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Milk fat microencapsulation using whey proteins
International Dairy Journal 9 (1999) 657}663
Milk fat microencapsulation using whey proteins M. Kieran Keogh*, Brendan T. O'Kennedy Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Republic of Ireland Received 28 March 1998; accepted 18 August 1999
Abstract The e!ects of homogenisation conditions (pressure and number of passes) and emulsion composition (fat, whey protein, lactose, salts and water) on the properties of emulsions and microencapsulated milk fat powders were measured. The emulsions were assessed by fat globule diameter and size stability over time. The properties of the powders measured were particle diameter, reconstituted emulsion fat globule diameter, free fat, surface fat and fat oxidation. As expected, increasing homogenisation pressure reduced the fat globule diameter and increasing the number of homogenisation passes reduced the diameter of the largest globules. Increasing fat and salts reduced fat globule diameter stability after 2 homogenisation passes but the reduction in stability was less after 4 passes. Increasing the lactose : whey protein concentrate ratio reduced free fat and fat globule aggregation after powder reconstitution, but not the surface fat. The higher level of fat increased the surface fat on powder particles and the level of oxidation during storage. ( 1999 Elsevier Science Ltd. All rights reserved. Keywords: Microencapsulation; Homogenisation; Composition; Oxidation
1. Introduction Microencapsulation involves dispersing and "xing sub-micron-sized particles in a solid particle matrix. Techniques suitable for the microencapsulation of fat, such as high-pressure homogenisation for dispersing fat globules and spray drying for "xation of the globules, are readily available in the dairy industry. The solid matrix or wall material of the powder particles should protect the microparticles or core material against deterioration (Rosenberg & Lee, 1993; Rosenberg & Young, 1993; Young, Sarda, & Rosenberg, 1993a, b). Any microparticle can be encapsulated, but when the core material is fat, part of the wall material must have emulsifying properties and be capable of dehydration. The milk protein products sodium caseinate and whey protein concentrate have excellent emulsifying and dehydration properties. While sodium caseinate has a relatively uniform composition, commercial whey protein concentrates contain 35}75% or more protein and whey protein isolates have at least 90% protein. A critical parameter of the powder is the extent of fat on the surface on the particles, which can be measured by
* Corresponding author. E-mail address: [email protected] (M.K. Keogh)
electron spectroscopy for chemical analysis (ESCA) (FaK ldt, Bergensta> hl & Carlsson, 1993). It has been shown that surface fat does not correlate well to solvent extractable free fat, since the latter also includes some nearsurface fat (Buchheim, 1982). Surface fat but not free fat by solvent extraction (Buma, 1971) was shown to be related to the oxidative stability of cholesterol in fat powders (Granelli, Fa> ldt, Appelqvist & Bergensta> hl, 1996). Some authors do not seem to distinguish clearly between free fat and surface fat, but associate the latter with the free fat which can be removed from the surface of the powder particles during drying. It was suggested (van Boekel & Walstra, 1991; see also FaK ldt & Bergansta> hl, 1995) that the extent of surface fat resulted from emulsion instability leading to coalescence. Coalescence instability in emulsions is assumed to increase the di!usion of fat onto the powder surface during drying (FaK ldt & Bergansta> hl, 1995). It should be noted that fat globule aggregation and increase in emulsion viscosity usually precede fat coalescence (Walstra, 1986) leading to higher free fat and surface fat in powders. Emulsion fat globule diameter and size stability over time, therefore, seem to be of fundamental importance for successful microencapsulation. The advantages of microencapsulation are that milk fat can be converted to a stable powder form and better powder #ow properties may be obtained if the extent of
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fat on the surface of the dried particles can be minimised. Low levels of fat on the surface could result in the production of powders with higher fat content using conventional dryers and of fat powders that are more stable to oxidation. The aim of this research was to determine the e!ects of homogenisation factors and composition (fat, whey protein, lactose and salts) on the formation and stability of emulsions and encapsulated fat powder particles and to relate these to the properties of the powder.
