when milk was heated at 1503C or when the air in the headspace was replaced by N . The stabilising e!ect of ca!eic acid was reduced when stability was assessed as the temperature required to cause instantaneous coagulation, i.e., a maximum and minimum were evident. The thermal degradation/transformation of ca!eic acid in milk was a zero-order reaction with an activation energy of &40 kJ mol\ and Q value of &1.3 from 130}1403C; when the air in the headspace was replaced by N , thermal degradation/transformation of ca!eic acid was reduced. The addition of ca!eic acid resulted in a reduction of the reactive lysine and sulphydryl content and inhibited the dissociation of i-casein-rich protein from the casein micelles in milk on heating. It is postulated that on heating in milk, ca!eic acid is thermally oxidised to quinones which then interact with nucleophilic amino acid residues to inhibit i-casein dissociation from the casein micelle. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Milk; Heat stability; Polyphenols; Ca!eic acid
1. Introduction Polyphenols are common constituents in plants, in which they probably exist in conjugated and/or glycosylated forms. They are probably produced as secondary metabolites and act as natural detergents to grazing animals and as natural antimicrobial agents (Haslam & Lilley, 1988; Haslam, 1998). Recently, polyphenols have received attention for their ability to inhibit the oxidation of human low-density lipoproteins, to enhance the stability of b-lactoglobulin foams and to extend the shelf life of processed meat products by inhibiting oxidative rancidity (Sarker, Wilde & Clark, 1995; Landau & Yang, 1997; Chung, Wei & Johnson, 1998; Meyer, Heinoen & Frankel, 1998). The ability of polyphenol-rich extracts from a variety of plant sources, e.g., tea, co!ee, cocoa, wine, aloe vera and oak leaves and bark, and also individual polyphenols, e.g., ferulic acid, ca!eic acid, ellagic acid and epigallocatechingallate, to enhance the heat stability of skimmed milk
and concentrated milk has been reported (O'Connell, Fox, Tan-Kintia & Fox, 1998; O'Connell & Fox, 1999a, b). Ca!eic acid [3-(3,4-dihyroxyphenyl) 2-propenic acid, also referred to as 3,4-dihydroxycinnamic acid], the most e!ective polyphenol examined, markedly increased heat stability at 5.5 mmol l\. It was suggested (O'Connell et al., 1998; O'Connell & Fox, 1999a) that the stabilising e!ect of polyphenols on the heat stability of milk is due partially to calcium chelation. This communication reports on other possible mechanisms by which ca!eic acid may increase the heat stability of milk. 2. Materials and methods All chemicals used were reagent grade. The dialysis tubing used (code No. T88-T108) was obtained from Medicell International (London N1 1LX, UK). The whey protein isolate used was obtained from BIPRO (Davisco Foods Int. Inc, Le Sueur, MN 56058). 2.1. Milk supply
* Corresponding author. Tel.: #353-21-902362; fax: #353-21270001. E-mail address: [email protected] (P.F. Fox)
Raw milk, obtained from a local dairy, was defatted by centrifugation at 2000 g for 20 min at 203C, followed by
0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 1 2 4 - 7
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"ltration through glass wool to remove fat particles. The skimmed milk was stored at 43C until required (not more than 3 days).
2.2. Ewect of caweic acid on some physicochemical properties of milk The e!ect of ca!eic acid on the heat stability (as determined by the heat coagulation time-pH pro"le, HCTpH), alcohol stability, rennet coagulation and the calcium ion concentration of milk were determined by the methods described by O'Connell et al. (1998). The heat coagulation temperature-pH (HCTemp) pro"le of skimmed milk was determined by the method of Miller and Sommer (1940), with the temperature required to cause coagulation in 4 min (including a come-up time of 2 min) being recorded as the heat coagulation temperature (HCTemp) (Miller & Sommer, 1940).
