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Physicochemical characterization of iron-supplemented skim milk
hr. Dcrir~ Journul 7 (1997) 141.148 0 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain PII:
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Physicochemical Characterization of Ironsupplemented Skim Milk FrkdCric Gaucheron,* Yvon Le Graet, Karine Raulot and Michel Piot Luhorutoire
de Rechrrchm
de Technologie LuitikJ,
(Received
Institut Nutional de lu Recherche Agronomique, Runes Cedex, Frunca 28 June 1996; accepted
8 November
65, YWJ de Suint-Brieuc.
35042
1996)
ABSTRACT The physicochemical characterization and study of technological properties of skim milk samples supplemented with FeC& or FeC13 were carried out. The iron concentrations ranged from 0 to 1.5 mM. For both salts used, iron was mainly bound to the micellar phase with a probable change of casein structure. In parallel, the mineral distribution and the casein micelle hydration changed differently as a function of the initial oxidation state of iron (Fe”’ or Fe’+). After acidification (between 6.7 and 4.0) or after heat treatments (95”CP15 and 30min), no release of iron was found. Rennet coagulation parameters (clotting time, aggregation time and curd firmness) of the different iron-supplemented skim milk samples were more modified when fortifications were performed with FeClz than with FeCI-(. The results were discussed in relation with proteins and minerals modifications. 0 1947 Elsevier Science Ltd
the meal which can increase or decrease the absorption of iron and the physiological state of the tested subject. For Carmichael et al. (1975), the gastrointestinal absorption of iron bound to purified caseins or to casein micelles was similar or higher than those obtained with ferrous salts. Hurrell rt al. (1989) reported that caseins are responsible for the low bioavailability of iron. Zhang and Mahoney (1989a) indicated that the bioavailability of iron incorporated into Cheddar cheese is similar to that of FeS04. Besides the biochemical and nutritional studies, few applications of technological dairy products fortification are described. Zhang and Mahoney (1989b, 1990, 1991) show that Cheddar fortification (40-50 mg kg-‘) is possible without affecting its quality. Reddy and Mahoney (1992) indicate that ferric chloride at a concentration of 0.2mM does not significantly affect the rennet clotting time. However, the effect of FeC13 on the coagulation of renneted micelles seems to relate to the thermal history of milk because ferric chloride decreases rennet clotting time when it is added to uncooled raw milk or to milk before pasteurisation but increases rennet clotting time when it is added to cooled raw milk or to milk after pasteurization. The effect of iron fortification of yogurt on its manufacture, the growth of lactic acid and spoilage bacteria, and on lipid oxidation reveals that iron-supplemented yogurt is a suitable vehicle for delivering iron to consumers (Hekmat et ul., 1994). The aims of this study were to prepare, characterize and appreciate the technological properties of ironsupplemented skim milk samples with two different iron salts: FeC12 and FeC13. They were added to skim milk samples in an iron concentration range of O1.5 mM. The physicochemical modifications (proteins and mineral distribution) were determined and related to the modifications of technological properties.
INTRODUCTION Milk is relatively poor in iron (0.2-0.4 mg L-‘) and this trace element is essential in animal and human nutrition. Iron participates to the structures of cytochromes and several enzymes. It is also a component of haem in haemoglobin and of myoglobin in which it plays an important role in the transport, storage and utilization of oxygen. Iron deficiency results in anaemia, which affects about 30% of the world’s population (especially women and teenagers). An iron fortification of milk and of dairy products could be considered as a potential approach to prevent anaemia. Iron, when added as FeC12 to skim milk, 85% is bound to caseins (72, 21 and 4% are bound to us-, /& and K-caseins, respectively) (Demott and Dincer, 1976). The strong affinity of caseins for iron is recognized and clustered phosphoseryl residues attributable to (Manson and Cannon, 1978; Hegenauer et ul., 1979; Gaucheron et al., 1995). The formation of iron-casein complexes induces the oxidation of iron from the ferrous to the ferric state (Manson and Cannon, 1978; Emery, 1992). In these complexes, iron is strongly bound to phosphoseryl residues because no release of iron from iron-supplemented caseinate was found after its acidification (between pH 6.5 and 4.0), after heat treatments (50, 70 and 90°C for 15 min) or in the presence of 20 mM Na2HP04 (Gaucheron et al., 1996a). Results concerning iron bioavailability and tissue distribution are conflicting (Kwock et al., 1984; Jackson and Lee, 1992) because these characteristics are not easy to evaluate. Indeed, they depend on different factors such as the chemical form of iron added (ferric, ferrous) (chloride, sulfate, lactate, gluconate, citrate), the presence of other components in *Corresponding
author. 141
142
F. Gaucheron
MATERIALS AND METHODS Milk Bulk whole milk (2OL), collected and stored at 4°C (Triballat, Noyal-sur-Vilaine, France), was warmed and skimmed at 40°C (30min). Then, the skim milk obtained was cooled to 20°C and sodium azide (w/w) (Fluka Chemie, Buchs, Switzerland) was added to 0.02% to prevent bacterial growth. Iron salts FeC12.4H20 and FeC13.6H20 were purchased from Merck (Darmstadt, Germany). The stock solutions of iron (50mM) were prepared just before their addition to skim milk samples. The pH values of these stock solutions were 3.5 and 1.9 for FeCl* and FeC13, respectively. Preparation of iron-supplemented skim milk samples Skim milk samples (500mL) were enriched, at room temperature, with both iron salts to produce final iron concentrations ranging from 0 to 1.5mM (previous experiments showed that protein precipitation occurred when iron concentration added to milk was about 8 mM). Deionized water was added to compensate for volume changes in milk. These mixtures were stirred vigorously to ensure rapid and complete mixing. Before analyses or technological treatments (acidification, heat treatments or rennet coagulation), the different iron-supplemented skim milk samples were left standing overnight at room temperature. Preparations of iron-supplemented skim milk samples and subsequent analyses were carried out in triplicate. Physicochemical characterization of iron-supplemented skim milk samples The spectrofluorimetric measurements were made on an LS 50B Perkin Elmer spectrofluorimeter (Saint Quentin en Yvelines, France). Firstly, the intrinsic fluorescence was determined on skim milk samples after a l/70 dilution into 50mM Tris HCl, pH 7.6. The excitation and emission wavelengths were 295 and 350nm. For both these wavelengths, slit widths were 2.5nm. Secondly, the exposure of hydrophobic groups was measured by the fluorescent probe binding method of Bonomi et aE. (1988). Fluorophor 8-aniline naphtalen 1-sulphonate (ANS) (Sigma Chemical Co, St Louis, USA) was used. When interacting with hydrophobic sites of proteins, its fluorescence parameters were an excitation wavelength of 390nm and an emission wavelength of 480nm. The emission and excitation slits were both set at 2.5 nm band width. The different milk samples (1 mL) were diluted with 50mM Tris HCl buffer, at pH 7.6, to a final volume of lOmL, and titrated at room temperature with an aqueous solution of ANS (final concentration between 0 and 300~~~) until no further increase in fluorescence was observed. Analysis of binding data allowed us to determine values of the maximum fluorescence attainable at saturating ANS concentration (F,,,, i.e. asymptotic value of titration curve).
et al.