2. Materials and methods 2.1. Ingredients Whey protein isolate (WPI) was obtained from Davisco International, (Le Sueur, MN, USA), and a whey protein concentrate (WPC-35) from Carbery Milk Products, Ballineen, Co. Cork, Ireland. Anhydrous milk fat (AMF) was obtained from Dairygold, Mallow, Co. Cork, Ireland. This AMF was produced during summer, was less than 1 month old and was within the IDF speci"cations (IDF, 1977). Lactose was obtained from Avonmore Foods plc, Ballyragget, Co. Kilkenny, Ireland. The chloride salts of sodium, potassium, calcium and magnesium used were Analar grade (BDH, Poole, Dorset, UK). All water used throughout was deionised. 2.2. Preparation of the emulsions Salts, lactose and whey proteins were sequentially added to deionised water using a Silverson mixer (Model AXR, Silverson Machines Ltd., Chesham, UK) for 5 min, adjusted to give a "nal pH of 7.0$0.1 and centrifuged at 503C at 2000 g]10 min to facilitate de-foaming. When large batches were prepared for pilot-scale drying, the aqueous phase was cooled and kept at 43C overnight to allow the foam to collapse. The aqueous phases contained di!erent levels and ratios of WPI, WPC-35, lactose and salts at total solids concentrations ranging 24}27%. Milk fat, preheated to dissolve all crystals, was mixed into the aqueous system using a Silverson mixer at the lowest speed to disperse the oil phase to give total emulsion solids in the 30}47% range. The coarse emulsion was then homogenised at 45 MPa for 2 or 4 passes at 503C using a Gaulin Mini-Lab homogeniser (APV, Silkeborg, Denmark, 60 kg h~1) before spray drying. During multiple-pass homogenisation, the temperature of the emulsions increased from 50 to about 583C after the fourth pass. 2.3. Spray drying The emulsions were spray dried in a pilot-scale Anhydro &Lab dryer' (Anhydro, Copenhagen, Denmark) us-
ing two #uid nozzle atomisation at a throughput rate of 40 kg h~1. The inlet air temperature was 1603C and the outlet temperature was 803C. 2.4. Fat globule diameter The fat globule diameter of the emulsions was measured using a Malvern Mastersizer X (Malvern Instruments, Malvern, UK) using an MSX15 small volume sample presentation unit. The instrument uses an approximation of the Mie-scattering thoery, which utilises the refractive index of the dispersed phase and its absorption. A relative refractive index n /n "1.095 and an 0*- 8!5%3 absorption value of 0.1 were used in the calculations. A 2 mW He}Ne laser beam (633 nm) and a 300 RF lens (size range 0.05}879 lm) were used for the measurements. The results were measured as the volume weighted diameter of the lower decile of the number of the fat globules D(v, 0.1), the volume weighted median diameter D(v, 0.5) and the volume weighted diameter D(v, 0.9) of the upper decile of the number of fat globules. Only the D(v, 0.9) value is reported here because it was found to be the most sensitive indicator of homogenisation e$ciency and emulsion stability. One sample of each diluted emulsion was repeatedly measured until an equilibrium value was reached. For the determination of the reconstituted powder fat globule size, the powders were suspended in deionised water at 603C at a rate of 10 g/100 g. 2.5. Storage of powders and assessment of oxidative stability The microencapsulated fat powders were stored in vacuum-treated aluminium foil sachets at 163C. The peroxide value (PV) was measured by the IDF sanctioned method (IDF, 1991). For taste assessment (Stone & Sidel, 1993), powders were suspended in deionised water at a rate of 8 g/100 g using a domestic blender (BraK un, FrankfuK rt/M, Germany), allowed to de-aerate and served to a panel of minimum 7 members at room temperature. 2.6. Analysis of free and surface fat The free fat content of the powders was determined by extraction with CCl (A/S Niro Atomizer, 1978). 4 Measurements of the surface composition of the powders were carried out by electron spectroscopy for chemical analysis (ESCA) (FaK ldt et al., 1993) at the Institute for Surface Chemistry, Stockholm, Sweden. In this method, samples are placed under very high vacuum (10~7 Torr) in an AXIS HS photoelectron spectrometer (Kratos Analytical, Manchester, UK), where they are irradiated with X-ray photons of a well-de"ned energy. This causes a complete transfer of the photon's energy to an atomic or molecular orbital electron. Where the electron binding energy is lower than the photon energy, the electron is
M.K. Keogh, B.T. O+Kennedy / International Dairy Journal 9 (1999) 657}663
emitted from the atom with a kinetic energy equal to the di!erence between the photon energy and the binding energy minus the spectrometer work function. Since the total binding energy is characteristic for each element and orbital, an analysis of the emitted photoelectrons allows an identi"cation of the elements of the nearsurface region (&10 nm depth). Each result is a mean of three analyses.
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Table 1 Composition of whey protein isolate and concentrate used Product
WPI WPC-35
Composition (%)
pH of a 10% soln
Protein
Na
K
Ca
Mg
89.9 35.4
0.59 0.62
0.11 1.87
0.13 0.71
0.02 *
7.3 6.6
2.7. Data analysis Experiments where 1}2 variables were studied were carried out in replicate while experiments with 3 variables presented in Tables 5 and 6 below were statistically designed. The results presented in Table 5 were derived from a linear design of 3 variables]2 levels]2 replicates. A quadratic design was used for the results presented in Table 6. The ECHIPTM statistical package used (Wheeler, 1989) recommended 15 trials and 5 replicates for these 3-variable designs. The replicate standard deviation (Rep SD) given in the Tables, is a measure of the replicate error, while residual standard deviation (Res SD) is a measure of the model error. The F-test compares the model error to replicate error as (Res SD)2/(Rep SD)2. The replicate SD should not be more than roughly twice the residual SD for "ve degrees of freedom in the F-test for a quadratic design. In order to be signi"cant, the e!ect of a variable must be roughly twice the residual SD for "ve degrees of freedom in a quadratic design.