2.5. Determination of total protein and N-acetyl neuraminic acid (NANA) The total protein and 12% trichloroacetic acid-insoluble NANA content of ultracentrifugal supernatants (90,000 g for 1 h at 203C) from milk at pH values in the range of 6.5}7.3 and heated at 903C for 10 min, with and without the addition of ca!eic acid to 5.5 mmol l\, were determined by the macro-Kjeldhal (AOAC, 1975) and thiobarbituric acid (Warren, 1959) methods, respectively. 2.6. Determination of zeta potential The zeta potential of control and experimental milks at pH 6.7, diluted 1 : 250 with SMUF (Jenness & Koops, 1962), was determined using a Malvern Zetamaster instrument (Malvern Instruments Ltd, Malvern, UK) at an applied voltage of 120 V and a modulation frequency of 250 Hz. The instrument was calibrated using a standard provided by Malvern Instruments.
2.3. Preparation of whey protein-free milk Whey protein-free milk was prepared by dispersing casein micelles, obtained by ultracentrifugation (90,000 g for 1 h at 203C), in synthetic milk ultra"ltrate without lactose (SMUF; Jenness & Koops, 1962), followed by exhaustive dialysis against bulk milk (100 ml against &2;4 l of bulk milk) for 48 h at 43C to allow for the addition of lactose and urea to levels similar to that in milk.
2.7. Measurement of casein micelle size The size distribution of casein micelles in control and experimental milks, heated at pH 6.7, was determined by photon correlation spectroscopy using a Malvern Zetamaster instrument. Milks were diluted with SMUF (3.4 ll milk ml\), "ltered through Whatman No. 40 "lter paper (Whatman International, Maidstone, UK) and analysed in triplicate.
2.4. Determination of available lysine, sulphydryl groups and hydroxymethyl furfural (HMF) content
2.8. Measurement of crosslinking of milk proteins on heating
Available sulphydryl groups and HMF levels in control and experimental milks were determined by the methods of Patrick and Swaisgood (1976) and Fink and Kessler (1986), respectively. Reactive lysine was determined by the method of Yaylayan, Huyghues-Despointes and Polydorides (1992): 0.10 ml of milk were diluted with 4 ml of 0.2 M potassium tetraborate and 1 ml of 0.1 M EDTA. One ml of #uorescamine (10 mg ml\ dissolved in acetone) was added and rapidly mixed. Fluorescence was measured at an excitation wavelength of 390 nm and an emission wavelength of 475 nm and reactive lysine was determined from a standard curve of lysine versus #uorescence.
Na-caseinate solutions (3.0%, w/v, in SMUF) at pH 6.7, with or without ca!eic acid at 5.5 mmol l\, were heated at 1303C for up to 35 min. The solutions were cooled and diluted 30 : 1 with H O and the absorbance at 440 nm determined. 2.9. Determination of relative viscosity The relative viscosity of control and experimental skimmed milks was determined at room temperature using an Ostwald U-tube viscometer (size C BS/U, Technico, Gallenkamp, Leicestershire, LE11 OTR, England) and expressed as relative viscosity using the equation:
flow time of sample;specific gravity of milk;specific gravity of H O Relative viscosity" flow time of H O
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2.10. Determination of caweic acid concentration in heated milk The concentration of ca!eic acid in heated milk was determined by RP-HPLC. Milks (containing 5.5 mmol l\ca!eic acid) were ultra"ltrated using Centriprep centrifugal concentrators with a nominal cut-o! value of 10 kDa (Amicon Inc, Beverly, MA, USA). The ultra"ltrate was diluted 1 : 5 with millipore water (Millipore Corp, Milford, USA) and 20 ll were injected onto a Nucleosil C RP-HPLC column (4.5;250 mm, 5 mm, 300 As pore size). RP-HPLC was performed using a Waters HPLC system (Waters, Millipore Corp, Milford, USA), consisting of a Waters 717 autosampler, a Waters 600 programmable pump and a Varian/Nainin UV detector (Varian Associates, Walnut Creek, CA, USA) set at 280 nm interfaced to a Dell Dimension PC running on Waters Millennium 2.10 software. The solvent used was methanol : H O : formic acid (20 : 79.7 : 0.3, v/v/v) at a #ow rate of 1 ml min\. 