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High pressure-induced gel formation of milk and whey concentrates
0 PII:
SO260-8774(98)00048-X
High Pressure-induced
Gel Formation of Milk and Whey Concentrates
M.-H. Famelart,U* L. Chapron, “INRA,
Jorrrrd of’hm/ 0z,y;nrenn~ 36 ( IYYX) 13Y- If14 1008 Elscvier Science Limited. All rights I-csetvcd Printed in Great Britain 0?60-X774iYX $lY.llO f’l.00
M. Piot,” G. BruEh & C. Durier”
Laboratoire
de Recherches de technologie Laitikre, 65, rue de St Brieuc, 35042 Rennes Cedex, France “ENSAR, 65, rue de St Brieuc, 35042 Rennes Cedex, France ’ INRA, Laboratoire de Biomktrie, Route de St Cyr, 78026 Versailles Cedex, France (Received
19 September
1997; accepted
12 March 1998)
ABSTRACT Gels j?om milk concentrates [milk and caseinate powder; ultrafiltration (UF) and microfiltration (MF)] an d wh ey concentrates (UF) were obtained with high pressure (200 and 400 MPa, 10 and 30 min). The effects of protein concentration (66-114gkg -’ for milk and 97-1273 kg-’ for whey), NaCl addition (0-8g kg -I), sodium citrate addition (O-4 g kg-‘) and pH (5.2-6.6 jar milk and 7-9 for whey) were studied with a Box-Behnken design. Milk .supplemented with caseinate powder did not lead to gel formation by pressure. For UF or MF milk concentrates, a pH decrease towards 5.9 and a protein content increase led to firmer pressure-set gels. Gels of whey concentrates were obtained only at pH 9. A pressure increase from 200 to 400 MPa led to firmer gels, while a protein content increase did not. 0 1998 Elsevier Science Limited. All rights reserved
INTRODUCTION High pressure processes have been increasingly applied to proteins, either for proteolysis and enzyme inactivation, or texture modification, aggregation or gelation. High pressure is known to affect only non-covalent interactions, i.e. intra- or interVan der Waals and electrostatic molecular hydrogen bonding, hydrophobic, interactions. According to Messens et al. (1997), only electrostatic and hydrophobic interactions are sensitive to pressure. *To whom correspondence
should be addressed. 149
150
M.-H. Fumelurt et al.
Protein gelation is the main process involved in food texture properties. Various food protein systems such as fish and animal meat, soybean protein dispersions, egg yolk and white, and whey protein gel after a heat treatment. Recently, hydrostatic pressure has been successfully applied to these food protein systems (Hayashi et al., 1989; Okamoto et al., 1990; Van Camp & Huyghebaert, 1995b). Hence, high hydrostatic pressure treatment is a potential tool for the gelation of food protein systems, as an alternative to heat treatments. It can be used to create new products or texture without any detrimental thermal modifications (Messens et al., 1997). According to the unique study on concentrated milk gelation under high pressure (Kumeno et al., 1993) high pressure treatment at 300-600 MPa for 5 min of freezeconcentrated milk at 25 wt% solid content leads to gelation. Using powdered milk or freeze-dried milk even at 25 wt% solid content or higher, does not lead to gel formation. A pressure increase from 300 to 600 MPa leads to stronger gels. According to Van Camp & Huyghebaert (1995a), a whey protein concentrate (WPC) reconstituted in a 50 mM phosphate buffer at pH 7 treated at 400 MPa for 30 min will gel. These pressure-set gels were compared to heat-set gels (Van Camp & Huyghebaert, 1995b). An increase in protein concentration from 110 to 180 g 1-I leads to a gel strength increase (Van Camp & Huyghebaert, 1995a), but in Van Camp & Huyghebaert (1995b), the increase in moduli was reduced between 150 and 180 g 1-l. According to Zasypkin et al. (1996), the rigidity of pressure-set gels from P-lactoglobulin (/3-lg) did not change with the protein concentration increase from 190 to 210 g kg-‘. An increase in the pressure level from 200 to 400 MPa leads to a gel strength increase, apart from the WPC at 150 g 1-l protein concentration, whose gel strength is constant between 300 and 400 MPa (Van Camp & Huyghebaert, 1995a). The effect of pH on pressure-set gels from WPC reconstituted in 50 mM buffer salts has been studied at 140 g 1-l protein concentration after a 400 MPa treatment for 30 min (Van Camp & Huyghebaert, 1995a). The gel strength increases from pH 5 to 8 and is constant between pH 8 and 9. In the absence of buffer salts, the strength increases between pH 5 and 6, and remains almost constant for higher pH values (Van Camp & Huyghebaert, 1995a). In the present paper, the effect of high pressure treatment of milk and whey concentrates on gel formation was studied. Various physicochemical conditions were tested to determine their impact on pressure-induced gelation. The physicochemical factors were pH, protein content, sodium citrate and NaCl concentration. Their levels were chosen for their ability to induce limited changes in protein structures, which can modify protein interactions and hence, gel firmness. Previous papers on WPC gels obtained after high pressure treatment only deal with factors studied one by one. For milk concentrates the effects of these factors have not been studied. The objectives were first, to determine the optimum parameters by use of a suitable experimental design, and secondly to better understand the protein-protein interactions within the gels. MATERIALS
AND METHODS
Dairy concentrates Values of nitrogen matters either for powders or concentrates Kjeldahl method with 6.38 as the converting factor.
were obtained
by the
High pressure-induced gels q f’duity corlcerltrutes
lil
Milk concentrates Milk was reconstituted from low heat skim milk powder (INRA, France) at 10 g of dry matter for 110 g of final milk and was supplemented with sodium caseinate (Armor protkines, St Brice en Cog& France) to reach protein concentrations of 9.3, 116 and 140 g kg- ‘. This was obtained with a weight ratio of sodium caseinate to milk powder of 0.77, 1.04 and 1.31, respectively. Raw skim milk (Triballat, Noyal sur Vilaine, France) was ultrafiltered or microfiltered on a 2S37 module with Ml membranes (Techsep, 100000 - 150000 Daltons nominal molecular weight cutoff [nmco], 1.6 m’) and with Ml4 membranes (Techsep, pore diameter 0.14 pm, 1.6 m*), respectively. The mean permeate and final retentate at a volume reduction factor of 5-5.6 were collected. Dilution of the final retentate with the mean permeate was performed to prepare three concentrates wii h protein contents (total nitrogen matter-non protein nitrogen matter) of about 93, 116 and 140 g kg-‘. Sodium azide (0.2 g kg-‘) was added to each concentrate. Accurate values for nitrogen matters are given in Table 1. Two ultrafiltrations (UF) and two microtiltrations (MF) were performed, but as compositions of the retentates were not significantly different, values for only one experiment are given. Whey concentrate Emmental cheese whey (CoopCrative laitikre Europienne-Montauban de Bretagne, France) was ultrafiltered on a DDS module with plane membranes of 10000 Daltons nmco (RO division). Concentrates at 130, 1.50 and 170 g kg-’ protein were prepared as for milk concentrates. Sodium azide (0.2 g kg- ‘) was added to each concentrate. Accurate values for nitrogen matter contents are given in Table 1. Values of non protein nitrogen matters for the whey concentrate were higher than for milk concentrates. This was due to the higher volume reduction factor and the reduced nmco of the membrane used in the case of whey concentration.
TABLE 1
Composition of Dairy Concentrates
in g kg
’
Theoretical protein
iri,tal nitrogerl mutter
Non proteitl nitrogen mutter
Protein
93 llh I 40
90.22
111.82
2.47 2.29 2.41
x7.:75 109.53 132.57
2.30
77.w 104.20 I2 1.Oh I 13.74 129.04 147.01
Total milk concentrate (ultrafiltration)
Cascin micelle concentrate (microfiltration)
134.98
93 116
79.9 106.35 124.15 120.64 136.15 155.76
140 Whey
concentrate
130 IS0 I 70
2.15 2.19 6.90 7.11 7.85
Total nitrogen matter = total nitrogen x 6.38. Non protein nitrogen matter = non acid) x 6.38. Protein = total nitrogen matter-non
protein
nitrogen
protein nitrogen
(i.e.
matter.
soluble
in
12%
Trichloroaccric
152
M-H. Famelart et al.