3. Results Table 1 shows the composition of the WPI and WPC35 used. The WPI contained 89.9 g protein and 2.7 g lactose versus 35.4 g and 50.4 g lactose/100 g in the WPC-35. Table 2 shows the e!ects of added salts and 2 or 4 homogenisation passes on the fat globule diameter in emulsions stabilised by WPI, after storage for 2 and 22 min. The WPI contained the same level of salts/g protein as WPC-35 when 1.90% salts were added. Using 2 passes only, the fat globule diameter D(v, 0.9) value measured after 2 min increased from 0.76 to 1.72 lm as the salt level increased to 0.63%, but fat globule diameter also increased with time at salt levels of 0.31% and above. At 1.90% salts addition, the emulsions were unstable and had much larger droplet size distributions which were bimodal. When 4 homogenisation passes were used, the D(v, 0.9) values did not increase with time at salt levels up to 0.63%, but the emulsions were unstable at the 1.90% level of salt addition. Table 3 shows an analogous situation where WPI/WPC-35 mixtures were used in proportions ranging from 100 : 0% to 0 : 100%. A D(v, 0.9) value of 1.0 lm was exceeded after 2 passes and the emulsion became unstable over time when 50 or 100% of the WPI was replaced by WPC-35. The D(v, 0.9)
Table 2 E!ect of added salts and 2 or 4 homogenisation passes on the fat globule diameter D(v, 0.9) in emulsions! stabilised by WPI, after standing for 2 and 22 min Added salts (%)
Fat globule diameter D(v, 0.9) lm 2 passes
0.00 0.10 0.20 0.31 0.63 1.90
4 passes
2 min
22 min
2 min
22 min
0.76 0.98 0.91 1.33 1.72 Unstable
0.80 1.04 0.99 1.61 2.42 n.m."
0.54 0.74 0.64 0.78 0.62 Unstable
0.54 n.m." n.m." 0.84 0.66 n.m."
! Containing 15% fat, 12% WPI, 12% lactose. " n.m."not measured.
Table 3 E!ect of the WPI : WPC-35 ratio and 2 or 4 homogenisation passes on the fat globule diameter D(v, 0.9) in emulsions!, after standing for 2 and 22 min WPI : WPC-35 ratio
Fat globule diameter D(v, 0.9) lm 2 passes
100 : 0 85 : 15 75 : 25 50 : 50 0 : 100
4 passes
2 min
22 min
2 min
22 min
0.76 0.81 0.83 1.10 Unstable
0.80 0.85 0.83 1.21 Unstable
0.54 0.60 0.63 0.71 0.76
0.54 0.61 0.65 0.73 0.78
! Containing 15% fat, 12% WPI : WPC-35, 12% lactose.
value remained stable over time after 4 passes for all proportions of WPC-35 up to 100%. This contrasts with the behaviour of WPI-stabilised emulsions in the presence of 1.90% added salts, where emulsion instability was observed. Table 4 shows the e!ect of the composition of emulsions, after 2 and 4 homogenisation passes, on the fat globule diameter after 2 and 22 min. With an emulsion containing fat, WPC-35 and lactose levels of 11, 12 and 12%, the D(v, 0.9) value remained below 1.6 lm for 22 min after 2 passes and below 0.6 lm after 4 passes. When
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Table 4 E!ect of the composition of emulsions after 2 and 4 homogenisation passes on the fat globule diameter after 2 and 22 min Fat globule diameter D(v, 0.9) lm
Composition (%) Fat
11 15 11
WPC-35
12 12 24
Lactose
12 12 0
2 passes
4 passes
2 min
22 min
2 min
22 min
1.50 3.34 1.26
1.58 2.46 11.65
0.55 0.76 0.61
0.57 0.78 14.53
Table 5 E!ect of fat, whey protein source and number of homogenisation passes on the emulsion D(v, 0.9), powder free fat and surface fat values Term
Emulsion D(v, 0.9)
Powder Free fat
(lm)
Surface fat
(Sig!) (%)
Linear Constant# 0.77 Fat (%)$ !0.12 *" Whey protein !0.10 * powder% Homogenis- !0.25 ** ation passes& Res SD' 0.10 * Rep SD) 0.10 *
(Sig)
(%)
(Sig)
2.6 13.2 *** !10.2 ***
46.1 8.1 * !4.1 *
!1.5 *
!2.9 *
1.5 * 1.6 *
5.0 * 7.0 *
! Sig denotes signi"cance; *"P40.05; **"P40.01; ***"P4 0.001. " Not signi"cant. #Constant is the response value of the model at the centred value of all the variables. $ Fat (%); 26 or 40 in powder. % Whey protein powder; 50 : 50 WPC-35 : WPI, or WPC-35 only. & Homogenisation passes; 2 or 4. ' Res SD is a measure of the model error. ) Rep SD is a measure of the replication error.