3. Results 3.1. Ewect of caweic acid on the heat stability of milk under diwerent experimental conditions As shown in Fig. 1a, ca!eic acid at 5.5 mmol l\ markedly enhanced the heat stability of milk. The apparent activation energy (E ) for the heat-induced coagulation of milk, with or without ca!eic acid at 5.5 mmol l\, was 174 and 142 at pH 6.7 and 198 and 140 kJ mol\ at pH 6.9, respectively (calculated from data in Table 1 as described by Parker & Dalgleish, 1977). The Q values for heat-induced coagulation was una!ected by the addition of ca!eic acid at all temperature ranges except for temperatures from 140}1503C, the signi"cance of which is discussed later. The stabilising e!ect of ca!eic acid was markedly reduced when the assay temperature was increased to 1503C and a minimum was reintroduced, i.e., a type A HCT-pH pro"le was evident (Fig. 1b). The presence of a shallow minimum in milk containing ca!eic acid was also apparent in the HCTemp-pH pro"le (Fig. 1c). Heat stability as determined by the HCTemp-pH pro"le is, essentially, a measure of the temperature required to cause instantaneous coagulation (Miller & Sommer, 1940). The method was developed to study coagulation under conditions where heat-induced changes, such as thermal oxidation of lactose, dephosphorylation and covalent polymerization, did not play a signi"cant role. The presence of a minimum in the HCTemp-pH pro"le of milk containing ca!eic acid indicates that the stabilizing mechanism of ca!eic acid involves some type of heat-induced reaction, and that at 1503C, coagulation occurs before ca!eic acid can undergo this reaction and exert its stabilizing e!ect.
525
The e!ect of ca!eic acid was eliminated if air in the headspace of the assay tubes was replaced by N (Fig. 1d). When an aqueous solution of ca!eic acid (5.5 mmol l\) was heated at 1403C for 15 min, lyophilized and the product added to milk at a weight basis equivalent to 5.5 mmol l\ ca!eic acid, it increased the heat stability of milk, regardless of the composition of the headspace (Fig. 1e). To investigate whether ca!eic acid interacts with milk proteins (either whey proteins or casein), 100 ml of milk containing 5.5 mmol l\ ca!eic acid were dialysed against 2;4 l of bulk milk for 48 h at 43C and the HCT-pH pro"le determined (Fig. 2a). The stabilising e!ect of ca!eic was lost on dialysis, presumably due to the di!usion of ca!eic acid into the bulk milk. When skimmed milk (50 ml) was dialysed against 500 ml milk containing 5.5 mmol l\ ca!eic acid, it assumed a HCTpH pro"le similar to that of milk containing ca!eic acid (Fig. 2b). These results suggest that most of the ca!eic acid in the experimental milk was dialysable and presumably had not interacted with the milk proteins at 43C. It should be noted that hydrophobic bonding, which has been proposed to be the principal binding force between proteins and polyphenols, is very weak at the temperature at which dialysis was carried out (&43C). The concentration of ca!eic acid in heated milk (1403C for 0}25 min) was determined in order to ascertain whether it interacted with some milk constituent or was degraded when heated at the assay temperature. The concentration of ca!eic acid in ultra"ltrate from milk containing 5.5 mmol l\ decreased from 5.36 to 2.31 mmol l\ on heating for 25 min (Table 2). Degradation/transformation of ca!eic acid in milk was a zero order reaction with respect to time and had an activation energy of&40 kJ mol\ and a Q value of 1.3 from 130 to 1403C. Degradation/transformation of ca!eic acid was reduced if air in the headspace was replaced by N (Table 2). When considered in conjunction with the data in Fig. 1d, it would appear that the heat-induced degradation/transformation of ca!eic acid is a prerequisite for its stabilising e!ect and that oxygen is required for the degradation/transformation of ca!eic acid. The low heat dependence for the degradation/transformation of ca!eic acid (E &40 KJ mol\ and Q value of&1.3) compared to heat-induced coagulation (E &140 KJ mol\ and Q value of &2}3) may explain why the e!ect of ca!eic acid on the heat stability of milk is less marked at 150 than at 1403C, i.e., at 1503C coagulation occurs before su$ciently enough ca!eic acid has been degraded/transformed to the active stabilizing agent. 3.2. Ewect of caweic acid on the calcium ion concentration of milk Polyphenols are capable of chelating metals (Radhakrishnan & Sivaprasad, 1980; McDonald, Mila &
526
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Fig. 1. E!ect of ca!eic acid at 5.5 mmol l\ or heat-trated ca!eic acid at 0.1%, w/v, on the HCT-pH pro"le of milk; control (air headspace) (}䢇}), control (N headspace) (}*}), with ca!eic acid (air headspace) (}䊏}), with ca!eic acid (N headspace) (}䊐}), heat-treated ca!eic acid (air headspace) (}䉱}), heat-treated ca!eic acid (N headspace) (}䉭}).