Preparation of samples for pressurization For some tests in the experimental design, crystalline NaCl was added at concentrations of 0, 4 and 8 g kg-’ and 5 g sodium citrate solution (56 g kg- ’ or 112 g kg-’ sodium citrate at pH 5.2) or 5 g water were added to 120 g of concentrates. The mix was stirred vigorously with a Polytron mixer at 10000 rpm during pH adjustment with 1 M NaOH or HCl to avoid aggregation over a period of 15 min and the final weight was adjusted to 140 g with deionized water. Thus, final citrate concentration was 0, 2 or 4 g kg-’ and final protein concentrations of the solutions before pressurization were as indicated in Table 2. Solutions were stored at 20°C overnight with magnetic stirring and the pH was eventually corrected while stirring with a Polytron mixer. Each solution was divided into two polyethylene vials of 16 ml without headspace. These were placed into two separate plastic bags and were pressurized separately the same day under the same conditions and were stored overnight at 20°C. Pressurization Milk concentrates were pressurized at 400 MPa and at 20°C for 10 min. Whey concentrates were pressurized at 200 and 400 MPa for 30 min at 20°C. Pressurization was performed in a 3 I pressure cell (120 mm diameter x 300 mm height, ACB, GEC Alstrom, Nantes, France). The maximum available pressure was 700 MPa. Compression times at 200 and 400 MPa were 85 and 155 s, respectively and decompression time was 30 s. Pressurized samples were stored for 1 or 2 h at room temperature prior to analysis. Firmness measurement A stainless steel rod (6.3 mm diameter) connected to a 10 N load cell of a 4501 Instron Universal testing machine with the Series IX software (Instron) penetrated 15 mm into the resultant gels or viscous solutions directly into the vials at 20 mm min-‘. Gel firmness (FZo MPa or F,,,,,,) was calculated as the ‘Young’s modulus’, using the initial sample height of 55 mm and the Cauchy strain (A& with I,, the height of the sample at t = 0 and AZ the compression) and therefore the natural logarithm of firmness was calculated. When ln(firmness) values less than 8.5 Pa were observed, then the samples were ungelified. When liquids were obtained after pressurization, i.e. for milk supplemented with caseinate, the time for the pressurized liquid to flow from a 10 ml pipette was used as a test for viscosity increase. The ratio R of flow time for pressurized sample to flow time for unpressurized was used for statistical analysis. Experimental design A Box-Behnken design (Box and Behnken, 1960) was used for each type of concentrate (Table 2). The symbols used for protein, NaCl, citrate and pH factors were prot, na, tit and pH, respectively, and their levels are in Table 2. The design was constructed into three groups that were realized in the order: group 1, then 2 and 3. Inside the groups, the order was randomized. The two separate pressurizations gave two repetitions. The SAS statistical package was used for anova analysis of
High pressure-induced
gels
qf duiry
IS.1
concentrutes
TABLE 2
The Box-Behnken Design and the Levels used in the Experimental Design. Experiments were Carried out in the Order Group 1. then 2 and 3, and the Random Order Inside Each Group as Given in the Last Column. Final Concentrations are in g kg ’ Group
riumher
Prot
NuCl
Citrutf
P 1-I
I
order
0 0 0 0
0 0 0 0
-I
Hurldotn
-I -I I
-I I 0 0 0 0 0 -I -I I 1 0 0 0 0 0 -I -1 I I 0
Protein concentration
Prot
NaCl concentration Citrate concentration PH
na tit PH
Milk UF Milk MF Whey UF
Milk UF, MF Whey UF
75 66 97 0 0 5.2 7
94 89 Ill 4 2 5.Y 8
114 IO4 127 8 4 6.6 9
M.-H. Famelart et al.
154
ln(firmness), called ln(F,,,,, MPa) or ln(FdoOMPa). Only the significant factors (p10.01) were retained in equations and in the deduced surface response curves. RESULTS Milk concentrates Caseinate-supplemented
milk
Pressurizing reconstituted caseinate-supplemented milk did not lead to gel formation. At 200 MPa, the linear model relating the ratio R and the coded levels of significant factors explained 58% of the sum of squares (SS), while at 400 MPa, only 25% of SS were explained. The main result is that, at 200 MPa, a five-fold increase in flow time was obtained at pH 5.2, without citrate addition, while other conditions were ineffective for ratio changes, even at 400 MPa. Milk concentrates
As the composition of the retentates and the regression equations in the duplicate experiments were not significantly different, only one experiment with the UF retentate and one experiment with the MF retentate are presented here. For some conditions, pressurization of milk concentrates led to gel formation. Gel firmness values were affected by the levels of the studied factors. The selected regression equation for the UF milk concentrate using the combination of levels - 1,0 and + 1 for the factors prot, tit and pH was: ln(F 4(HIMPa)= 10.2382 + 1.3422*prot - 1.3042*cit - l.O865*pH -0.3229*cit’ -2.3561 *pH* + 0.6947*prot*cit This equation for the MF milk concentrate
-0.9055*prot*pH*
was:
ln(F,,,,, MPa)= 10.8508 + 1.3008’”prot -0.9533*cit -2.4329*pH2
+ I. 1905*cit*pH.
+0.6189*prot*cit
- 1.29 16*pH - 0.5495*cit2
-0.7377*prot*pH
+ 1.115 1*cit*pH.
These models explained 98.0% and 92.3% of SS, for UF and MF, respectively. For the two kinds of milk concentrates, the same factors were selected and the coefficients were very close. The protein level had a positive effect, while the pH and the citrate level acted negatively. The ln(firmness) was not influenced by NaCl, in the studied range. The pH showed a large quadratic effect, while some interactions between pH, citrate and protein level were significant, as shown in the above equations. The variation of ln(F,,,,, MPa) as determined by these models, together with experimental values are shown in Figs 1 and 2, for the three levels of citrate contents. The experimental data was very close to the values given by the model. The curves clearly showed that the increase in citrate concentration was not favorable to the firmness, except for the case of high protein content at pH 6.6, where a slight increase of gel firmness was noted with citrate addition (Figs 1 and 2). An increase in the protein level led to firmer gels, and intermediate values of pH around pH 5.9 were the optimum for gel firmness. An increase in the protein content led to a reduction from pH 5.9 to about 5.4-5.6 of the pH value of maximum firmness, while the addition of citrate led to an increase in this optimum pH value. For example, without citrate addition, the pH value for maximum firmness changed from 5.70 to 5.55 and to 5.44 as the protein increased, irrespective of
High pressure-induced gels
qf dnir)
concentrutev
1.i5
a
5 4
I
I
31
13 12 _
~..__.------'--.-__...
1111.~'~
b I'--..
__-----a
--
*.
.*.
-\
..
-.
**.. *.
;
13
C
12 11
IO
g a -
9
2
8
a
LL s
7
5
6 5 4 3 52
5.4
56
58
6
6.2
64
6.6
PH
Fig. 1. Influence of pH, protein and citrate concentration on In(gel firmness). Gels were obtained by pressurization (400 MPa, 10 min, 20°C) of an ultrafiltration concentrate (Techsep membrane Ml) from raw skim milk. Protein concentrations were:--r--: 7Sgkg I;- -*-: 94g kg ‘;----:114 g kg ‘. a: Og kg ’ added citrate: h: 2 g kg- ’ added citrate; c: 4 g kg ’ added citrate. Gels were obtained when In(gcl firmness) was greater than 8.5.
M.-H. Famelati et al.
156
____-----.._.___
b -... f __
___-----
--._ 1. -.
‘,
.A
*.
*.
13
C
12
4 3 52
5.4
5.6
5.8
6
6.2
6.4
6.6
PH
Fig. 2. Influence of pH, protein and citrate concentration on ln(ge1 firmness). Gels were obtained by pressurization (400 MPa, 10 min, 20°C) of a microfiltration concentrate (Techsep membrane M14) from raw skim milk. Protein concentrations were:--+-: 66 g kg - ‘;- -o-: 89 g kg-‘;----: 104 g kg-‘. a: Og kg-’ added citrate; b: 2g kg-’ added citrate; c: 4 g kg-’ added citrate. Gels were obtained when ln(ge1 firmness) was greater than 8.5.
157
High pressure-induced gels qf dairy concentrates
membrane used (Figs 1 and 2, Table 3). At the medium protein level, this pH value was around 5.55, 5.73 and 5.91 for citrate contents of 0, 2 and 4 g kg- ‘, respectively. Comparison of the results for UF and MF led to the conclusion that, although protein contents in MF concentrates were lower (Table 2) firmness was greater for the MF concentrates, in particular at citrate levels of 2 or 4 g kg- ‘. Anyway, the variations of gel firmness with the studied factors were not so different for the two types of concentrates, and the pH intervals were very close. The pH intervals where gels were obtained for fixed levels of protein and citrate contents are given in Table 3. Again, it shows the benefit of citrate as gels were obtained than 100 g kg-‘,
addition at a protein concentration greater for higher pH values in the presence of
sodium citrate. For example, upper pH limiting values of 6.45 and 6.47 for 2 and 4 g kgg’ sodium citrate, were found respectively, with 114 g kgg’ of protein, compared to 6.38 without citrate. At the lower protein level with citrate addition, no gels were formed over the whole range of pH studied. Whey concentrate Only at pH 9, whey concentrate gels were obtained regression equations obtained were:
at 200 and 400 MPa and the
TABLE 3
Values Calculated from the Regression Equation for Milk Concentrates at Fixed Levels of Protein and Citrate. The pH Limiting Values, Where Pressure-set Gels were Obtained, Maximum Gel Firmness (F,,,,, MPamax) and the pH Value at Maximum Firmness are Indicated. Pressurization was Performed at 400 MPa for IO min at 20°C. Concentrations are in
Citrate concentration UF
0
2
4
MF
0
2
Protein concentration IS 94 114 75 94 114 7.5 94 114 66 x9 104 66 89 104
4 :: 104 “Not calculated.