Caseins, a-lactalbumin and /?-lactoglobulin of milk containing 1.5 mM iron were separated by reversedphase (RP) HPLC on a C4 column as described by Jaubert and Martin (1992). RP-HPLC analyses of proteins are commonly performed after sample dissolution in a buffer containing a reducing agent (e.g. 2-mercaptoethanol or dithiothreitol) to improve the chromatographic separation (Jaubert and Martin, 1992). In our case, as both these chemical compounds have an affinity for elemental iron, this chemical reduction was not performed. Eluted peaks were detected by absorbance at 214nm. In parallel, ironmodified a,,-casein was applied to the C4 column. The cc,,-casein was purified as described by Brignon et al. (1977) and modified to have about 1 mol iron mole” casein. The pH value was measured with a Titroprocessor 686 pH meter (Metrohm, Herisau, Switzerland). The milk aqueous phase was obtained either by ultrafiltration on Centriflo CF 25 (molecular mass cutoff: 25,000 Da; Amicon, Epernon, France) after centrifugation at 500g for 1 h or by ultracentrifugation at 77,000 g (Beckman L-8-55 ultracentrifuge, Gagny, France) for 2 h at 20°C. From the wet pellet obtained by ultracentrifugation, the determination of casein micelle hydration was carried out. Briefly, the drained casein pellet was weighed and then dried at 103°C for 7 h to remove the water of hydration. The difference between the weight before and after drying, expressed as g of water per g of dry casein micelle pellet, was taken as the water of hydration. Iron and calcium concentrations in ultrafiltrates and in ultracentrifugal supernatants were evaluated by atomic absorption spectrometry (Varian AA300 spectrometer, Les Ulis, France) (Bruli: et al., 1974). Inorganic phosphate and citrate concentrations in ultrafiltration permeates were determined by ion chromatography (Gaucheron et al., 1996bj. A laser granulometer (N4MD Coulter, Coultronics, Margency, France) was used to evaluate the average diameter of particles at 20°C. The milk sample was diluted into 20mM imidazole/HCl buffer, pH 6.6, containing 5 mM CaC12 and 50 mM NaCl, stabilised for 1Omin inside the cell holder, and a measurement of light intensity scattered at 90” for 600s was carried out. Acidification of skim milk samples containing 1.5 mM of iron The effects of acidification by 1 M HCl (pH from 6.7 to 4.0) on skim milk samples containing 1.5 mM of iron were studied. After acidification, samples were left standing overnight at room temperature. Then, pH values were again controlled. A control sample (without iron) was tested under the same experimental conditions. Heat treatments of skim milk samples containing 1.5 mM of iron The effects of heat treatments (15 and 30 min at 95°C) on skim milk samples containing 1.5 mM of iron were studied: for this, 1OmL of milk sample in sealed Pyrex tube were submerged in an oil-bath and agitated during heating. Three hours before heating, the pH of
Iron-supplemented skim milk samples was adjusted to 6.70 with 1 M NaOH and readjusted if necessary just before heating. A control sample (without iron) was tested under the same experimental conditions. Rennet coagulation and determination peptide release (CMP)
of caseinomacro-
The rennet clotting time, the aggregation time and the curd firmness were determined by using a Formagraph (Foss Electric, Paris, France) (McMahon and Brown, 1982). Three hours before measurements, pH values of milk samples heated at 30°C were adjusted to 6.70 with 1 M NaOH and readjusted if necessary just before rennet addition. Thus, the effects that we observed were due solely to iron salts. A 0.5% rennet solution (Hansen, Saint Germain les Arpajons, France) was used. The rate of chymosin hydrolysis was determined on the basis of released CMP, as described by Lconil and Molle (1991). Experiments were carried out with no modified skim milk and with iron-supplemented skim milk samples containing 1.5mM of iron ions. The CMP exclusion determined by concentration was chromatography (chromatographic column ProgelTMTSK G3000 SW, Supelco, Saint Germain-en-Laye, France) (personal communication, Nutrinov, Rennes, France). The CMP was eluted by a solution containing 30% acetonitrile and 0.1 o/o trifluoroacetic acid. Detection was performed by absorbance at 214nm. The CMP was identified and quantified by comparison with a CMP standard prepared in our laboratory.
RESULTS
AND DISCUSSION
Physicochemical skim milk
characterization
of iron-supplemented
Distribution of’ iron Table 1 indicates the concentrations and the percentages of iron found in the aqueous phase ultracentrifugation or either by obtained ultrafiltration. Whatever concentration and nature of salt the percentage of iron in iron used, ultracentrifugal supernatants was always higher than those obtained in ultrafiltrates. The percentage of iron slightly decreased in the iron concentration range used: between 11 .O to 9.3% or 9.8 to 7.8% in the ultracentrifugal supernatants of iron-supplemented skim milk samples with FeC12 and FeC13, respectively, and between 5.6 to 4.5% or 3.6 to 3.1% in the ultrafiltrates of iron-supplemented skim milk samples with FeC12 and FeC13, respectively. Thus, when iron
143
was added to milk in the concentration range of 0 to 1.5 mM, 89% and more of iron were bound to the colloidal phase (Table 1). The proportionality between the binding of iron to the colloidal phase and the added concentration suggests that the binding sites were not saturated and had the same affmity for iron ions in the iron concentration range used. Presumably, the iron salts used did not precipitate in the aqueous phase of skim milk because their addition (final iron concentration 1.5 mM) to milk ultrafiltrate induced no precipitation (results not shown). Moreover, as described by Gaucheron et al. (1996a), the formation of large hydroxide complexes which eventually precipitate and are not able to pass through the ultrafiltration membrane was unlikely. The important modification of the colloidal phase with FeQ is in agreement with the results of Demott and Dincer (1976). However, the several possible forms of micellar iron (associations of iron ions with caseins or/and with colloidal calcium phosphate or/and with citrate molecules) were not distinguishable because it is impossible by differential centrifugation or column fractionation techniques to obtain casein micelles, colloidal calcium phosphate and colloidal citrate as separate fractions. The presence of whey proteins, in ultracentrifugal supernatant, able to bind iron (c(lactalbumin, b-lactoglobulin, lactoferrin) (Baumy and Brule, 1988; Nagasako et al., 1993) explains the higher percentages of iron ions found in this aqueous phase (Table 1) even though these proteins are absent in milk ultrafiltrates. Iron ions, in the ultrafiltrates, are free or bound to small molecules, named LOW Molecular Weight fraction by Fransson and Lonnerdal (1983) which are able to pass through the ultrafiltration membrane. These small molecules, not identified in this study, can be citrate, organic and inorganic phosphate, lactose (Bachran and Bernhard, 1980) sulfur-containing compounds or erotic acid. Mod$cations qf’prateins Firstly, the intrinsic fluorescence decreased as a function of iron concentration (Fig. 1A) and traduced conformatronal change of milk proteins. Secondly, the structural modification of milk proteins was confirmed because the fluorescence binding curves as a function of ANS concentration were different in the presence or absence of iron ions. The F,,, values decreased as a function of iron concentration (Fig. IS), but no significant difference between iron-supplemented skim milk samples with FeCl2 or FeC17 was observed. RPHPLC analyses showed differences between ironsupplemented skim milk samples and unmodified skim milk (Fig. 2). In the absence of iron ions (Fig. 2A), five fractions were detected (noted I to V).
Table 1. Concentrations and Percentages of Iron in the Aqueous Phase of Iron-supplemented Phases were Obtained by Ultracentrifugation or by Ultrafiltration. Control Corresponds Control Total iron (mM) ultracentrifugable ultrafiltrable
iron
iron (PM)
(PM)
(“XI)
(%)
0 0 0
Skim Milk Samples. The Aqueous to the Unsupplemented Milk. FeC13
FeClz 0.50 (E%) 28 (5.6%)
1.oo 96 (9.6%) 49 (4.9%)
1so 140 (9.3%) 67 (4.5%)
0.50 49 (9.8%) 18 (3.6%)
1.oo 87 (8.7%) 35 (3.5%)
1.50 117 (7.8%) 47 (3.1%)
F. Gaucheron et
144
56’ 0
I
0.5
1
1.5
2
Iron concentration (mM) Fig. 1. Influence of iron concentration on the intrinsic fluorescence intensity (A) and on the exposure of hydrophobic regions (B) of milk proteins. The accuracies were i 5 unit (A) and & 2% (B), respectively. (*) FeClz and (+ ) FeC&.