Scalbert, 1996). Ca!eic acid at 5.5 mmol l\ reduced the concentration of Ca> in unheated milk from 3.25 to 2.55 mM as determined using a calcium selective electrode (Table 3). Readjustment of the calcium ion concentration to the value of the control by adding 1.0 M CaCl
reduced, but did not eliminate, the stabilising e!ect of ca!eic acid (Fig. 3), suggesting that chelation of calcium is only partially responsible for the stabilising e!ect of ca!eic acid on the heat stability of milk. This conclusion is supported by the fact that addition of 5.5 mmol l\
J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536
527
Table 1 E!ect of ca!eic acid at 5.5 mmol l\ on the heat coagulation times and Q values of milk at di!erent temperatures System
Q for coagulation at temperature
Heat coagulation time, min at 1253C
1303C
Control, 60.0$3.5 39.1$4.5 pH 6.7 Control, 21.4$5.4 12.1$1.2 pH 6.9 With ca!eic 112.8$3.0 69.1$1.6 acid, pH 6.7 With ca!eic 127$6.5 77.1$2.1 acid, pH 6.9
1353C
1403C
1453C
1503C
125}1353C
130}1403C
135}1453C
140}1503C
25.2$4.2
15.6$2.2
10.2$1.1
5.7$0.9
2.38
2.51
2.47
2.74
7.3$2.3
3.7$1.1
2.5$0.8
1.9$0.2
2.93
3.27
2.92
1.95
47.2$0.7
31.0$1.5
17.6$2.3
3.4$0.5
2.39
2.23
2.68
9.12
60.1$3.2
35.4$2.6
14.8$1.2
2.5$0.7
2.12
2.18
4.06
14.16
Table 2 Concentration of ca!eic acid in ultra"ltration permeates from milk containing 5.5 mmol l\ ca!eic acid and heated at 1403C with air or nitrogen in the headspace (mean$SD, n"3) Heating, min at 1403C
Concentration (mM)
0 5 10 15 20 25
Air
Nitrogen
5.36$0.11 4.58$0.60 4.22$0.34 3.81$0.41 3.08$0.19 2.31$0.54
5.36$0.11 5.24$0.06 5.17$0.09 4.93$0.24 N.D. N.D.
N.D.: not determined.
ca!eic acid had little e!ect on the ethanol stability or rennet coagulation of milk, both of which are strongly in#uenced by [Ca>] (Table 3).
& Jenness, 1984; van Boekel, Nieuwenhuijse & Walstra, 1989), is partially responsible for the heat-induced coagulation of milk at pH values outside the minimum, due to covalent polymerization. Ca!eic acid reduced the concentration of HMF, which is an index of Maillard reaction, formed in milk on heating, but only after 12 min of heating (Fig. 4). Interaction of ca!eic acid with lysine residues may also increase heat stability in a manner analogous to formaldehyde, i.e., by preventing the dissociation of i-caseinrich protein from the casein micelles by cross-linking proteins (Holt, Muir & Sweetsur, 1978; Singh & Fox, 1985). The ability of ca!eic acid to crosslink milk proteins on heating was determined by measuring the turbidity of diluted Na caseinate solutions at 440 nm; turbidity increased (Fig. 5a) markedly in the presence of ca!eic acid, suggesting that the ability of ca!eic acid to increase heat stability may be due to crosslinking of proteins. Ca!eic acid also increased casein micelle size upon heating (Fig. 5b), which may be due to crosslinking.