Lower pH limiting value
Upper pH limiting value
F4,,, MR, max
6.38 6.38 6.38 6.14 6.36 6.45 No gel 6.07 6.47 6.47 6.49 6.52 6.29 6.42 6.52 No gel 6.28 6.53
4.44 x lOA 12.06 x 104 40.03 x 10” 0.66 x lo4 2.97 x 10’ 13.32 x lo4 No gel 0.54 x IO” 4.62 x 10” 4.90 x lo4 13.32 x lo4 40.03 x lo4 1.33 x 1o4 5.42 x IO” 26.83 x 10” No gel I .09 x IO4 8.08 x IO4
(Pa)
pH of ma.x firmness 5.70 5.56 5.43 I 5.74 --‘I I 5.91 --I’ 5.66 5.55 5.45 --*I 5.71 ‘I 5.92
M.-H. Famelart et al.
158
In(F 2ooMPa)= 6.690 + 2.875*pH + 2.884*pH2 -O.O657*na-0.229*na*pH and: In(F 4ooMP;,)= 6.698 + 2.966*pH -O.l12*na
-O.O93*cit + 2.966*pH2 -0.337*na*pH
- 0.278*cit*pH More than 99% of SS were explained by these models. Apart from very small effects of NaCI, and citrate addition, the gelification was mainly determined by pH values, with a large quadratic effect. The increase in protein content at the tested levels, from 97 to 127 g kg-‘, did not lead to any significant firmness increase. The curves effects of NaCl and of In(F200 MPa) or In(hotj MPa ) (Fig. 3) show the small negative citrate addition at 200 and 400 MPa on the gel firmness of pressure-set whey protein gels. The increase of pressure between 200 and 400 MPa led to a firmness increase of whey protein gels from 3.4 x lo5 to 6.9 x 10’ Pa in the absence of NaCl and citrate (Table 4). Pressure-set gels of whey concentrates were firmer than those of milk concentrates (for example by comparison at about 95 g kg-’ of protein content).
DISCUSSION Milk concentrates Pressurization of concentrated milk obtained by UF or MF at 400 MPa for 10 min with a protein concentration ranging from 66 to 114 g kg-’ induced gelation without addition of any substance such as rennet. Gel firmness was strongly increased by a little acidification towards pH 5.7-5.9. At pH 6.6 and at the upper level of protein content, sodium citrate addition increased the gel firmness. At the highest protein content, sodium citrate addition also resulted in gel formation at higher pH values than without citrate. This underlined the complementary roles of acidification and citrate addition on mineral dissociation from the casein micelle (Pyne, 1962; Shalabi & Fox, 1982; Dalgleish & Law, 1989; Le Graet & BrulC, 1993). This suggested the positive role of a partial mineral dissociation from the colloidal phase on this pressure-induced gelation. The pH for maximum gel firmness was found to decrease with an increase in protein content. This can be consistent with the ionic strength increase. The ionic strength increase leads to a reduction of the isoelectric pH value. More crucially, the mineralization level of casein micelles increases with the protein concentration by ultrafiltration. Ionization regression and demineralization in a UF retentate are complete at a lower pH value compared to milk (Le Graet, unpublished results; BrulC & Fauquant, 1981). Acidification to lower pH values than 5.7-5.9, for example 5.2, led to softer gels or did not allow gel formation. The demineralization of the micelle structure due to acidification or citrate addition must not be too substantial. Samples composed of milk supplemented with sodium caseinate did not lead to gel formation. Both results led to the conclusion that a colloidal structure close to milk or the presence of minerals in the colloidal phase seemed to be necessary to the gelation process. Native phosphocaseinate (NPC) reconstituted in water at the same protein content as in this study is mainly composed of casein micelles suspended in water (Famelart et al., 1996). Pressure treatment at 400 MPa for 10 min of NPC at pH 6.5 and 5.9
High pressure-induced
gels
I so
of dniry concetrtratrs
14 13
8
n a” H
w
IL
-E J
12 11 10 9 8 7 6 5 65
7
7.5
a PH
a.5
9
14
9.5
b
13 12 z 9
11
m %
10
u! F
a
-I
9 7 6 5 14 13 12 11 10 9 a 7 6
d
65
7
75
a PH
a5
9
95
Fig. 3. Influence of pH, pressure level and NaCl and citrate concentration on In(gel mmness). Gels were obtained by pressurization, (200 or 400 MPa, 30 min, 20°C) of an (RO division, 10000 Daltons) of Emmental ultrafiltration concentrate from 97 to 127 g kg ---: 4 g kg -‘----: 8 g kg ‘.a: cheese whey. Added NaCl were:-r-: 0 g kg :added citrate; c: 400 MPa, 2 g kgg’ added citrate: d: 200 MPa; b: 400,MPa, Og kg added citrate. Gels were obtained when In(gel firmness) was greater than 400 MPa, 4 g kg 8.5.
M-H.
160
Famelati et al.
TABLE 4 Values Calculated from the Regression Equation of the Maximum Firmness (F400MPamax) for Pressure-set Gels of whey Protein Concentrates, at pH 9, for Fixed Levels of NaCl and
Citrate Contents. Pressurization was Performed at 200 and 400 MPa for 30 min. Concentrations are in g kg-’ Pressure
200 MPa 400 MPa
Citrate concentration
- a - a a ? 2 4
NaCl concentration
0 4 8 0 4 8 0 4 8 0 4 8
F+w MPu max (Pa)
3.41 2.55 1.89 6.94 4.42 2.83 4.79 3.06 1.95 3.30 2.11 1.34
x x x x x x x x x x x x
lo5 lo5 lo5 lo5 lo5 lo5 lo5 lo’ 10’ lo5 10’ lo5
a This factor was not significant.
led to gelation (results not presented), which showed that pressure-induced gelation was not dependent upon the mineral content of the soluble phase. Moreover, NaCl addition was not a significant factor at its tested levels, suggesting that the increase in ionic strength did not modify the gel firmness. Results were very close for UF and MF concentrates, because the UF membrane used has rather large pores. With a higher ratio of caseins/whey proteins, MF concentrates were more efficient in increasing firmness, because whey proteins did not lead to any gelation at these pH values. The reason for the gel firmness increase with acidification at pH 5.7-5.9 remained unclear. Acidification of concentrated milk firstly involves the negative charge reduction of casein micelles due to ionization regression, and secondly, it involves the depletion of colloidal minerals. It also involves micelle structural modifications that are likely to be related to calcium phosphate removal from particles. The complementary actions of acidification and citrate addition are more consistent with the last two processes. Amongst the weak structural modifications due to acidification of milk, the only properties that behave like the gel firmness are casein micelle solvation and dynamic viscosity (Tarodo de la Fuente & Alais, 1975; Snoeren et al., 1984; Creamer, 1985; Famelart et al., 1996). The initial hydration and viscosity reduction with acidification is probably due to the charge reduction or to the collapse of the outer hairy layer (Banon & Hardy, 1992). The optimal pH value for gel firmness appeared to be the pH for minimal micellar voluminosity in milk, with lower steric hindrance. The effect of high pressure on protein systems like milk micelles has been studied previously by numerous authors (see Omar & Abou El-Nour, 1995). High pressure treated-milk becomes translucent because of the solubilization of colloidal calcium phosphate and the dissociation of casein micelles into chains or clusters of casein
High pressure-induced gels
qf dairy concentrates
161
subunits (Johnston et al., 1992a,b; Shibauchi et al., 1992; Buchheim & Prokopek, 1992; Desobry-Banon et al., 1994; Buchheim et al., 1996; Gaucheron et al., 1997). These modifications are accompanied by an increase in hydrophobic residue exposure (Johnston et al., 1992a,b; Gaucheron et al., 1997). The combination of micelle disruption into smaller aggregates, the charge reduction and the collapse of any protruding residues can allow gelation, because of the high casein content, and the short interaction distances. The nature of the interactions between caseins may be hydrophobic, or electrostatic, as the casein negative charge is highly reduced at pH 5.9, probably with non specific sites onto subunit surfaces, but more research is required. Whey concentrate
Pressurizing whey concentrates at protein concentrations ranging from 97 to 127 g kg-’ and at pressures from 200 to 400 MPa for 30 min led to gel formation, provided that the pH has been previously adjusted to 9. Van Camp & Huyghebaert (1995a) found that at pH 7 and 400 MPa for 30 min, gel formation occurs at a minimum protein content of 120 g kg-‘, but their concentrates were prepared by dissolving powder into 50 mM phosphate buffer or distilled water, while the current gels were obtained at the native whey ionic strength of about 0.08 M. Van Camp & Huyghebaert (1995a) also studied the effect of pH between 4 and 9 for concentrates dissolved in water or buffer, but at greater concentrations than in this study ( 140 g l_ ’ against 97-127 g l- ‘). In phosphate buffer, maximum gel strength was at pH 8-9, while in water, it was 7-9. No gel formation occurred at acid pH values. This gel formation in the alkaline pH region, i.e. far from the isoelectric pH value (around pH 5), together with the fact that the addition of N-ethylmaleimide (NEM), a S-H blocking agent, prevented the gel formation (Van Camp & Huyghebaert, 1995a) and confirms the role of S-H groups in this process. Studies on fl-lg and x-lactalbumine (r-la) denaturation under high pressure (Hinrichs et al., 1996) show an irreversible denaturation of p-lg and x-la at 350 and 450 MPa, respectively. Based on the same assumptions as the effect of heat treatment, high pressure treatment probably lead first to a dissociation of p-lg dimers into monomers through the weakening of hydrophobic interactions, then to the protein unfolding and finally to the formation of aggregates. Dumay et al. (1994) found that at pH 7, unfolding of p-lg by a 450 MPa treatment was followed by aggregation. This denaturation appears to be partly reversible with time. At pH 7, denaturation of p-lg begins at 50 MPa, while the midpoint of denaturation was found at 175 MPa (Dufour et al., 1994). According to Tanaka et al. (1996) at pH 6.8 and 400 MPa, p-lg dimers are formed through intermolecular reaction of single free S-H groups, and NEM prevents this covalent binding. High pressure treatment applied to the whey concentrates of this study at pH 5.2-8 did not lead to gel formation (results from pH values 5.2 to 7 are not shown). This suggested that the pH value was crucial for high pressure gelation of whey concentrates. High pressure probably led to exposure of hydrophobic residues of globular proteins and of the buried free S-H groups of /I-lg, by unfolding of the globular structure. The reactivity of S-H groups is highly increased at alkaline pH values and exchanges between S-H and S-S groups can take place because of the proximity to cysteine pK value (PK = 9.5 at 25°C according to Walstra and Jenness, 1984). Intermolecular protein-protein interactions such as hydrophobic, ionic or
162
M.-H. Famelart et al.