Fraction I contained K- and a,*-caseins. Because of low concentrations (3 and 4g L-‘, respectively), heterogeneity (the presence of various glycoforms of rc-casein) and the absence of reduction treatment, the chromatographic separation of both these proteins was incomplete. Fractions II and III contained aSi-casein and c(lactalbumin, respectively. Fraction IV contained the A’ and A2 genetic variants of p-casein and fraction V, the A and B genetic variants of /I-lactoglobulin. The genetic variants of /I-casein and of /I-lactoglobulin differ in their amino acid sequences (Visser et al., 1991). In the presence of FeCl2 or FeCl3 (1.5 mM), the chromatographic profiles were modified (arrows on the chromatogram of Fig. 2B and 2C) because the c(,icasein chromatographic areas (14.4 and 16.2% of total area in the presence of 1.5 mM FeClz and FeCls, respectively vs 22.8% in the absence of iron) and retention times (18.25 min in the presence of 1.5 mM iron vs 19.7min in the absence of iron) were changed. These modifications were confirmed by chromatographic injection of iron-modified a,,-casein (Fig. 2D). Thus, cr,i-casein was more modified than other caseins. Retention times and chromatographic areas of tllactalbumin and /&lactoglobulin were not modified. Moreover, whichever iron salt added, the modifications of chromatographic profiles were qualitatively and quantitatively similar. The decreases in intrinsic fluorescence intensities and to the added iron in F,,,,, values proportional
al.
concentration indicated structural modifications of milk proteins because when a protein is disorganised, hydrophobic segments can be differently exposed. From the chromatographic analyses of caseins, of CIlactalbumin and of p-lactoglobulin (Fig. 2) we demonstrated that iron element is bound mainly to asIresidues per casein, which has 7-9 phosphoseryl molecule (Leonil et al., 1995). The iron binding to presidues per casein, which has five phosphoseryl molecule (Leonil et al., 1995), and aSz-casein, which is the most phosphorylated of caseins (lo-13 phosphoseryl residues per molecule (Leonil et al., 1995)), is also possible but was not observed. The preferential modification of a,i-casein supports the results of Demott and Dincer (1976). The complex formation that takes place at phosphoseryl residues would change the protein structures in such a manner that a,,-casein would be differently exposed to the modified silica matrix and consequently decrease the chromatographic retention time. Reddy and Mahoney (199 l), using ultraviolet and fluorescence spectroscopy, also showed a conformational change in bovine aSIcasein on interaction with Fe (III). In these complexes, iron is probably bound to casein molecules via the oxygen of the phosphoseryl residues by coordination link (Hegenauer et al., 1979). However, iron ions may also bind to casein molecules via tyrosine, glutamic and aspartic residues. Iron complexation to carboxyl groups in a bovine serum albumin digest was observed by Shears et al. (1987). In these complexes with caseins, iron would adopt a tetrahedral coordination structure as observed with ferric iron saturated phosvitin (Webb et al., 1973). Thus, from fluorescence and chromatographic studies and as observed by Gaucheron et al. (1996a), changes in the local environment of caseins can be deduced. A casein conformational change in the presence of iron ions and/or an association of casein molecules via iron bridges can be proposed. These changes of protein structures seem to be independent of the initial oxidation state of iron added (Fe2+ or Fe3+). Mod$cations
of mineral distribution
In the absence of iron ions, the pH value of skim milk was 6.73. Addition of iron ions to skim milk resulted in pH decreases (Fig. 3) which were significantly greater with FeC13 than with FeClz. These pH decreases were related to the acidities of iron solutions and to exchanges between iron ions added and micellar bound Hf. The characterization of iron-phosphopeptide /I-casein (l-25) complexes by electrospray-ionization mass spectrometry (Gaucheron et al., 1995) showed a release of three protons for one bound iron atom. The consequences of pH decrease are displacements of calcium, inorganic phosphate and citrate ions from the colloidal phase to the aqueous phase (Chaplin, 1984; Dalgleish and Law, 1989; Le Graet and Brule, 1993). In our case, the calcium concentration in the aqueous phase was slightly decreased when skim milk was enriched with FeCl2 and unmodified when FeC13 was used (Fig. 4A). The inorganic phosphate concentration in the aqueous phase decreased linearly and independently of the iron salt used (Fig. 4B). Thus, the micellar phase was enriched with inorganic phosphate ions and some calcium ions had probably been exchanged between
Iron-,supplemented
skim milk
145
II
fA
a,,-CN
IV
0 06
Fe-a,,-CN
a
a-01
11
n
I
CA 004
IC
0 02 0 00
c
II
D
01
Fe-a,,-CN L
0 08 0 06 //A__
0 04 002 ZL 0 0
m
z
“7
w
2
::
Time (min) Fig. 2. Reversed-phase chromatographic profiles of milk proteins in the absence of iron (A) or in the presence of FeCl? (B) or FeCI, (C) at the concentration of 1.5 mM. Chromatographic profile (D) is for a,,-casein containing -I mole of iron mole casein-‘. Separation under linear gradient elution conditions used acetonitrile and triiluoroacetic acid (TFA). Solution A was 0. I % TFA in double-distilled water (v/v); solution B was 0. I”/0 TFA in 80/20 acetonitrilejdouble-distilled water (v,/v). Proteins (5Opg) were from 37 to 63% in separated, on a Cq column at 4O’C and at a flow rate of I mL min.‘, by increasing solution B concentration 30 min.
micellar phase and the aqueous phase. Similar displacements of phosphate ions from the aqueous phase to the micellar phase were observed after or magnesium additions to milk (van calcium Hooydonk e/ czl., 1986). The citrate concentration in the aqueous phase decreased as a function of iron concentration (Fig. 4C). To explain the decrease, several reasons can be evoked. Firstly, the formation of iron-citrate complexes which would be displaced from the aqueous to the micellar phase could occur. the
6.7
Mod~ficaiions
+ I
6.65
a
!
1
‘-. *,
/
\
6.6
6,66/
, 0
0.5
Precipitation of iron citrate was unlikely because addition of iron ions to milk ultrafiltrate did not lead to precipitation of iron (result not shown). Secondly, direct binding of iron to micellar phase could induce a solubilization of calcium ions from the micellar phase to the aqueous phase and then, formation of calciumcitrate and calcium-phosphate which could go again to the micellar phase. Thirdly, the formation of soluble not detectable by ion iron-citrate complexes chromatography coupled to a conductimetric detection is also possible (persona1 communication, Dionex, Jouy-en-Josas, France). Further work is necessary to a better understanding of iron addition consequences to the citrate system.
“‘1
1
Iron concentration
) 1.5
2
(mM)
Fig. 3. Influence of iron concentration on the pH of skim milk. The accuracy was ?E0.01 unit. (e) FeClz and (+ ) FeCls.
of’casein
micelles
In spite of protein modifications (Fig. 2) and of changes in mineral distribution after iron addition (Fig. 4), the average diameters of particles were constant and unmodified in the absence or in the presence of iron ions. The values were about I80 i: 5 nm for all skim milk samples (results not shown). On the contrary, the casein micelle hydration as a function of concentration of the two different iron salts decreased (Fig. 5). The decreases, which were significantly more pronounced in the presence of FeC13 than in the presence of FeC&, traduced a solvent exposure change of amino acid side-chains and of peptide bonds and consequently a release of water present into cavities located into the hydrophobic core.
F. Gaucheron et al.
146
1.
_I 0
0.6
1
2
1.6
Iron concentration (mM) Fig. 5. Influence of iron concentration on the casein micelle hydration. The accuracy was ?L0.02 g. (0) FeC12 and (+) FeCl3.
C -1
samples was lower than those found in the aqueous phase of unmodified milk. At the final pH (-4.0), the calcium and inorganic phosphate concentrations were exactly the same in the absence or in the presence of 1.5mM iron. These solubilizations correspond to a protonation of acidic groups of caseins (carboxyls, phosphates) and consequently to a direct solubilization of the micellar
Fig. 4. Influence of iron concentration
on the calcium (A), inorganic phosphate (B) and citrate (C) concentrations in the aqueous phase of skim milk obtained by ultrafiltration. The accuracies of calcium, inorganic phosphate and citrate concentrations were fl%. (0) FeC12 and ( + ) FeC13.
2 -
21.5
B
* !z.