3.3. Ewect of caweic acid on the reactive lysine residues content of milk
3.4. Ewect of caweic acid on the sulphydryl content of milk
Ca!eic acid reduced the lysine content of heated milk by&10%; again, replacement of air in the headspace by N eliminated this e!ect (Table 4). Blocking of e-amino groups of lysine may be important in inhibiting the Maillard reaction, which, it has been suggested (Walstra
Addition of ca!eic acid (5.5 mmol l\) reduced the sulphydryl content of a heated milk (953C for 10 min), Table 4 [note: milk was heated at 953C for 10 min and not at assay temperature because at the latter temperature the decrease of cysteine is so rapid that it is not
Table 3 E!ect of ca!eic acid (5.5 mmol l\) on some properties of skimmed milk at 213C (with the exception of rennet coagulation time (RCT) which was determined at 313C). (mean$SD, n"3) System
Control With ca!eic acid
[Ca>], mM
3.25$0.22 2.55$0.13
RCT, min
6.98$0.92 6.75$0.25
Milk was heated at 1303C for 10 min.
Ethanol stability, pH
Relative viscosity
Zeta potential, mV
6.5
7.0
Unheated
Heated
Unheated
Heated
65.0$0 67.5$0
99.18$1.44 98.33$1.44
1.60 1.69
1.70 1.73
!15.6$0.5 !15.2$0.5
!18.7$1.2 !18.7$0.7
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Fig. 2. E!ect of dialysis of (a) skimmed milk containing ca!eic acid at 5.5 mmol l\ against bulk on its HCT-pH pro"le; control (}䢇}), dialysed control (}*}), with ca!eic acid (}䊏}), with ca!eic acid and dialysed (}䊐}); (b) of skimmed milk agianst an excess milk containing ca!eic acid at 5.5 mmol l\, control (}䉱}), with ca!eic acid (}䉬}), control milk dialysed against milk containing 5.5 mmol l\ ca!eic acid (}䉭}).
possible to analyse]. Loss of cysteine residues was not a!ected by the composition of the headspace (Table 4). A decrease of cysteine content of milk proteins, by chemical oxidation, markedly enhanced the heat stability of milk (Singh & Fox, 1987). Decreasing of the sulphydryl content inhibits or disrupts the formation of whey protein-i-casein complexes and consequently the dissociation of i-casein-rich protein from the casein micelles at pH values'pH 6.8 is inhibited (Singh & Fox, 1987). The
ability of ca!eic acid to inhibit heat-induced dissociation of protein and i-casein was shown by measuring the protein content and N-acetyl neuraminic acid content of ultracentrifugal supernatants of heated milk with and without the addition of ca!eic acid (90,000 g for 1 h at 203C) (Fig. 6). The possibility that ca!eic acid eliminates the minimum in the HCT-pH pro"le of milk by interacting with the cysteine group of b-lactoglobulin and other whey
J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536
529
Fig. 3. E!ect of ca!eic acid at 5.5 mmol l\ on the HCT-pH pro"le of skimmed milk, with and without supplementation with CaCl ; control (}䢇}), with ca!eic acid (}䊏}), with ca!eic acid and calcium activity readjusted to level of control (}䊐}).
Fig. 4. E!ect of ca!eic acid at 5.5 mmol l\ on the concentration of hydroxymethylfurfural in heated milk; control (}䢇}), with ca!eic acid (}䊏}).
proteins is supported by the fact that addition of whey protein isolate (WPI) to ca!eic acid-containing milk (5.5 mmol l\) reintroduced the minimum in the HCTpH pro"le (Fig. 7a). However, it should be noted that preheat treatment at 903C for 10 min prior to addition of ca!eic acid did not eliminate the stabilising in#uence of ca!eic acid (Fig. 8). Pre-heating under such conditions
should result in complex formation between b-lactoglobulin and i-casein and consequently if the exclusive stabilising mechanism of ca!eic acid was by inhibition of complex formation via reduction of suphydryl groups it would be expected to have little e!ect on preheated milk (Fig. 8). Also, the addition of ca!eic acid to whey protein-free milk enhanced stability (Fig. 7b).