hydrogen bonding and covalent bonding through S-H and S-S interchanges were probably responsible for the gel formation. According to Van Camp and Huyghebaert (1995a), a protein concentration increase from 110 to 180 g kg-’ at pH 7 leads to a seven-fold increase in the gel strength. But in some studies, a plateau region in gel strength was reached; above 150 g kg -’ at pH 7 for WPC pressure-set gels (Van Camp & Huyghebaert, 1995b) or above 170 g kgg’ at pH 7 for pressure-set gels of p-lg (Zasypkin et al., 1996). In the current findings, protein content increase did not lead to gel firmness increase, which means that no more bonding and interactions occurred as the protein content increased. Between these studies and the current one, protein concentrations are smaller, effects are mainly calculated from gels obtained at pH 9 instead of pH 7 and ionic strengths are different. Van Camp and Huyghebaert (1995a) have underlined the major role of the protein environment; at pH 6, pressure-set gel strengths are 19, 45 and 63 mJ in phosphate buffer, water and BIS-tris buffer, respectively. These differences are probably due to the pressure sensitivity of the aqueous phases of concentrates, and consequently to the pH values reached under pressure. Pressure increase led to firmer gels (Table 4). Hinrichs et al. (1996) found that denaturation of /?-lg increases with the pressure level from 150 to 800 MPa, while a pressure increase generally leads to an increase in the gel strength (Van Camp & Huyghebaert, 1995a). Other food proteins (soy, meat, fish and egg proteins) lead to pressure-induced gels with a higher strength when increased pressures are applied (Hayashi et al., 1989; Okamoto et al., 1990). The studied ionic strength varied through NaCl addition from about 0.08 to 0.22 M, and had a slight negative effect. NaCl addition from 0 to 0.1 M at pH 7-8 leads to an increase in the strength of heat-set WPC gels and to a decrease above 0.2 M NaCl (Mulvihill & Kinsella, 1988; Kinsella & Whitehead, 1989; Gault & Fauquant, 1992). The ionic strength in the current study is in the region of the decrease of heat-set gel firmness, because of shielding. Heat gelation of p-lg at alkaline pH values in the presence of salts leads to soft white coarse gels, with large aggregates (Mulvihill & Kinsella, 1988). A small calcium addition is known to increase heat-set gel firmness (Mulvihill & Kinsella, 1988). In the current study, citrate addition led to a decrease in gel firmness. The reduction of the available calcium content by citrate chelation (Gardner, 1975) can account for this.
CONCLUSIONS The approach and use of the experimental design in the study of milk protein gelation by pressurization has shown the main effect of the pH factor, either if the reasons are different; micelles encountering a structural modification or dissociation with acidification at pH 5.7-5.9, while alkalinization at pH 9 of WPC was required. Anyway, in both systems, gel formation was possible, dependent on the environmental conditions; high protein content, with citrate addition when working at high pH values or without sodium citrate addition at pH 5.7-5.9 led to gel formation at 400 MPa for 10 min at 20°C of milk micelles, while low salinity led to gel formation at pH 9 of WPC treated at 200 or 400 MPa for 30 min at 20°C. Optimization of gel formation has been successfully performed by the design approach, with a simultaneous comprehension of the studied factors. High pressure could be available for gel
High pressure-induced
gels of dairy concentrates
163
production from milk protein concentrates, without heat treatment and any enzyme or product addition. More fundamental knowledge on the nature of the interactions is nevertheless required.
ACKNOWLEDGEMENTS This work was supported
by Bretagne
Biotechnologie
Aliment.
REFERENCES Banon, S. & Hardy, J. (1992). A colloidal approach
of milk acidification by Glucono-deltalactone. Journal of Dairy Science, 75, 935-941. Box, G.E.P. & Behnken, D.W. (1960). Some new three-level designs for the study of quantitative variables. Technometrics, 2, 455-475. BrulC, G. & Fauquant, J. (1981). Mineral balance in skim-milk and retentate: effect of physicochemical characteristics of the aqueous phase. Journal of Dairy Research, 48, 9 l-97. Buchheim, W. & Prokopek, D. (1992). Treatment of milk and other foods by high hydrostatic pressure. European Dairy Magazine, 4, 4-7. Buchheim, W., Schrader, K., Morr, C.V., Frede, E. & Schutt, M. (1996). Effects of high pressure on the protein, lipid and mineral phase of milk. In Heat Treatments and Alternative Methods, IDF, vol. 9602, pp. 202-213 Creamer, L.K. (1985). Water absorption by renneted casein micelles. Milchwissenschaft, 40, 589-591.
Dalgleish, D.G. & Law, A.J.R. (1989). pH-Induced dissociation of bovine casein micelles. Il. Mineral solubilization and its relation to casein release. Journal of Dairy Research, 56, 727-735.
Desobry-Banon, S., Richard, F. & Hardy, J. (1994). Study of acid and rennet coagulation of high pressurized milk. Journal of Dairy Science, 77, 3267-3274. Dufour, E., Hui Bon Hoa, G. & Haertle, T. (1994). High-pressure effects on fi-lactoglobulin interactions with ligands studied by fluorescence. Biochimica et Biophysics Acta, 1206. 166-172.
Dumay, E.M., Kalichevsky, gation of fi-lactoglobulin
M.T. & Cheftel, J.C. (1994). High-pressure unfolding and aggreand the baroprotective effects of sucrose. Journal of Agricultural and Food Chemistry, 42, 1861- 1868. Famelart, M.H., Lepesant, F., Gaucheron, F., Le Graet, Y. & Schuck, P. (1996). pH-Induced physicochemical modifications of native phosphocaseinate suspensions: influence of aqueous phase. Lait, 76, 445-460. Gardner, W.H. (1975). Acidulants in food processing. In Handbook of Food Additives, eda T.E. Furia, chap. 5, CRC Press, Cleveland, OH, pp. 225-270. Gaucheron, F., Famelart, M.H., Mariette, F., Michel, F. & Le Graet, Y. (1997). Combined effects of temperature and high-pressure treatment on physicochcmical characteristics of skim milk. Food Chemistry, 59,439-447. Gault, P. & Fauquant, J. (1992). Aptitude a la gelification thermique de la /j-lactoglobuline: influence du pH, de I’environnement ionique et de la presence des autres proteines du lactoserum. Lair, 72, 491-S 10. Hayashi, R., Kawamura, Y., Nakasa, T. & Okinaka, 0. (1989). Application of high prcssurc to food processing: pressurization of egg white and yolk, and properties of gels formed. Agn’cultural and Biological Chemistry, 53, 2935-2939.
Hinrichs, J., Rademacher, B. & Kessler, H.G. (1996). Reaction denaturation of whey proteins. Milchwissenchaft, 51, 504-508.