E
19.6 -
*+
0 ‘3 Technological properties of iron-supplemented milk samples
5 g
It was also important to investigate the stability of iron-supplemented milk samples because they could be used as drinks, yogurts or cheeses, but also as food additives and thus undergo various treatments such as acidification, heat treatments and rennet coagulation. Acidification Whatever the nature of the iron salt used, iron was never solubilized in the studied pH range (from 6.7 to 4.0) (results not shown). The solubilization curves for calcium (Fig. 6A) and inorganic phosphate ions (Fig. 6B) as a function of pH were similar in the absence or in the presence of iron ions. However, before acidification, the inorganic phosphate concentration in the aqueous phase of iron-supplemented skim milk
0
s
15.6 17.5 -
-Ii
I
0,s I 3.5
4
4.6
s
6.6
6
6.6
7
PH
Fig. 6. Effect of acidification of iron-supplemented
skim milk samples (containing 1.5 mM iron) on concentrations of calcium (A) and inorganic phosphate (B) in the aqueous phase obtained by ultrafiltration. The accuracies of calcium and inorganic phosphate concentrations were f 1%. (0) control, ( + ) FeC12, and (.) FeC13.
Iron-supplemcwted
calcium phosphate (Chaplin, 1984; Dalgleish and Law, 1989; Le Graet and Brulk, 1993). From these results, we can suggest that there is no association of iron ions to colloidal phosphate because iron ions were not solubilized at the same time as calcium and inorganic phosphate ions. The absence of iron solubilization, as also observed during acidification of iron-supplemented caseinate in the same pH range (Gaucheron PI al., 1996a), indicates that this element would not be released of caseins by chemical or biological acidification of ironsupplemented milk or iron-supplemented dairy products. experiments have shown that during Previous acidification of yogurts, no release of iron in the aqueous phase was observed. Heut
147
skim milk 25 I
Al
tveatment,s
Heat treatments (15 or 30min at 95’C) of ironsupplemented skim milk samples (containing 1.5mM of iron) induced no visible heat instability: no coagulation or aggregation was observed and the RP-HPLC profiles were similar before and after heating (results not shown). Moreover, no transfer of iron ions from the micellar phase to the aqueous phase was found because the iron concentrations in ultrafiltration permeates of heated and no heated skim milk samples were less than 5% of the total concentration (results not shown). An absence of iron solubilization was also observed during heat treatments of iron-supplemented caseins (Gaucheron ez u/., 1996a). Rennet
J C:I
coagulution
Green and Marshall (1977), Marshall and Green (1980), Green (I 982), and Pierre (1983) have described acceleration of casein micelle rennet coagulation by divalent cations and polycations. Their interpretation was a neutralization of the negative charges of casein enhancement of hydrophobic and an micelles interactions between particles during aggregation and gel reticulation. On the contrary, van Hooydonk et al. (1986) described a decrease in the renneting process when zinc and copper ions were added to milk in the concentration range of 0 to 3mM. In our case, iron salts (especially FeC12) added to skim milk had the same surprising effects than those observed by van Hooydonk et al. (1986) because decreases in casein micelle rennet coagulations were observed. Figure A, B and C show the influence of iron concentration on the clotting time, aggregation time and on the curd firmness, respectively. The results were significantly different for both iron salts. Increases in FeCl* concentrations induced: (i) strong increases in clotting times; (ii) increases in the aggregation times; and (iii) decreases in the curd firmness. Increases in FeC13 concentrations induced slight increases in clotting time whereas the aggregation time increased and the curd firmness was slightly decreased. In parallel, the initial rates and the total amounts of CMP released after rennet addition to supplemented skim milk containing 1.5 mM of iron were not different to those observed with no iron-supplemented skim milk (results not shown). Thus, the primary phase of rennet coagulation was not affected by the presence of iron. These results suggest that: (i) chymosin was probably not modified by iron ions; (ii) xi-casein, which has one or two phosphoseryl residues per molecule (Lkonil et ul., 1995), was unmodified; and (iii) the preferential
A 0
0.5
1
iron Concentration
1.5
2
(mM)
Fig. 7. Influence of iron concentration on the rennet coagulation time (RCT) (A), on the aggregation time (B) and on the curd firmness ( + ) FeC13.
(C). The accuracies
were AI5%. (e) FeC& and
binding of iron to cr,,-casein did not affect the enzymatic hydrolysis of rc-casein. So, the secondary (aggregation of paracasein micelles) and tertiary (gel reticulation) phases could be altered by modifications of molecular interactions between paracasein micelles in the presence of iron ions. The modifications with (especially can FeC12) correspond to conformational changes of caseins detected by RPHPLC (Fig. 2B) or to physicochemical modifications of micellar calcium and inorganic phosphate (Fig. 4A, B and C) with formation of charged areas or to modifications on the structure of water associated with proteins (Fig. 5) or a combination of the three.
ACKNOWLEDGEMENTS The authors particle size.
thank
F. Michel for the determination
of
F. Gaucheron et al.
148 REFERENCES
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Fransson, G. B. and Lonnerdal, B. (1983) Distribution of trace elements and mineral in human and cow’s milk. Pediatric Research, 17, 912-915.
Gaucheron, F., Molle, D., Leonil, J. and Maubois, J. L. (1995) Selective determination of phosphopeptide /I-CN (1-25) in a /I-casein digest by adding iron: characterization by liquid chromatography with on-line electrospray ionization mass spectrometric detection. Journal ofChromatography B, 664, 1933200.
Gaucheron, F., Famelart, M. H. and Le Graet, Y. (1996a) Iron-supplemented caseins: preparation, physicochemical characterization and stability. Journal of Dairy Research, 63, 233-243.
Gaucheron, F., Le Graet, Y., Piot, M. and Boyaval, E. (1996b) Determination of anions of milk by ion chromatography. Lait, 76, 433-443.
Green, M. L. and Marshall, R. J. (1977) The acceleration by cationic materials of the coagulation of the casein micelles by rennet. Journal of Dairy Research, 44, 521-531. Green, M. L. (1982) Mode of binding of ionic materials to casein micelles. Journal of Dairy Research, 49, 99-105. Hegenauer, J., Saltman, P., Ludwig, D., Ripley, L. and Ley, A. (1979) Iron-supplemented cow milk. Identification and spectral properties of iron bound to casein micelles. Journal of Agricultural and Food Chemistry, 27, 12941301.
Hekmat, S., Weimer, B. and McMahon, D. J. (1994) Effect of iron fortification of yogurt on its manufacture, growth of lactic acid and spoilage bacteria, and lipid oxidation. Journal of Dairy Science, 77, 11 l-1 12 Suppl. 1, Abst. 19.6. Hurrell, R. F., Lynch, S. R., Trinidad, T. P., Dassenko, S. A. and Cook, J. D. (1989) Iron absorption in humans as influenced by bovine milk proteins. American Journal of Clinical Nutrition, 49, 546-552.
Jackson, S. and Lee, K. (1992) The effect of dairy products on iron availability. Critical Reviews in Food Science and Nutrition, 31, 259-210.
A. and Martin,
P. (1992) Reverse-phase
1461.
Le Graet, Y. and Brule, G. (1993) Les equilibres mineraux du lait: influence du pH et de la force ionique. Lait, 73, 51-60. Leonil, J. and Molle, D. (1991) A method for determination of glycomacropeptide by cation-exchange fast performance liquid chromatography and its use for following the action of chymosin in milk. Journal af Dairy Research, 58, 321328.
Lionil, J., MO&, D., Gaucheron, F., Arpino, P., Guenot, P. and Maubois, J. L. (1995) Analysis of major bovine milk proteins by on-line high-performance liquid chromatography and electrospray ionization-mass spectrometry. Lair, 75, 193-210.
Manson, W. and Cannon, J. (1978) The reaction of c(,i- and bcasein with ferrous ions in the presence of oxygen. Journal of Dairy Research, 45, 59-67.
Journal of Clinical Nutrition, 28, 487492.