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Fig. 5. E!ect of ca!eic acid 0 (}䢇}), 5.5 (}*}), 11.0 (}䊏}) or 22.0 (}䊐}) mmol l\ on (a) turbidity of Na-caseinate solution and (b) casein micelle size in milk.
3.5. Ewect of caweic acid derivatives and related compounds on the heat stability of milk The molecular reactivity of polyphenols is generally attributed to the presence of hydroxyl groups (Spencer et al., 1988; Haslam & Lilley, 1988). Indeed, replacement of the hydroxyl groups of ca!eic acid by methoxy groups, i.e., 3,4-dimethoxycinnamic acid, eliminated the stabilizing e!ect of ca!eic acid (Fig. 9). In an attempt to determine which speci"c functional group(s) may be responsible for the ability of ca!eic acid to enhance the heat
stability of milk, the e!ects of derivatives of ca!eic acid and related compounds on the heat stability of milk were examined (Fig. 9). Benzene had no e!ect on the heat stability. Phenol (monohydroxybenzene) had no e!ect on the heat stability of milk, nor did the monohydroxyl derivative of ca!eic acid, coumaric acid, regardless of the isomer. Introduction of a second hydroxyl group on the benzene ring a!ected the HCT-pH pro"le of skimmed milk but this e!ect was dependent on the position of the second hydroxyl group. Catechol (1,2-benzenediol) and
J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536 Table 4 E!ect of ca!eic acid at 5.5 mmol l\ on the free cysteine and lysine content of milk with and without heating (953C for 10 min at pH 6.7, with air or nitrogen in the headspace). (mean$SD, n"3) System
Cysteine (mM)
Lysine (mM)
Control, unheated Control, heated (air) Control, heated (N ) With ca!eic acid, unheated With ca!eic acid, heated (air) With ca!eic acid, heated (N )
0.012$0.003 0.032$0.004 0.032$0.003 0.008$0.002 0.012$0.001 0.013$0.002
26.52$0.66 26.13$0.80 25.48$0.78 25.27$0.26 23.62$0.63 26.12$1.26
hydroquinone (1,4-benzenediol) converted a type A milk to a type B milk, while resorcinol (1,3-benzenediol) had little e!ect on the heat stability of milk, suggesting that positioning of the hydroxyl groups at the ortho or para position is necessary for the stabilizing e!ect of polyphenols. Supporting this assertion, 1,2-naphthalene enhanced heat stability while 1,3-naphthalene had little e!ect. Saturation of the aromatic ring eliminated the stabilizing e!ect, e.g., the saturated derivative of catechol, 1,2-cyclohexanediol, did not a!ect the heat stability of milk. However, ca!eic acid had a much greater e!ect on the heat stability of milk than catechol, which destabilized milk in the region of the maximum. The stabilizing e!ect of ca!eic acid at the maximum in the HCT-pH pro"le may be attributed to the chain on C-1. This would appear
531
to be supported by the fact that ferulic acid, which contains the same C-1 moiety as ca!eic acid, stabilized milk in the region of the maximum but had a destabilizing e!ect at the pH of the minimum and on the alkaline side of the minimum, presumably because it does not possess a second hydroxyl group. This e!ect is analogous to other mono phenolic compounds containing a carboxylic acid moiety, e.g., vanillic acid (O'Connell & Fox, 1999b). The e!ect of these monophenolic compounds (ferulic acid and vanillic acid) was similar to that of other acids, e.g., tartaric acid and citric acid (Mohammad & Fox, 1983). Perhaps it is the carboxylic acid moiety of monophenols that stabilises milk in the region of the maximum by chelation of calcium. Modi"cation of the carboxylic acid moiety at C-1 of ca!eic acid had a profound e!ect on the ability of diphenols to increase the heat stability of milk. 3,4-Dihydroxypropiophenone or 3,4-dihydroxyphenyl-acetic acid did not eliminate the minimum in the HCT-pH pro"le. The double bond in the C-1 chain of ca!eic acid did not appear to be important, as hydroca!eic acid also stabilised milk. 3,4-Dihydroxybenzaldehyde, which differs from ca!eic acid in that it has an aldehyde group, rather than propenic acid, at C-1, markedly increased the heat stability of milk. However, the signi"cance of this result is unclear considering that aldehyde groups alone also increase the heat stability of milk (Holt et al., 1978; Singh & Fox, 1985). However, vanillin (4-hydroxy 3methoxy benzaldehyde) had no e!ect on the heat stability of milk.