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Johnston, D.E., Austin, B.A. & Murphy, R.J. (1992a). The effects of high pressure treatment of skim milk. In High pressure and Biotechnology, eds C. Balny, R. Hayashi, K. Heremans & P. Masson, Colloq. INSERM, John Libbey Eurotext Ltd, pp. 243-247. Johnston, D.E., Austin, B.A. & Murphy, R.J. (1992b). Effects of high hydrostatic pressure on milk. Milchwissenschaft, 47, 760-763. Kinsella, J.E. & Whitehead, D.M. (1989). Proteins in whey: chemical, physical, and functional properties. Advances in Food and Nutrition Research, 33, 343-438. Kumeno, K., Nakahama, N., Honma, K., Makino, T. & Watanabe, M. (1993). Production and characterization of a pressure-induced gel from freeze-concentrated milk. Bioscience, Biotechnology and Biochemistry, 57, 750-752.
Le Graet, Y. & Brule, G. (1993). Les Cquilibres mineraux du lait: influence du pH et de la force ionique. Lait, 73, 51-60. Messens, W., Van Camp, J. & Huyghebaert, A. (1997). The use of high pressure to modify the functionality of food proteins. Trends in Food Science and Technology, 8, 107-112. Mulvihill, D.M. & Kinsella, J.E. (1988). Gelation of j?-lactoglobulin: effects of sodium chloride and calcium chloride on the rheological and structural properties of gels. Journal of Food Science, 53,231-236. ~ Okamoto, M., Kawamura, Y. & Hayashi, R. (1990). Application of high pressure to food processing: textural comparison of pressure- and heat-induced gels of food proteins. Agricultural and Biological Chemistry, 54, 183-189.
Omar, M.M. & Abou El-Nour, tion in dairy processing. In Technology, Cairo, Egypt, 4-6 Pyne, G.T. (1962). Reviews of
A.M. (1995). High hydrostatic Proceedings
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Shalabi, S.I. & Fox, P.F. (1982). Influence
of pH on the rennet
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Shibauchi, Y., Yamamoto, H. & Sagara, Y. (1992). Conformational change of casein micelles by high pressure treatment. In High Pressure and Biotechnology, eds C. Balny, R. Hayashi, K. Heremans & P. Masson, Colloq. INSERM, John Libbey Eurotext Ltd., pp. 239-242. Snoeren, T.H.M., Klok, H.J., Van Hooydonk, A.C.M. & Damman, A.J. (1984). The voluminosity of casein micelles. Milchwissenschaft, 39, 461-463. Tanaka, N., Tsurui, Y., Kobayashi, I. & Kunugi, S. (1996). Modification of the single unpaired sulfhydryl group of P-lactoglobulin under high pressure and the role of intermolecular S-S exchange in the pressure denaturation. International Journal of Biological Macromolecules,
Tarodo
19, 63-68.
de la Fuente,
B. & Alais, C. (1975). Solvation
of casein in bovine milk. Journal of
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Van Camp, J. & Huyghebaert, A. (1995a). High pressure-induced gel formation of a whey protein and haemoglobin protein concentrate. Lebensmittel- Wissenschaft und -Technologie, 28,111-117.
Van Camp, J. & Huyghebaert, A. (1995b). A comparative rheological study of heat and high pressure induced whey protein gels. Food Chemistry, 54, 357-364. Walstra, P. & Jenness, R. (1984). Dairy Chemistry and Physics. Wiley, New York. Zasypkin, D.V., Dumay, A. & Cheftel, J.C. (1996). Pressure- and heat-induced gelation of mixed fl-lactoglobulinlxanthan solutions. Food Hydrocolloids, 10,203-211.
SO260-8774(98)00048-X
High Pressure-induced
Gel Formation of Milk and Whey Concentrates
M.-H. Famelart,U* L. Chapron, “INRA,
Jorrrrd of’hm/ 0z,y;nrenn~ 36 ( IYYX) 13Y- If14 1008 Elscvier Science Limited. All rights I-csetvcd Printed in Great Britain 0?60-X774iYX $lY.llO f’l.00
M. Piot,” G. BruEh & C. Durier”
Laboratoire
de Recherches de technologie Laitikre, 65, rue de St Brieuc, 35042 Rennes Cedex, France “ENSAR, 65, rue de St Brieuc, 35042 Rennes Cedex, France ’ INRA, Laboratoire de Biomktrie, Route de St Cyr, 78026 Versailles Cedex, France (Received
19 September
1997; accepted
12 March 1998)
ABSTRACT Gels j?om milk concentrates [milk and caseinate powder; ultrafiltration (UF) and microfiltration (MF)] an d wh ey concentrates (UF) were obtained with high pressure (200 and 400 MPa, 10 and 30 min). The effects of protein concentration (66-114gkg -’ for milk and 97-1273 kg-’ for whey), NaCl addition (0-8g kg -I), sodium citrate addition (O-4 g kg-‘) and pH (5.2-6.6 jar milk and 7-9 for whey) were studied with a Box-Behnken design. Milk .supplemented with caseinate powder did not lead to gel formation by pressure. For UF or MF milk concentrates, a pH decrease towards 5.9 and a protein content increase led to firmer pressure-set gels. Gels of whey concentrates were obtained only at pH 9. A pressure increase from 200 to 400 MPa led to firmer gels, while a protein content increase did not. 0 1998 Elsevier Science Limited. All rights reserved
INTRODUCTION High pressure processes have been increasingly applied to proteins, either for proteolysis and enzyme inactivation, or texture modification, aggregation or gelation. High pressure is known to affect only non-covalent interactions, i.e. intra- or interVan der Waals and electrostatic molecular hydrogen bonding, hydrophobic, interactions. According to Messens et al. (1997), only electrostatic and hydrophobic interactions are sensitive to pressure. *To whom correspondence
should be addressed. 149
150
M.-H. Fumelurt et al.
Protein gelation is the main process involved in food texture properties. Various food protein systems such as fish and animal meat, soybean protein dispersions, egg yolk and white, and whey protein gel after a heat treatment. Recently, hydrostatic pressure has been successfully applied to these food protein systems (Hayashi et al., 1989; Okamoto et al., 1990; Van Camp & Huyghebaert, 1995b). Hence, high hydrostatic pressure treatment is a potential tool for the gelation of food protein systems, as an alternative to heat treatments. It can be used to create new products or texture without any detrimental thermal modifications (Messens et al., 1997). According to the unique study on concentrated milk gelation under high pressure (Kumeno et al., 1993) high pressure treatment at 300-600 MPa for 5 min of freezeconcentrated milk at 25 wt% solid content leads to gelation. Using powdered milk or freeze-dried milk even at 25 wt% solid content or higher, does not lead to gel formation. A pressure increase from 300 to 600 MPa leads to stronger gels. According to Van Camp & Huyghebaert (1995a), a whey protein concentrate (WPC) reconstituted in a 50 mM phosphate buffer at pH 7 treated at 400 MPa for 30 min will gel. These pressure-set gels were compared to heat-set gels (Van Camp & Huyghebaert, 1995b). An increase in protein concentration from 110 to 180 g 1-I leads to a gel strength increase (Van Camp & Huyghebaert, 1995a), but in Van Camp & Huyghebaert (1995b), the increase in moduli was reduced between 150 and 180 g 1-l. According to Zasypkin et al. (1996), the rigidity of pressure-set gels from P-lactoglobulin (/3-lg) did not change with the protein concentration increase from 190 to 210 g kg-‘. An increase in the pressure level from 200 to 400 MPa leads to a gel strength increase, apart from the WPC at 150 g 1-l protein concentration, whose gel strength is constant between 300 and 400 MPa (Van Camp & Huyghebaert, 1995a). The effect of pH on pressure-set gels from WPC reconstituted in 50 mM buffer salts has been studied at 140 g 1-l protein concentration after a 400 MPa treatment for 30 min (Van Camp & Huyghebaert, 1995a). The gel strength increases from pH 5 to 8 and is constant between pH 8 and 9. In the absence of buffer salts, the strength increases between pH 5 and 6, and remains almost constant for higher pH values (Van Camp & Huyghebaert, 1995a). In the present paper, the effect of high pressure treatment of milk and whey concentrates on gel formation was studied. Various physicochemical conditions were tested to determine their impact on pressure-induced gelation. The physicochemical factors were pH, protein content, sodium citrate and NaCl concentration. Their levels were chosen for their ability to induce limited changes in protein structures, which can modify protein interactions and hence, gel firmness. Previous papers on WPC gels obtained after high pressure treatment only deal with factors studied one by one. For milk concentrates the effects of these factors have not been studied. The objectives were first, to determine the optimum parameters by use of a suitable experimental design, and secondly to better understand the protein-protein interactions within the gels. MATERIALS
AND METHODS
Dairy concentrates Values of nitrogen matters either for powders or concentrates Kjeldahl method with 6.38 as the converting factor.