Chaplin, L. C. (1984) Studies on micellar calcium phosphate: composition and apparent solubility product in milk over a wide pH range. Journal of Dairy Research, 51, 251-257. Dalgleish, D. G. and Law, A. J. R. (1989) pH-induced dissociation of bovine casein micelles II. Mineral solubilization and its relation to casein release. Journal af Dairy
Jaubert,
analysis of goat caseins. Identification of c(,i and c(,zgenetic variants. Lait, 72, 235-241. Kwock, R. O., Keen, C. L., Hegenauer, J., Saltman, P., Hurley, L. S. and Lonnerdal, B. (1984) Retention and distribution of iron added to cow’s milk and human milk as various salts and chelates. Journal of Nutrition, 114, 1454-
HPLC
Marshall, R. J. and Green, M. L. (1980) The effect of the chemical structure of additives on the coagulation of casein micelle suspensions by rennet. Journal of Dairy Research, 47, 359-369.
McMahon, D. J. and Brown, R. J. (1982) Evaluation of Formagraph for comparing rennet solutions. Journal of Dairy Science, 65, 1639-1642. Nagasako, Y., Saito, H., Tamura, Y., Shimamura, S. and Tomita, M. (1993) Iron-binding properties of bovine lactoferrin in iron-rich solution. Journal of Dairy Science, 76, 18761881. Pierre, A. (1983) Influence de la modification de la charge des micelles de caseine sur le taux du castinomacroneotide lib&e par la presure au moment de la coagulation hu lait. Lait, 63, 217-229.
Reddy, I. M. and Mahoney, A. W. (1991) A study of the interaction of Fe(II1) with cc,,-casein using ultraviolet and fluorescence spectroscopy. Journal of Dairy Science, 74, Supplement 1. Abstract 6745. Reddy, I. M. and Mahoney, A. W. (1992) Effect of ferric chloride on chymosin hydrolysis and rennet clotting time of milk. Journal of Dairy Science, 75, 2648-2658. Shears, G. E., Ledward, D. A. and Neale, R. J. (1987) Iron complexation to carboxyl groups in a bovine serum albumin digest. International Journal of Food Science and Technology, 22, 265-272.
van Hooydonk, A. C. M., Hagedoorn, H. G. and Boerrigter, I. J. (1986) The effect of various cations on the renneting of milk. Netherlands Milk and Dairy Journal, 40, 369-390. Visser, S., Slanger, C. and Rollema, H. S. (1991) Phenotyping of bovine milk proteins by reversed-phase high-performance liquid chromatography. Journal of Chromatography, 548, 361-370.
Webb, J., Multani, J. S., Saltman, P., Beach, N. A. and Gray, H. B. (1973) Spectroscopic and magnetic studies of iron (III) phosvitins. Biochemistry, 12, 1797-1802. Zhang, D. and Mahoney, A. W. (1989a) Bioavaibility of ironmilk-protein complexes and fortified Cheddar cheese. Journal of Dairy Science, 72, 2845-2855.
Zhang, D. and Mahoney, A. W. (1989b) Effect of iron fortification on quality of Cheddar cheese. Journal of Dairy Science, 72, 322-332.
Zhang, D. and Mahoney, A. W. (1990) Effect of iron fortification on quality of Cheddar cheese - 2. Effects of aging and fluorescence light on pilot scale cheeses. Journal of Dairy Science, 73, 2252-2258.
Zbang, D. and Mahoney, A. W. (1991) Iron fortification of process Cheddar cheese. Journal of Dairy Science, 74,353358.
ELSEVIER
SO958-6946(96)00054-4
O958-6946!97/$17.00
+ 0.00
Physicochemical Characterization of Ironsupplemented Skim Milk FrkdCric Gaucheron,* Yvon Le Graet, Karine Raulot and Michel Piot Luhorutoire
de Rechrrchm
de Technologie LuitikJ,
(Received
Institut Nutional de lu Recherche Agronomique, Runes Cedex, Frunca 28 June 1996; accepted
8 November
65, YWJ de Suint-Brieuc.
35042
1996)
ABSTRACT The physicochemical characterization and study of technological properties of skim milk samples supplemented with FeC& or FeC13 were carried out. The iron concentrations ranged from 0 to 1.5 mM. For both salts used, iron was mainly bound to the micellar phase with a probable change of casein structure. In parallel, the mineral distribution and the casein micelle hydration changed differently as a function of the initial oxidation state of iron (Fe”’ or Fe’+). After acidification (between 6.7 and 4.0) or after heat treatments (95”CP15 and 30min), no release of iron was found. Rennet coagulation parameters (clotting time, aggregation time and curd firmness) of the different iron-supplemented skim milk samples were more modified when fortifications were performed with FeClz than with FeCI-(. The results were discussed in relation with proteins and minerals modifications. 0 1947 Elsevier Science Ltd
the meal which can increase or decrease the absorption of iron and the physiological state of the tested subject. For Carmichael et al. (1975), the gastrointestinal absorption of iron bound to purified caseins or to casein micelles was similar or higher than those obtained with ferrous salts. Hurrell rt al. (1989) reported that caseins are responsible for the low bioavailability of iron. Zhang and Mahoney (1989a) indicated that the bioavailability of iron incorporated into Cheddar cheese is similar to that of FeS04. Besides the biochemical and nutritional studies, few applications of technological dairy products fortification are described. Zhang and Mahoney (1989b, 1990, 1991) show that Cheddar fortification (40-50 mg kg-‘) is possible without affecting its quality. Reddy and Mahoney (1992) indicate that ferric chloride at a concentration of 0.2mM does not significantly affect the rennet clotting time. However, the effect of FeC13 on the coagulation of renneted micelles seems to relate to the thermal history of milk because ferric chloride decreases rennet clotting time when it is added to uncooled raw milk or to milk before pasteurisation but increases rennet clotting time when it is added to cooled raw milk or to milk after pasteurization. The effect of iron fortification of yogurt on its manufacture, the growth of lactic acid and spoilage bacteria, and on lipid oxidation reveals that iron-supplemented yogurt is a suitable vehicle for delivering iron to consumers (Hekmat et ul., 1994). The aims of this study were to prepare, characterize and appreciate the technological properties of ironsupplemented skim milk samples with two different iron salts: FeC12 and FeC13. They were added to skim milk samples in an iron concentration range of O1.5 mM. The physicochemical modifications (proteins and mineral distribution) were determined and related to the modifications of technological properties.