Fig. 6. E!ect of pH on the level of protein and 12% TCA-insoluble NANA in the ultracentrifugal supernatant (90,000 g for 1 h at 203C) of heated milk (903C for 10 min) with and without 5.5 mmol l\ ca!eic acid; control: protein (}䊏}), experimental protein (2䊐2), control, NANA (}䢇}), experimental NANA (2*2).
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Fig. 7. E!ect of ca!eic acid at 5.5 mmol l\ on the HCT-pH pro"le of (a) skimmed milk with and without WPI supplementation at 1.0%, w/v; control (}䢇}), control with WPI (}*}), ca!eic acid (}䊏}), with ca!eic acid and WPI (}䊐}); (b) of whey-protein free milk; control (}䢇}), with ca!eic acid (}䉱}).
Quinic acid (1,3,4,5-tetrahydroxycyclohexanecarboxylic acid) at 0.1}0.4%, w/v, markedly destabilised milk throughout the pH range 6.3}7.3. The e!ect of tri or tetra hydroxycinnamic acids (i.e., ca!eic acid with additional hydroxyl groups) on the heat stability of milk would be of interest. Unfortunately, these compounds are not available. The e!ect of derivatives of ca!eic acid and related compounds on the heat stability of milk suggests that the presence of two hydroxyl groups at ortho or para positions and the aromatic ring are prerequisites. The pres-
ence of a carboxylic group in the C-1 also appears to be important. 3.6. Ewect of quinones on the heat stability of milk 1,4-Benzoquinone, the quinone derivative of hydroquinone (1,4-benzenediol) which converted a type A milk to a type B HCT-pH pro"le, stabilized skimmed milk (Fig. 9) regardless of the headspace in the test tube (air or N ). 1,2-Naphthoquinone, the quinone derivative of 1,2-naphthalene, also stabilized milk
J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536
533
Fig. 8. E!ect of preheating (903C for 10 min) on the heat coagulation time-pH pro"le of skimmed milk with and without ca!eic acid at 5.5 mmol l\; control (}䢇}), control (preheated) (}*}), with ca!eic acid (}䊏}), with ca!eic acid (preheated) (}䊐}).
(Fig. 9) regardless of the composition of the head space.