were obtained
by the
High pressure-induced gels q f’duity corlcerltrutes
lil
Milk concentrates Milk was reconstituted from low heat skim milk powder (INRA, France) at 10 g of dry matter for 110 g of final milk and was supplemented with sodium caseinate (Armor protkines, St Brice en Cog& France) to reach protein concentrations of 9.3, 116 and 140 g kg- ‘. This was obtained with a weight ratio of sodium caseinate to milk powder of 0.77, 1.04 and 1.31, respectively. Raw skim milk (Triballat, Noyal sur Vilaine, France) was ultrafiltered or microfiltered on a 2S37 module with Ml membranes (Techsep, 100000 - 150000 Daltons nominal molecular weight cutoff [nmco], 1.6 m’) and with Ml4 membranes (Techsep, pore diameter 0.14 pm, 1.6 m*), respectively. The mean permeate and final retentate at a volume reduction factor of 5-5.6 were collected. Dilution of the final retentate with the mean permeate was performed to prepare three concentrates wii h protein contents (total nitrogen matter-non protein nitrogen matter) of about 93, 116 and 140 g kg-‘. Sodium azide (0.2 g kg-‘) was added to each concentrate. Accurate values for nitrogen matters are given in Table 1. Two ultrafiltrations (UF) and two microtiltrations (MF) were performed, but as compositions of the retentates were not significantly different, values for only one experiment are given. Whey concentrate Emmental cheese whey (CoopCrative laitikre Europienne-Montauban de Bretagne, France) was ultrafiltered on a DDS module with plane membranes of 10000 Daltons nmco (RO division). Concentrates at 130, 1.50 and 170 g kg-’ protein were prepared as for milk concentrates. Sodium azide (0.2 g kg- ‘) was added to each concentrate. Accurate values for nitrogen matter contents are given in Table 1. Values of non protein nitrogen matters for the whey concentrate were higher than for milk concentrates. This was due to the higher volume reduction factor and the reduced nmco of the membrane used in the case of whey concentration.
TABLE 1
Composition of Dairy Concentrates
in g kg
’
Theoretical protein
iri,tal nitrogerl mutter
Non proteitl nitrogen mutter
Protein
93 llh I 40
90.22
111.82
2.47 2.29 2.41
x7.:75 109.53 132.57
2.30
77.w 104.20 I2 1.Oh I 13.74 129.04 147.01
Total milk concentrate (ultrafiltration)
Cascin micelle concentrate (microfiltration)
134.98
93 116
79.9 106.35 124.15 120.64 136.15 155.76
140 Whey
concentrate
130 IS0 I 70
2.15 2.19 6.90 7.11 7.85
Total nitrogen matter = total nitrogen x 6.38. Non protein nitrogen matter = non acid) x 6.38. Protein = total nitrogen matter-non
protein
nitrogen
protein nitrogen
(i.e.
matter.
soluble
in
12%
Trichloroaccric
152
M-H. Famelart et al.
Preparation of samples for pressurization For some tests in the experimental design, crystalline NaCl was added at concentrations of 0, 4 and 8 g kg-’ and 5 g sodium citrate solution (56 g kg- ’ or 112 g kg-’ sodium citrate at pH 5.2) or 5 g water were added to 120 g of concentrates. The mix was stirred vigorously with a Polytron mixer at 10000 rpm during pH adjustment with 1 M NaOH or HCl to avoid aggregation over a period of 15 min and the final weight was adjusted to 140 g with deionized water. Thus, final citrate concentration was 0, 2 or 4 g kg-’ and final protein concentrations of the solutions before pressurization were as indicated in Table 2. Solutions were stored at 20°C overnight with magnetic stirring and the pH was eventually corrected while stirring with a Polytron mixer. Each solution was divided into two polyethylene vials of 16 ml without headspace. These were placed into two separate plastic bags and were pressurized separately the same day under the same conditions and were stored overnight at 20°C. Pressurization Milk concentrates were pressurized at 400 MPa and at 20°C for 10 min. Whey concentrates were pressurized at 200 and 400 MPa for 30 min at 20°C. Pressurization was performed in a 3 I pressure cell (120 mm diameter x 300 mm height, ACB, GEC Alstrom, Nantes, France). The maximum available pressure was 700 MPa. Compression times at 200 and 400 MPa were 85 and 155 s, respectively and decompression time was 30 s. Pressurized samples were stored for 1 or 2 h at room temperature prior to analysis. Firmness measurement A stainless steel rod (6.3 mm diameter) connected to a 10 N load cell of a 4501 Instron Universal testing machine with the Series IX software (Instron) penetrated 15 mm into the resultant gels or viscous solutions directly into the vials at 20 mm min-‘. Gel firmness (FZo MPa or F,,,,,,) was calculated as the ‘Young’s modulus’, using the initial sample height of 55 mm and the Cauchy strain (A& with I,, the height of the sample at t = 0 and AZ the compression) and therefore the natural logarithm of firmness was calculated. When ln(firmness) values less than 8.5 Pa were observed, then the samples were ungelified. When liquids were obtained after pressurization, i.e. for milk supplemented with caseinate, the time for the pressurized liquid to flow from a 10 ml pipette was used as a test for viscosity increase. The ratio R of flow time for pressurized sample to flow time for unpressurized was used for statistical analysis. Experimental design A Box-Behnken design (Box and Behnken, 1960) was used for each type of concentrate (Table 2). The symbols used for protein, NaCl, citrate and pH factors were prot, na, tit and pH, respectively, and their levels are in Table 2. The design was constructed into three groups that were realized in the order: group 1, then 2 and 3. Inside the groups, the order was randomized. The two separate pressurizations gave two repetitions. The SAS statistical package was used for anova analysis of
High pressure-induced
gels
qf duiry
IS.1
concentrutes
TABLE 2
The Box-Behnken Design and the Levels used in the Experimental Design. Experiments were Carried out in the Order Group 1. then 2 and 3, and the Random Order Inside Each Group as Given in the Last Column. Final Concentrations are in g kg ’ Group
riumher
Prot
NuCl
Citrutf
P 1-I
I
order
0 0 0 0
0 0 0 0
-I
Hurldotn
-I -I I
-I I 0 0 0 0 0 -I -I I 1 0 0 0 0 0 -I -1 I I 0
Protein concentration
Prot
NaCl concentration Citrate concentration PH
na tit PH
Milk UF Milk MF Whey UF
Milk UF, MF Whey UF
75 66 97 0 0 5.2 7
94 89 Ill 4 2 5.Y 8
114 IO4 127 8 4 6.6 9
M.-H. Famelart et al.
154
ln(firmness), called ln(F,,,,, MPa) or ln(FdoOMPa). Only the significant factors (p10.01) were retained in equations and in the deduced surface response curves. RESULTS Milk concentrates Caseinate-supplemented
milk
Pressurizing reconstituted caseinate-supplemented milk did not lead to gel formation. At 200 MPa, the linear model relating the ratio R and the coded levels of significant factors explained 58% of the sum of squares (SS), while at 400 MPa, only 25% of SS were explained. The main result is that, at 200 MPa, a five-fold increase in flow time was obtained at pH 5.2, without citrate addition, while other conditions were ineffective for ratio changes, even at 400 MPa. Milk concentrates
As the composition of the retentates and the regression equations in the duplicate experiments were not significantly different, only one experiment with the UF retentate and one experiment with the MF retentate are presented here. For some conditions, pressurization of milk concentrates led to gel formation. Gel firmness values were affected by the levels of the studied factors. The selected regression equation for the UF milk concentrate using the combination of levels - 1,0 and + 1 for the factors prot, tit and pH was: ln(F 4(HIMPa)= 10.2382 + 1.3422*prot - 1.3042*cit - l.O865*pH -0.3229*cit’ -2.3561 *pH* + 0.6947*prot*cit This equation for the MF milk concentrate
-0.9055*prot*pH*
was:
ln(F,,,,, MPa)= 10.8508 + 1.3008’”prot -0.9533*cit -2.4329*pH2
+ I. 1905*cit*pH.
+0.6189*prot*cit
- 1.29 16*pH - 0.5495*cit2
-0.7377*prot*pH
+ 1.115 1*cit*pH.
These models explained 98.0% and 92.3% of SS, for UF and MF, respectively. For the two kinds of milk concentrates, the same factors were selected and the coefficients were very close. The protein level had a positive effect, while the pH and the citrate level acted negatively. The ln(firmness) was not influenced by NaCl, in the studied range. The pH showed a large quadratic effect, while some interactions between pH, citrate and protein level were significant, as shown in the above equations. The variation of ln(F,,,,, MPa) as determined by these models, together with experimental values are shown in Figs 1 and 2, for the three levels of citrate contents. The experimental data was very close to the values given by the model. The curves clearly showed that the increase in citrate concentration was not favorable to the firmness, except for the case of high protein content at pH 6.6, where a slight increase of gel firmness was noted with citrate addition (Figs 1 and 2). An increase in the protein level led to firmer gels, and intermediate values of pH around pH 5.9 were the optimum for gel firmness. An increase in the protein content led to a reduction from pH 5.9 to about 5.4-5.6 of the pH value of maximum firmness, while the addition of citrate led to an increase in this optimum pH value. For example, without citrate addition, the pH value for maximum firmness changed from 5.70 to 5.55 and to 5.44 as the protein increased, irrespective of
High pressure-induced gels
qf dnir)
concentrutev
1.i5
a
5 4
I
I
31
13 12 _
~..__.------'--.-__...