INTRODUCTION Milk is relatively poor in iron (0.2-0.4 mg L-‘) and this trace element is essential in animal and human nutrition. Iron participates to the structures of cytochromes and several enzymes. It is also a component of haem in haemoglobin and of myoglobin in which it plays an important role in the transport, storage and utilization of oxygen. Iron deficiency results in anaemia, which affects about 30% of the world’s population (especially women and teenagers). An iron fortification of milk and of dairy products could be considered as a potential approach to prevent anaemia. Iron, when added as FeC12 to skim milk, 85% is bound to caseins (72, 21 and 4% are bound to us-, /& and K-caseins, respectively) (Demott and Dincer, 1976). The strong affinity of caseins for iron is recognized and clustered phosphoseryl residues attributable to (Manson and Cannon, 1978; Hegenauer et ul., 1979; Gaucheron et al., 1995). The formation of iron-casein complexes induces the oxidation of iron from the ferrous to the ferric state (Manson and Cannon, 1978; Emery, 1992). In these complexes, iron is strongly bound to phosphoseryl residues because no release of iron from iron-supplemented caseinate was found after its acidification (between pH 6.5 and 4.0), after heat treatments (50, 70 and 90°C for 15 min) or in the presence of 20 mM Na2HP04 (Gaucheron et al., 1996a). Results concerning iron bioavailability and tissue distribution are conflicting (Kwock et al., 1984; Jackson and Lee, 1992) because these characteristics are not easy to evaluate. Indeed, they depend on different factors such as the chemical form of iron added (ferric, ferrous) (chloride, sulfate, lactate, gluconate, citrate), the presence of other components in *Corresponding
author. 141
142
F. Gaucheron
MATERIALS AND METHODS Milk Bulk whole milk (2OL), collected and stored at 4°C (Triballat, Noyal-sur-Vilaine, France), was warmed and skimmed at 40°C (30min). Then, the skim milk obtained was cooled to 20°C and sodium azide (w/w) (Fluka Chemie, Buchs, Switzerland) was added to 0.02% to prevent bacterial growth. Iron salts FeC12.4H20 and FeC13.6H20 were purchased from Merck (Darmstadt, Germany). The stock solutions of iron (50mM) were prepared just before their addition to skim milk samples. The pH values of these stock solutions were 3.5 and 1.9 for FeCl* and FeC13, respectively. Preparation of iron-supplemented skim milk samples Skim milk samples (500mL) were enriched, at room temperature, with both iron salts to produce final iron concentrations ranging from 0 to 1.5mM (previous experiments showed that protein precipitation occurred when iron concentration added to milk was about 8 mM). Deionized water was added to compensate for volume changes in milk. These mixtures were stirred vigorously to ensure rapid and complete mixing. Before analyses or technological treatments (acidification, heat treatments or rennet coagulation), the different iron-supplemented skim milk samples were left standing overnight at room temperature. Preparations of iron-supplemented skim milk samples and subsequent analyses were carried out in triplicate. Physicochemical characterization of iron-supplemented skim milk samples The spectrofluorimetric measurements were made on an LS 50B Perkin Elmer spectrofluorimeter (Saint Quentin en Yvelines, France). Firstly, the intrinsic fluorescence was determined on skim milk samples after a l/70 dilution into 50mM Tris HCl, pH 7.6. The excitation and emission wavelengths were 295 and 350nm. For both these wavelengths, slit widths were 2.5nm. Secondly, the exposure of hydrophobic groups was measured by the fluorescent probe binding method of Bonomi et aE. (1988). Fluorophor 8-aniline naphtalen 1-sulphonate (ANS) (Sigma Chemical Co, St Louis, USA) was used. When interacting with hydrophobic sites of proteins, its fluorescence parameters were an excitation wavelength of 390nm and an emission wavelength of 480nm. The emission and excitation slits were both set at 2.5 nm band width. The different milk samples (1 mL) were diluted with 50mM Tris HCl buffer, at pH 7.6, to a final volume of lOmL, and titrated at room temperature with an aqueous solution of ANS (final concentration between 0 and 300~~~) until no further increase in fluorescence was observed. Analysis of binding data allowed us to determine values of the maximum fluorescence attainable at saturating ANS concentration (F,,,, i.e. asymptotic value of titration curve).
et al.
Caseins, a-lactalbumin and /?-lactoglobulin of milk containing 1.5 mM iron were separated by reversedphase (RP) HPLC on a C4 column as described by Jaubert and Martin (1992). RP-HPLC analyses of proteins are commonly performed after sample dissolution in a buffer containing a reducing agent (e.g. 2-mercaptoethanol or dithiothreitol) to improve the chromatographic separation (Jaubert and Martin, 1992). In our case, as both these chemical compounds have an affinity for elemental iron, this chemical reduction was not performed. Eluted peaks were detected by absorbance at 214nm. In parallel, ironmodified a,,-casein was applied to the C4 column. The cc,,-casein was purified as described by Brignon et al. (1977) and modified to have about 1 mol iron mole” casein. The pH value was measured with a Titroprocessor 686 pH meter (Metrohm, Herisau, Switzerland). The milk aqueous phase was obtained either by ultrafiltration on Centriflo CF 25 (molecular mass cutoff: 25,000 Da; Amicon, Epernon, France) after centrifugation at 500g for 1 h or by ultracentrifugation at 77,000 g (Beckman L-8-55 ultracentrifuge, Gagny, France) for 2 h at 20°C. From the wet pellet obtained by ultracentrifugation, the determination of casein micelle hydration was carried out. Briefly, the drained casein pellet was weighed and then dried at 103°C for 7 h to remove the water of hydration. The difference between the weight before and after drying, expressed as g of water per g of dry casein micelle pellet, was taken as the water of hydration. Iron and calcium concentrations in ultrafiltrates and in ultracentrifugal supernatants were evaluated by atomic absorption spectrometry (Varian AA300 spectrometer, Les Ulis, France) (Bruli: et al., 1974). Inorganic phosphate and citrate concentrations in ultrafiltration permeates were determined by ion chromatography (Gaucheron et al., 1996bj. A laser granulometer (N4MD Coulter, Coultronics, Margency, France) was used to evaluate the average diameter of particles at 20°C. The milk sample was diluted into 20mM imidazole/HCl buffer, pH 6.6, containing 5 mM CaC12 and 50 mM NaCl, stabilised for 1Omin inside the cell holder, and a measurement of light intensity scattered at 90” for 600s was carried out. Acidification of skim milk samples containing 1.5 mM of iron The effects of acidification by 1 M HCl (pH from 6.7 to 4.0) on skim milk samples containing 1.5 mM of iron were studied. After acidification, samples were left standing overnight at room temperature. Then, pH values were again controlled. A control sample (without iron) was tested under the same experimental conditions. Heat treatments of skim milk samples containing 1.5 mM of iron The effects of heat treatments (15 and 30 min at 95°C) on skim milk samples containing 1.5 mM of iron were studied: for this, 1OmL of milk sample in sealed Pyrex tube were submerged in an oil-bath and agitated during heating. Three hours before heating, the pH of
Iron-supplemented skim milk samples was adjusted to 6.70 with 1 M NaOH and readjusted if necessary just before heating. A control sample (without iron) was tested under the same experimental conditions. Rennet coagulation and determination peptide release (CMP)
of caseinomacro-
The rennet clotting time, the aggregation time and the curd firmness were determined by using a Formagraph (Foss Electric, Paris, France) (McMahon and Brown, 1982). Three hours before measurements, pH values of milk samples heated at 30°C were adjusted to 6.70 with 1 M NaOH and readjusted if necessary just before rennet addition. Thus, the effects that we observed were due solely to iron salts. A 0.5% rennet solution (Hansen, Saint Germain les Arpajons, France) was used. The rate of chymosin hydrolysis was determined on the basis of released CMP, as described by Lconil and Molle (1991). Experiments were carried out with no modified skim milk and with iron-supplemented skim milk samples containing 1.5mM of iron ions. The CMP exclusion determined by concentration was chromatography (chromatographic column ProgelTMTSK G3000 SW, Supelco, Saint Germain-en-Laye, France) (personal communication, Nutrinov, Rennes, France). The CMP was eluted by a solution containing 30% acetonitrile and 0.1 o/o trifluoroacetic acid. Detection was performed by absorbance at 214nm. The CMP was identified and quantified by comparison with a CMP standard prepared in our laboratory.