4. Discussion From the data presented, it appears that the e!ect of ca!eic acid on the HCT-pH pro"le of skimmed milk is not due exclusively to one mechanism; rather, it appears to be a multifactorial e!ect. Chelation of calcium appears to be involved. Two possible binding mechanisms of calcium by ca!eic acid are presented in Fig. 10 (reactions 1 and 2). Interaction of ca!eic acid with proteins also appears to be important in the stabilizing e!ect of ca!eic acid. The ability of polyphenols to interact with proteins is established (Asquith, Uhlig, Mehansho, Putman, Carlson & Butler, 1987; Haslam & Lilley, 1988). Most of the literature suggests that hydrophobic and hydrogen bonding are responsible for polyphenol}protein interactions (Spencer et al., 1988; Haslam & Lilley, 1988). However, the heat stability assay is carried out at 1403C, at which hydrophobic and hydrogen bonding are negligible. The ability of polyphenols to interact covalently with proteins has also been suggested (Meek & Weiss, 1979; Beart, Lilley & Haslam, 1985). Beart et al. (1985) proposed that under certain conditions, e.g., enzymatic oxidation, polyphenols are converted to quinones which may then react with nucleophilic amino acids, e.g., sulphydryl or amino
groups. Horigome and Kandatsu (1968) reported a 3}11% reduction in the lysine content of casein when incubated with ca!eic acid and polyphenol oxidase. It is conceivable that heating at high temperatures may cause thermal oxidation of ca!eic acid to quinones (Fig. 10, reaction 3). Pyrolysis (2283C for 15 min) of ca!eic acid resulted in the production of strong reducing compounds (Guillot, Malmoe & Stadler, 1996). Possible binding modes of quinones with amino groups are shown in Fig. 10 (reactions 4 and 5); the possible implications of such interactions on the heat stability of milk has been discussed. Loss of cysteine residues may be due to interaction between products of ca!eic acid oxidation and cysteine (Fig. 10, reactions 5 and 6) or reduction of quinones to ca!eic acid resulting in a conversion of cysteine to cystine (Fig. 10, reaction 7). Alternatively, loss of cysteine may be due to direct complexation with ca!eic acid, forming cysteinyl-ca!eic acid (Fig. 10, reaction 8; Cillers & Singleton, 1990). The signi"cance of the interaction of ca!eic acid with sulphydryl residues in relation to heat stability has already been discussed. The likelihood that thermal oxidation of ca!eic acid to quinones is responsible for the ability of ca!eic acid to increase the heat stability of milk is supported by the fact that replacement of air in the headspace by N , i.e., removal of oxygen, inhibited thermal oxidation of ca!eic acid (Table 2) and consequently eliminated the stabilising e!ect of ca!eic acid (Fig. 1d). It is also noteworthy that heat-treated ca!eic acid (Fig. 1e) and quinones stabilised
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Fig. 9. E!ect of ca!eic acid and related compounds on the HCT-pH pro"le of skimmed milk (graphs are representative, i.e., not exact) (*) control, (- - -) with added phenolic compound.
milk, regardless of the composition of the headspace. The proposed mechanism, i.e., that heating converts ca!eic acid to quinone, may also explain why ca!eic acid stabilises milk while closely related compounds do not.
The fact that ca!eic acid, catechol, hydroquinone and 1,2-naphthalene exerted an e!ect on the heat stability of milk while resorcinol, 1,2-cyclohexanediol and 1,3naphthalene had little e!ect is likely to be due to the
J.E. O+Connell, P.F. Fox / International Dairy Journal 9 (1999) 523}536
535
Fig. 10. Possible molecular interactions of ca!eic acid and quinones formed upon oxidation with nucleophilic amino acids, lysine and cysteine.
inability of the latter group of compounds to oxidise to quinones. The e!ect of ca!eic acid on the heat stability of skimmed milk is of interest in that its addition markedly increases heat stability at relatively low concentrations, 5.5}20 mmol l\, without a!ecting other properties such as rennet coagulation time, alcohol stability, viscosity or zeta potential (Table 2). Secondly, since ca!eic acid is a naturally occurring constituent of plants and can be easily chemically synthesised (Horigome & Kandatzu, 1968), it may be considered as a &natural functional ingredient' in foods. Polyphenol-rich extracts of tea are currently being incorporated into sweet paste biscuits, milk drinks and chewing gum for nutritional reasons due to their attributed bene"cial health e!ects, e.g., anticarcinogenic, antimutagenic and antioxidative properties (Landau & Yang, 1997; Anon, 1997; Chung et al., 1998; Hennessy, 1998). The addition of phenolic compounds to meat products as antioxidants (Pratt & Hudson, 1990), to b-lactoglobulin foams as stabilizers (Sarker et al., 1995), to milk as antifungal agents (Rosenthal, Rosen & Bernstein, 1997) and to foods as inhibitors of browning reactions (Cillers & Singleton, 1990) has been advocated.
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