1111.~'~
b I'--..
__-----a
--
*.
.*.
-\
..
-.
**.. *.
;
13
C
12 11
IO
g a -
9
2
8
a
LL s
7
5
6 5 4 3 52
5.4
56
58
6
6.2
64
6.6
PH
Fig. 1. Influence of pH, protein and citrate concentration on In(gel firmness). Gels were obtained by pressurization (400 MPa, 10 min, 20°C) of an ultrafiltration concentrate (Techsep membrane Ml) from raw skim milk. Protein concentrations were:--r--: 7Sgkg I;- -*-: 94g kg ‘;----:114 g kg ‘. a: Og kg ’ added citrate: h: 2 g kg- ’ added citrate; c: 4 g kg ’ added citrate. Gels were obtained when In(gcl firmness) was greater than 8.5.
M.-H. Famelati et al.
156
____-----.._.___
b -... f __
___-----
--._ 1. -.
‘,
.A
*.
*.
13
C
12
4 3 52
5.4
5.6
5.8
6
6.2
6.4
6.6
PH
Fig. 2. Influence of pH, protein and citrate concentration on ln(ge1 firmness). Gels were obtained by pressurization (400 MPa, 10 min, 20°C) of a microfiltration concentrate (Techsep membrane M14) from raw skim milk. Protein concentrations were:--+-: 66 g kg - ‘;- -o-: 89 g kg-‘;----: 104 g kg-‘. a: Og kg-’ added citrate; b: 2g kg-’ added citrate; c: 4 g kg-’ added citrate. Gels were obtained when ln(ge1 firmness) was greater than 8.5.
157
High pressure-induced gels qf dairy concentrates
membrane used (Figs 1 and 2, Table 3). At the medium protein level, this pH value was around 5.55, 5.73 and 5.91 for citrate contents of 0, 2 and 4 g kg- ‘, respectively. Comparison of the results for UF and MF led to the conclusion that, although protein contents in MF concentrates were lower (Table 2) firmness was greater for the MF concentrates, in particular at citrate levels of 2 or 4 g kg- ‘. Anyway, the variations of gel firmness with the studied factors were not so different for the two types of concentrates, and the pH intervals were very close. The pH intervals where gels were obtained for fixed levels of protein and citrate contents are given in Table 3. Again, it shows the benefit of citrate as gels were obtained than 100 g kg-‘,
addition at a protein concentration greater for higher pH values in the presence of
sodium citrate. For example, upper pH limiting values of 6.45 and 6.47 for 2 and 4 g kgg’ sodium citrate, were found respectively, with 114 g kgg’ of protein, compared to 6.38 without citrate. At the lower protein level with citrate addition, no gels were formed over the whole range of pH studied. Whey concentrate Only at pH 9, whey concentrate gels were obtained regression equations obtained were:
at 200 and 400 MPa and the
TABLE 3
Values Calculated from the Regression Equation for Milk Concentrates at Fixed Levels of Protein and Citrate. The pH Limiting Values, Where Pressure-set Gels were Obtained, Maximum Gel Firmness (F,,,,, MPamax) and the pH Value at Maximum Firmness are Indicated. Pressurization was Performed at 400 MPa for IO min at 20°C. Concentrations are in
Citrate concentration UF
0
2
4
MF
0
2
Protein concentration IS 94 114 75 94 114 7.5 94 114 66 x9 104 66 89 104
4 :: 104 “Not calculated.
Lower pH limiting value
Upper pH limiting value
F4,,, MR, max
6.38 6.38 6.38 6.14 6.36 6.45 No gel 6.07 6.47 6.47 6.49 6.52 6.29 6.42 6.52 No gel 6.28 6.53
4.44 x lOA 12.06 x 104 40.03 x 10” 0.66 x lo4 2.97 x 10’ 13.32 x lo4 No gel 0.54 x IO” 4.62 x 10” 4.90 x lo4 13.32 x lo4 40.03 x lo4 1.33 x 1o4 5.42 x IO” 26.83 x 10” No gel I .09 x IO4 8.08 x IO4
(Pa)
pH of ma.x firmness 5.70 5.56 5.43 I 5.74 --‘I I 5.91 --I’ 5.66 5.55 5.45 --*I 5.71 ‘I 5.92
M.-H. Famelart et al.
158
In(F 2ooMPa)= 6.690 + 2.875*pH + 2.884*pH2 -O.O657*na-0.229*na*pH and: In(F 4ooMP;,)= 6.698 + 2.966*pH -O.l12*na
-O.O93*cit + 2.966*pH2 -0.337*na*pH
- 0.278*cit*pH More than 99% of SS were explained by these models. Apart from very small effects of NaCI, and citrate addition, the gelification was mainly determined by pH values, with a large quadratic effect. The increase in protein content at the tested levels, from 97 to 127 g kg-‘, did not lead to any significant firmness increase. The curves effects of NaCl and of In(F200 MPa) or In(hotj MPa ) (Fig. 3) show the small negative citrate addition at 200 and 400 MPa on the gel firmness of pressure-set whey protein gels. The increase of pressure between 200 and 400 MPa led to a firmness increase of whey protein gels from 3.4 x lo5 to 6.9 x 10’ Pa in the absence of NaCl and citrate (Table 4). Pressure-set gels of whey concentrates were firmer than those of milk concentrates (for example by comparison at about 95 g kg-’ of protein content).
DISCUSSION Milk concentrates Pressurization of concentrated milk obtained by UF or MF at 400 MPa for 10 min with a protein concentration ranging from 66 to 114 g kg-’ induced gelation without addition of any substance such as rennet. Gel firmness was strongly increased by a little acidification towards pH 5.7-5.9. At pH 6.6 and at the upper level of protein content, sodium citrate addition increased the gel firmness. At the highest protein content, sodium citrate addition also resulted in gel formation at higher pH values than without citrate. This underlined the complementary roles of acidification and citrate addition on mineral dissociation from the casein micelle (Pyne, 1962; Shalabi & Fox, 1982; Dalgleish & Law, 1989; Le Graet & BrulC, 1993). This suggested the positive role of a partial mineral dissociation from the colloidal phase on this pressure-induced gelation. The pH for maximum gel firmness was found to decrease with an increase in protein content. This can be consistent with the ionic strength increase. The ionic strength increase leads to a reduction of the isoelectric pH value. More crucially, the mineralization level of casein micelles increases with the protein concentration by ultrafiltration. Ionization regression and demineralization in a UF retentate are complete at a lower pH value compared to milk (Le Graet, unpublished results; BrulC & Fauquant, 1981). Acidification to lower pH values than 5.7-5.9, for example 5.2, led to softer gels or did not allow gel formation. The demineralization of the micelle structure due to acidification or citrate addition must not be too substantial. Samples composed of milk supplemented with sodium caseinate did not lead to gel formation. Both results led to the conclusion that a colloidal structure close to milk or the presence of minerals in the colloidal phase seemed to be necessary to the gelation process. Native phosphocaseinate (NPC) reconstituted in water at the same protein content as in this study is mainly composed of casein micelles suspended in water (Famelart et al., 1996). Pressure treatment at 400 MPa for 10 min of NPC at pH 6.5 and 5.9
High pressure-induced
gels
I so
of dniry concetrtratrs
14 13
8
n a” H
w
IL
-E J
12 11 10 9 8 7 6 5 65
7
7.5
a PH
a.5
9
14
9.5
b
13 12 z 9
11
m %
10
u! F
a
-I
9 7 6 5 14 13 12 11 10 9 a 7 6
d
65
7
75
a PH
a5
9
95
Fig. 3. Influence of pH, pressure level and NaCl and citrate concentration on In(gel mmness). Gels were obtained by pressurization, (200 or 400 MPa, 30 min, 20°C) of an (RO division, 10000 Daltons) of Emmental ultrafiltration concentrate from 97 to 127 g kg ---: 4 g kg -‘----: 8 g kg ‘.a: cheese whey. Added NaCl were:-r-: 0 g kg :added citrate; c: 400 MPa, 2 g kgg’ added citrate: d: 200 MPa; b: 400,MPa, Og kg added citrate. Gels were obtained when In(gel firmness) was greater than 400 MPa, 4 g kg 8.5.
M-H.
160
Famelati et al.