RESULTS
AND DISCUSSION
Physicochemical skim milk
characterization
of iron-supplemented
Distribution of’ iron Table 1 indicates the concentrations and the percentages of iron found in the aqueous phase ultracentrifugation or either by obtained ultrafiltration. Whatever concentration and nature of salt the percentage of iron in iron used, ultracentrifugal supernatants was always higher than those obtained in ultrafiltrates. The percentage of iron slightly decreased in the iron concentration range used: between 11 .O to 9.3% or 9.8 to 7.8% in the ultracentrifugal supernatants of iron-supplemented skim milk samples with FeC12 and FeC13, respectively, and between 5.6 to 4.5% or 3.6 to 3.1% in the ultrafiltrates of iron-supplemented skim milk samples with FeC12 and FeC13, respectively. Thus, when iron
143
was added to milk in the concentration range of 0 to 1.5 mM, 89% and more of iron were bound to the colloidal phase (Table 1). The proportionality between the binding of iron to the colloidal phase and the added concentration suggests that the binding sites were not saturated and had the same affmity for iron ions in the iron concentration range used. Presumably, the iron salts used did not precipitate in the aqueous phase of skim milk because their addition (final iron concentration 1.5 mM) to milk ultrafiltrate induced no precipitation (results not shown). Moreover, as described by Gaucheron et al. (1996a), the formation of large hydroxide complexes which eventually precipitate and are not able to pass through the ultrafiltration membrane was unlikely. The important modification of the colloidal phase with FeQ is in agreement with the results of Demott and Dincer (1976). However, the several possible forms of micellar iron (associations of iron ions with caseins or/and with colloidal calcium phosphate or/and with citrate molecules) were not distinguishable because it is impossible by differential centrifugation or column fractionation techniques to obtain casein micelles, colloidal calcium phosphate and colloidal citrate as separate fractions. The presence of whey proteins, in ultracentrifugal supernatant, able to bind iron (c(lactalbumin, b-lactoglobulin, lactoferrin) (Baumy and Brule, 1988; Nagasako et al., 1993) explains the higher percentages of iron ions found in this aqueous phase (Table 1) even though these proteins are absent in milk ultrafiltrates. Iron ions, in the ultrafiltrates, are free or bound to small molecules, named LOW Molecular Weight fraction by Fransson and Lonnerdal (1983) which are able to pass through the ultrafiltration membrane. These small molecules, not identified in this study, can be citrate, organic and inorganic phosphate, lactose (Bachran and Bernhard, 1980) sulfur-containing compounds or erotic acid. Mod$cations qf’prateins Firstly, the intrinsic fluorescence decreased as a function of iron concentration (Fig. 1A) and traduced conformatronal change of milk proteins. Secondly, the structural modification of milk proteins was confirmed because the fluorescence binding curves as a function of ANS concentration were different in the presence or absence of iron ions. The F,,, values decreased as a function of iron concentration (Fig. IS), but no significant difference between iron-supplemented skim milk samples with FeCl2 or FeC17 was observed. RPHPLC analyses showed differences between ironsupplemented skim milk samples and unmodified skim milk (Fig. 2). In the absence of iron ions (Fig. 2A), five fractions were detected (noted I to V).
Table 1. Concentrations and Percentages of Iron in the Aqueous Phase of Iron-supplemented Phases were Obtained by Ultracentrifugation or by Ultrafiltration. Control Corresponds Control Total iron (mM) ultracentrifugable ultrafiltrable
iron
iron (PM)
(PM)
(“XI)
(%)
0 0 0
Skim Milk Samples. The Aqueous to the Unsupplemented Milk. FeC13
FeClz 0.50 (E%) 28 (5.6%)
1.oo 96 (9.6%) 49 (4.9%)
1so 140 (9.3%) 67 (4.5%)
0.50 49 (9.8%) 18 (3.6%)
1.oo 87 (8.7%) 35 (3.5%)
1.50 117 (7.8%) 47 (3.1%)
F. Gaucheron et
144
56’ 0
I
0.5
1
1.5
2
Iron concentration (mM) Fig. 1. Influence of iron concentration on the intrinsic fluorescence intensity (A) and on the exposure of hydrophobic regions (B) of milk proteins. The accuracies were i 5 unit (A) and & 2% (B), respectively. (*) FeClz and (+ ) FeC&.
Fraction I contained K- and a,*-caseins. Because of low concentrations (3 and 4g L-‘, respectively), heterogeneity (the presence of various glycoforms of rc-casein) and the absence of reduction treatment, the chromatographic separation of both these proteins was incomplete. Fractions II and III contained aSi-casein and c(lactalbumin, respectively. Fraction IV contained the A’ and A2 genetic variants of p-casein and fraction V, the A and B genetic variants of /I-lactoglobulin. The genetic variants of /I-casein and of /I-lactoglobulin differ in their amino acid sequences (Visser et al., 1991). In the presence of FeCl2 or FeCl3 (1.5 mM), the chromatographic profiles were modified (arrows on the chromatogram of Fig. 2B and 2C) because the c(,icasein chromatographic areas (14.4 and 16.2% of total area in the presence of 1.5 mM FeClz and FeCls, respectively vs 22.8% in the absence of iron) and retention times (18.25 min in the presence of 1.5 mM iron vs 19.7min in the absence of iron) were changed. These modifications were confirmed by chromatographic injection of iron-modified a,,-casein (Fig. 2D). Thus, cr,i-casein was more modified than other caseins. Retention times and chromatographic areas of tllactalbumin and /&lactoglobulin were not modified. Moreover, whichever iron salt added, the modifications of chromatographic profiles were qualitatively and quantitatively similar. The decreases in intrinsic fluorescence intensities and to the added iron in F,,,,, values proportional
al.
concentration indicated structural modifications of milk proteins because when a protein is disorganised, hydrophobic segments can be differently exposed. From the chromatographic analyses of caseins, of CIlactalbumin and of p-lactoglobulin (Fig. 2) we demonstrated that iron element is bound mainly to asIresidues per casein, which has 7-9 phosphoseryl molecule (Leonil et al., 1995). The iron binding to presidues per casein, which has five phosphoseryl molecule (Leonil et al., 1995), and aSz-casein, which is the most phosphorylated of caseins (lo-13 phosphoseryl residues per molecule (Leonil et al., 1995)), is also possible but was not observed. The preferential modification of a,i-casein supports the results of Demott and Dincer (1976). The complex formation that takes place at phosphoseryl residues would change the protein structures in such a manner that a,,-casein would be differently exposed to the modified silica matrix and consequently decrease the chromatographic retention time. Reddy and Mahoney (199 l), using ultraviolet and fluorescence spectroscopy, also showed a conformational change in bovine aSIcasein on interaction with Fe (III). In these complexes, iron is probably bound to casein molecules via the oxygen of the phosphoseryl residues by coordination link (Hegenauer et al., 1979). However, iron ions may also bind to casein molecules via tyrosine, glutamic and aspartic residues. Iron complexation to carboxyl groups in a bovine serum albumin digest was observed by Shears et al. (1987). In these complexes with caseins, iron would adopt a tetrahedral coordination structure as observed with ferric iron saturated phosvitin (Webb et al., 1973). Thus, from fluorescence and chromatographic studies and as observed by Gaucheron et al. (1996a), changes in the local environment of caseins can be deduced. A casein conformational change in the presence of iron ions and/or an association of casein molecules via iron bridges can be proposed. These changes of protein structures seem to be independent of the initial oxidation state of iron added (Fe2+ or Fe3+). Mod$cations
of mineral distribution
In the absence of iron ions, the pH value of skim milk was 6.73. Addition of iron ions to skim milk resulted in pH decreases (Fig. 3) which were significantly greater with FeC13 than with FeClz. These pH decreases were related to the acidities of iron solutions and to exchanges between iron ions added and micellar bound Hf. The characterization of iron-phosphopeptide /I-casein (l-25) complexes by electrospray-ionization mass spectrometry (Gaucheron et al., 1995) showed a release of three protons for one bound iron atom. The consequences of pH decrease are displacements of calcium, inorganic phosphate and citrate ions from the colloidal phase to the aqueous phase (Chaplin, 1984; Dalgleish and Law, 1989; Le Graet and Brule, 1993). In our case, the calcium concentration in the aqueous phase was slightly decreased when skim milk was enriched with FeCl2 and unmodified when FeC13 was used (Fig. 4A). The inorganic phosphate concentration in the aqueous phase decreased linearly and independently of the iron salt used (Fig. 4B). Thus, the micellar phase was enriched with inorganic phosphate ions and some calcium ions had probably been exchanged between
Iron-,supplemented
skim milk
145
II
fA
a,,-CN
IV
0 06
Fe-a,,-CN
a
a-01
11
n
I
CA 004
IC
0 02 0 00
c
II
D
01
Fe-a,,-CN L
0 08 0 06 //A__
0 04 002 ZL 0 0
m
z
“7
w
2
::
Time (min) Fig. 2. Reversed-phase chromatographic profiles of milk proteins in the absence of iron (A) or in the presence of FeCl? (B) or FeCI, (C) at the concentration of 1.5 mM. Chromatographic profile (D) is for a,,-casein containing -I mole of iron mole casein-‘. Separation under linear gradient elution conditions used acetonitrile and triiluoroacetic acid (TFA). Solution A was 0. I % TFA in double-distilled water (v/v); solution B was 0. I”/0 TFA in 80/20 acetonitrilejdouble-distilled water (v,/v). Proteins (5Opg) were from 37 to 63% in separated, on a Cq column at 4O’C and at a flow rate of I mL min.‘, by increasing solution B concentration 30 min.
micellar phase and the aqueous phase. Similar displacements of phosphate ions from the aqueous phase to the micellar phase were observed after or magnesium additions to milk (van calcium Hooydonk e/ czl., 1986). The citrate concentration in the aqueous phase decreased as a function of iron concentration (Fig. 4C). To explain the decrease, several reasons can be evoked. Firstly, the formation of iron-citrate complexes which would be displaced from the aqueous to the micellar phase could occur. the
6.7
Mod~ficaiions
+ I
6.65
a
!