TABLE 4 Values Calculated from the Regression Equation of the Maximum Firmness (F400MPamax) for Pressure-set Gels of whey Protein Concentrates, at pH 9, for Fixed Levels of NaCl and
Citrate Contents. Pressurization was Performed at 200 and 400 MPa for 30 min. Concentrations are in g kg-’ Pressure
200 MPa 400 MPa
Citrate concentration
- a - a a ? 2 4
NaCl concentration
0 4 8 0 4 8 0 4 8 0 4 8
F+w MPu max (Pa)
3.41 2.55 1.89 6.94 4.42 2.83 4.79 3.06 1.95 3.30 2.11 1.34
x x x x x x x x x x x x
lo5 lo5 lo5 lo5 lo5 lo5 lo5 lo’ 10’ lo5 10’ lo5
a This factor was not significant.
led to gelation (results not presented), which showed that pressure-induced gelation was not dependent upon the mineral content of the soluble phase. Moreover, NaCl addition was not a significant factor at its tested levels, suggesting that the increase in ionic strength did not modify the gel firmness. Results were very close for UF and MF concentrates, because the UF membrane used has rather large pores. With a higher ratio of caseins/whey proteins, MF concentrates were more efficient in increasing firmness, because whey proteins did not lead to any gelation at these pH values. The reason for the gel firmness increase with acidification at pH 5.7-5.9 remained unclear. Acidification of concentrated milk firstly involves the negative charge reduction of casein micelles due to ionization regression, and secondly, it involves the depletion of colloidal minerals. It also involves micelle structural modifications that are likely to be related to calcium phosphate removal from particles. The complementary actions of acidification and citrate addition are more consistent with the last two processes. Amongst the weak structural modifications due to acidification of milk, the only properties that behave like the gel firmness are casein micelle solvation and dynamic viscosity (Tarodo de la Fuente & Alais, 1975; Snoeren et al., 1984; Creamer, 1985; Famelart et al., 1996). The initial hydration and viscosity reduction with acidification is probably due to the charge reduction or to the collapse of the outer hairy layer (Banon & Hardy, 1992). The optimal pH value for gel firmness appeared to be the pH for minimal micellar voluminosity in milk, with lower steric hindrance. The effect of high pressure on protein systems like milk micelles has been studied previously by numerous authors (see Omar & Abou El-Nour, 1995). High pressure treated-milk becomes translucent because of the solubilization of colloidal calcium phosphate and the dissociation of casein micelles into chains or clusters of casein
High pressure-induced gels
qf dairy concentrates
161
subunits (Johnston et al., 1992a,b; Shibauchi et al., 1992; Buchheim & Prokopek, 1992; Desobry-Banon et al., 1994; Buchheim et al., 1996; Gaucheron et al., 1997). These modifications are accompanied by an increase in hydrophobic residue exposure (Johnston et al., 1992a,b; Gaucheron et al., 1997). The combination of micelle disruption into smaller aggregates, the charge reduction and the collapse of any protruding residues can allow gelation, because of the high casein content, and the short interaction distances. The nature of the interactions between caseins may be hydrophobic, or electrostatic, as the casein negative charge is highly reduced at pH 5.9, probably with non specific sites onto subunit surfaces, but more research is required. Whey concentrate
Pressurizing whey concentrates at protein concentrations ranging from 97 to 127 g kg-’ and at pressures from 200 to 400 MPa for 30 min led to gel formation, provided that the pH has been previously adjusted to 9. Van Camp & Huyghebaert (1995a) found that at pH 7 and 400 MPa for 30 min, gel formation occurs at a minimum protein content of 120 g kg-‘, but their concentrates were prepared by dissolving powder into 50 mM phosphate buffer or distilled water, while the current gels were obtained at the native whey ionic strength of about 0.08 M. Van Camp & Huyghebaert (1995a) also studied the effect of pH between 4 and 9 for concentrates dissolved in water or buffer, but at greater concentrations than in this study ( 140 g l_ ’ against 97-127 g l- ‘). In phosphate buffer, maximum gel strength was at pH 8-9, while in water, it was 7-9. No gel formation occurred at acid pH values. This gel formation in the alkaline pH region, i.e. far from the isoelectric pH value (around pH 5), together with the fact that the addition of N-ethylmaleimide (NEM), a S-H blocking agent, prevented the gel formation (Van Camp & Huyghebaert, 1995a) and confirms the role of S-H groups in this process. Studies on fl-lg and x-lactalbumine (r-la) denaturation under high pressure (Hinrichs et al., 1996) show an irreversible denaturation of p-lg and x-la at 350 and 450 MPa, respectively. Based on the same assumptions as the effect of heat treatment, high pressure treatment probably lead first to a dissociation of p-lg dimers into monomers through the weakening of hydrophobic interactions, then to the protein unfolding and finally to the formation of aggregates. Dumay et al. (1994) found that at pH 7, unfolding of p-lg by a 450 MPa treatment was followed by aggregation. This denaturation appears to be partly reversible with time. At pH 7, denaturation of p-lg begins at 50 MPa, while the midpoint of denaturation was found at 175 MPa (Dufour et al., 1994). According to Tanaka et al. (1996) at pH 6.8 and 400 MPa, p-lg dimers are formed through intermolecular reaction of single free S-H groups, and NEM prevents this covalent binding. High pressure treatment applied to the whey concentrates of this study at pH 5.2-8 did not lead to gel formation (results from pH values 5.2 to 7 are not shown). This suggested that the pH value was crucial for high pressure gelation of whey concentrates. High pressure probably led to exposure of hydrophobic residues of globular proteins and of the buried free S-H groups of /I-lg, by unfolding of the globular structure. The reactivity of S-H groups is highly increased at alkaline pH values and exchanges between S-H and S-S groups can take place because of the proximity to cysteine pK value (PK = 9.5 at 25°C according to Walstra and Jenness, 1984). Intermolecular protein-protein interactions such as hydrophobic, ionic or
162
M.-H. Famelart et al.
hydrogen bonding and covalent bonding through S-H and S-S interchanges were probably responsible for the gel formation. According to Van Camp and Huyghebaert (1995a), a protein concentration increase from 110 to 180 g kg-’ at pH 7 leads to a seven-fold increase in the gel strength. But in some studies, a plateau region in gel strength was reached; above 150 g kg -’ at pH 7 for WPC pressure-set gels (Van Camp & Huyghebaert, 1995b) or above 170 g kgg’ at pH 7 for pressure-set gels of p-lg (Zasypkin et al., 1996). In the current findings, protein content increase did not lead to gel firmness increase, which means that no more bonding and interactions occurred as the protein content increased. Between these studies and the current one, protein concentrations are smaller, effects are mainly calculated from gels obtained at pH 9 instead of pH 7 and ionic strengths are different. Van Camp and Huyghebaert (1995a) have underlined the major role of the protein environment; at pH 6, pressure-set gel strengths are 19, 45 and 63 mJ in phosphate buffer, water and BIS-tris buffer, respectively. These differences are probably due to the pressure sensitivity of the aqueous phases of concentrates, and consequently to the pH values reached under pressure. Pressure increase led to firmer gels (Table 4). Hinrichs et al. (1996) found that denaturation of /?-lg increases with the pressure level from 150 to 800 MPa, while a pressure increase generally leads to an increase in the gel strength (Van Camp & Huyghebaert, 1995a). Other food proteins (soy, meat, fish and egg proteins) lead to pressure-induced gels with a higher strength when increased pressures are applied (Hayashi et al., 1989; Okamoto et al., 1990). The studied ionic strength varied through NaCl addition from about 0.08 to 0.22 M, and had a slight negative effect. NaCl addition from 0 to 0.1 M at pH 7-8 leads to an increase in the strength of heat-set WPC gels and to a decrease above 0.2 M NaCl (Mulvihill & Kinsella, 1988; Kinsella & Whitehead, 1989; Gault & Fauquant, 1992). The ionic strength in the current study is in the region of the decrease of heat-set gel firmness, because of shielding. Heat gelation of p-lg at alkaline pH values in the presence of salts leads to soft white coarse gels, with large aggregates (Mulvihill & Kinsella, 1988). A small calcium addition is known to increase heat-set gel firmness (Mulvihill & Kinsella, 1988). In the current study, citrate addition led to a decrease in gel firmness. The reduction of the available calcium content by citrate chelation (Gardner, 1975) can account for this.
CONCLUSIONS The approach and use of the experimental design in the study of milk protein gelation by pressurization has shown the main effect of the pH factor, either if the reasons are different; micelles encountering a structural modification or dissociation with acidification at pH 5.7-5.9, while alkalinization at pH 9 of WPC was required. Anyway, in both systems, gel formation was possible, dependent on the environmental conditions; high protein content, with citrate addition when working at high pH values or without sodium citrate addition at pH 5.7-5.9 led to gel formation at 400 MPa for 10 min at 20°C of milk micelles, while low salinity led to gel formation at pH 9 of WPC treated at 200 or 400 MPa for 30 min at 20°C. Optimization of gel formation has been successfully performed by the design approach, with a simultaneous comprehension of the studied factors. High pressure could be available for gel
High pressure-induced
gels of dairy concentrates
163
production from milk protein concentrates, without heat treatment and any enzyme or product addition. More fundamental knowledge on the nature of the interactions is nevertheless required.
ACKNOWLEDGEMENTS This work was supported
by Bretagne
Biotechnologie
Aliment.
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