1
‘-. *,
/
\
6.6
6,66/
, 0
0.5
Precipitation of iron citrate was unlikely because addition of iron ions to milk ultrafiltrate did not lead to precipitation of iron (result not shown). Secondly, direct binding of iron to micellar phase could induce a solubilization of calcium ions from the micellar phase to the aqueous phase and then, formation of calciumcitrate and calcium-phosphate which could go again to the micellar phase. Thirdly, the formation of soluble not detectable by ion iron-citrate complexes chromatography coupled to a conductimetric detection is also possible (persona1 communication, Dionex, Jouy-en-Josas, France). Further work is necessary to a better understanding of iron addition consequences to the citrate system.
“‘1
1
Iron concentration
) 1.5
2
(mM)
Fig. 3. Influence of iron concentration on the pH of skim milk. The accuracy was ?E0.01 unit. (e) FeClz and (+ ) FeCls.
of’casein
micelles
In spite of protein modifications (Fig. 2) and of changes in mineral distribution after iron addition (Fig. 4), the average diameters of particles were constant and unmodified in the absence or in the presence of iron ions. The values were about I80 i: 5 nm for all skim milk samples (results not shown). On the contrary, the casein micelle hydration as a function of concentration of the two different iron salts decreased (Fig. 5). The decreases, which were significantly more pronounced in the presence of FeC13 than in the presence of FeC&, traduced a solvent exposure change of amino acid side-chains and of peptide bonds and consequently a release of water present into cavities located into the hydrophobic core.
F. Gaucheron et al.
146
1.
_I 0
0.6
1
2
1.6
Iron concentration (mM) Fig. 5. Influence of iron concentration on the casein micelle hydration. The accuracy was ?L0.02 g. (0) FeC12 and (+) FeCl3.
C -1
samples was lower than those found in the aqueous phase of unmodified milk. At the final pH (-4.0), the calcium and inorganic phosphate concentrations were exactly the same in the absence or in the presence of 1.5mM iron. These solubilizations correspond to a protonation of acidic groups of caseins (carboxyls, phosphates) and consequently to a direct solubilization of the micellar
Fig. 4. Influence of iron concentration
on the calcium (A), inorganic phosphate (B) and citrate (C) concentrations in the aqueous phase of skim milk obtained by ultrafiltration. The accuracies of calcium, inorganic phosphate and citrate concentrations were fl%. (0) FeC12 and ( + ) FeC13.
2 -
21.5
B
* !z.
E
19.6 -
*+
0 ‘3 Technological properties of iron-supplemented milk samples
5 g
It was also important to investigate the stability of iron-supplemented milk samples because they could be used as drinks, yogurts or cheeses, but also as food additives and thus undergo various treatments such as acidification, heat treatments and rennet coagulation. Acidification Whatever the nature of the iron salt used, iron was never solubilized in the studied pH range (from 6.7 to 4.0) (results not shown). The solubilization curves for calcium (Fig. 6A) and inorganic phosphate ions (Fig. 6B) as a function of pH were similar in the absence or in the presence of iron ions. However, before acidification, the inorganic phosphate concentration in the aqueous phase of iron-supplemented skim milk
0
s
15.6 17.5 -
-Ii
I
0,s I 3.5
4
4.6
s
6.6
6
6.6
7
PH
Fig. 6. Effect of acidification of iron-supplemented
skim milk samples (containing 1.5 mM iron) on concentrations of calcium (A) and inorganic phosphate (B) in the aqueous phase obtained by ultrafiltration. The accuracies of calcium and inorganic phosphate concentrations were f 1%. (0) control, ( + ) FeC12, and (.) FeC13.
Iron-supplemcwted
calcium phosphate (Chaplin, 1984; Dalgleish and Law, 1989; Le Graet and Brulk, 1993). From these results, we can suggest that there is no association of iron ions to colloidal phosphate because iron ions were not solubilized at the same time as calcium and inorganic phosphate ions. The absence of iron solubilization, as also observed during acidification of iron-supplemented caseinate in the same pH range (Gaucheron PI al., 1996a), indicates that this element would not be released of caseins by chemical or biological acidification of ironsupplemented milk or iron-supplemented dairy products. experiments have shown that during Previous acidification of yogurts, no release of iron in the aqueous phase was observed. Heut
147
skim milk 25 I
Al
tveatment,s
Heat treatments (15 or 30min at 95’C) of ironsupplemented skim milk samples (containing 1.5mM of iron) induced no visible heat instability: no coagulation or aggregation was observed and the RP-HPLC profiles were similar before and after heating (results not shown). Moreover, no transfer of iron ions from the micellar phase to the aqueous phase was found because the iron concentrations in ultrafiltration permeates of heated and no heated skim milk samples were less than 5% of the total concentration (results not shown). An absence of iron solubilization was also observed during heat treatments of iron-supplemented caseins (Gaucheron ez u/., 1996a). Rennet
J C:I
coagulution
Green and Marshall (1977), Marshall and Green (1980), Green (I 982), and Pierre (1983) have described acceleration of casein micelle rennet coagulation by divalent cations and polycations. Their interpretation was a neutralization of the negative charges of casein enhancement of hydrophobic and an micelles interactions between particles during aggregation and gel reticulation. On the contrary, van Hooydonk et al. (1986) described a decrease in the renneting process when zinc and copper ions were added to milk in the concentration range of 0 to 3mM. In our case, iron salts (especially FeC12) added to skim milk had the same surprising effects than those observed by van Hooydonk et al. (1986) because decreases in casein micelle rennet coagulations were observed. Figure A, B and C show the influence of iron concentration on the clotting time, aggregation time and on the curd firmness, respectively. The results were significantly different for both iron salts. Increases in FeCl* concentrations induced: (i) strong increases in clotting times; (ii) increases in the aggregation times; and (iii) decreases in the curd firmness. Increases in FeC13 concentrations induced slight increases in clotting time whereas the aggregation time increased and the curd firmness was slightly decreased. In parallel, the initial rates and the total amounts of CMP released after rennet addition to supplemented skim milk containing 1.5 mM of iron were not different to those observed with no iron-supplemented skim milk (results not shown). Thus, the primary phase of rennet coagulation was not affected by the presence of iron. These results suggest that: (i) chymosin was probably not modified by iron ions; (ii) xi-casein, which has one or two phosphoseryl residues per molecule (Lkonil et ul., 1995), was unmodified; and (iii) the preferential
A 0
0.5
1
iron Concentration
1.5
2
(mM)
Fig. 7. Influence of iron concentration on the rennet coagulation time (RCT) (A), on the aggregation time (B) and on the curd firmness ( + ) FeC13.
(C). The accuracies
were AI5%. (e) FeC& and
binding of iron to cr,,-casein did not affect the enzymatic hydrolysis of rc-casein. So, the secondary (aggregation of paracasein micelles) and tertiary (gel reticulation) phases could be altered by modifications of molecular interactions between paracasein micelles in the presence of iron ions. The modifications with (especially can FeC12) correspond to conformational changes of caseins detected by RPHPLC (Fig. 2B) or to physicochemical modifications of micellar calcium and inorganic phosphate (Fig. 4A, B and C) with formation of charged areas or to modifications on the structure of water associated with proteins (Fig. 5) or a combination of the three.
ACKNOWLEDGEMENTS The authors particle size.
thank
F. Michel for the determination
of
F. Gaucheron et al.
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