the fat level was increased to 15%, the D(v, 0.9) values were higher in all cases, about 3.0 lm after 2 passes but below 0.8 lm after 4 passes. However, when lactose addition was omitted, signi"cant fat globule aggregation/coalescence occurred after 22 min after both 2 and 4 passes. Three series of microencapsulated milk fat powders were made to test the e!ects of emulsion composition and number of homogenisation passes on the resulting milk powder properties. The results are shown in Tables 5}7. Tables 5 and 6 show the e!ects on the response variables as each input variable changes from its low to its high value. The constant corresponds to the model response at the centred value of all the input variables. Because of this, the linear or main e!ect ranges from half the e!ect
value below the constant to half the e!ect value above the constant. The polynomial coe$cients of the models were determined but are not presented. In the "rst experiment (Table 5), milk fat and two whey protein powders (50 : 50 WPC-35 : WPI and WPC-35 only) were used to make 10% fat emulsions (25 and 38.5% solids) which were homogenised at 45 MPa using 2 and 4 passes. The emulsion fat globule diameter D(v, 0.9) value was not signi"cantly a!ected by the emulsion composition in this case, but was signi"cantly reduced by the higher number of homogenisation passes. After drying the emulsions (26 and 40% fat powders), the surface fat values were high, ranging from 38.4 to 52.7% and agreeing with values found for whey protein emulsion powders (FaK ldt, 1995). The free fat and surface fat values of the higher (40%) fat powders were higher by 13.2 and 8.1%, respectively. Increasing the level of WPC-35 from 50 to 100% (thus increasing the lactose level and reducing the whey protein level) reduced the free fat signi"cantly by 10.2%, but not the surface fat or emulsion globule D(v, 0.9) value. The higher number of homogenisation passes reduced the D(v, 0.9) value signi"cantly by 0.25 lm but not the free fat or surface fat. When WPC-35 alone was used to make these powders, a signi"cantly higher PV (3.4 vs 1.3 meq O kg~1 fat) was found at zero time than when 2 the WPC-35/WPI blend was used. This indicated that the fat present in the WPC-35 was already oxidised. The results of this experiment using WPC-35 could not therefore be used for testing for stability to oxidation. The lactose : whey protein ratio was then varied by addition of lactose to whey protein isolate (Table 6), but in this experiment, surface fat values were not measured. Milk fat and three blends of lactose : WPI (0 : 100, 33 : 67 and 50 : 50) were used to make 10% fat emulsions which were spray dried. The emulsion solids levels were 25 and 38.5%. In this experiment, the D(v, 0.9) values of the emulsions were signi"cantly reduced by 0.55 lm by increasing the lactose : WPI ratio, unlike the WPC-35/WPI blend which contains salts (Table 5). The reconstituted emulsion globule diameter decreased signi"cantly by 3.0 lm with increasing lactose : WPI ratio, a much greater decrease than for the emulsion globule diameter before drying (Table 6). On drying, the free fat levels of the powders (26 and 40% fat) were signi"cantly higher (by 29.1%) at the higher powder fat level and lower (by 65.1%) as the lactose : WPI ratio increased, (Table 6) as shown by other workers (Young et al., 1993a). Despite this very large decrease in free fat, the result from the previous experiment (Table 5) and other work (FaK ldt, 1995) indicated that surface fat did not decrease as lactose : WPI ratio increased, but increased signi"cantly only as fat increased. This could account for the higher PV of 3.85 (2.4#0.5]2.9) in the higher fat, but not in the higher lactose : WPI ratio powders after 2 months storage. A PV of 3.5 was associated with oxidised o!-#avour in whole milk powders stored at 603C (Tuohy, 1987).
M.K. Keogh, B.T. O+Kennedy / International Dairy Journal 9 (1999) 657}663
661
Table 6 E!ect of fat and lactose : WPI ratio on the emulsion D(v, 0.9) value and powder properties Term
Emulsion D(v, 0.9) (lm)
Linear Interaction Quadratic
Constant# Fat$ Lactose : WPI% Fat x Lactose : WPI Lactose : WPI'2 Res SD Rep SD
0.30 !0.18 !0.55 0.60 0.20 0.09 0.10
Powder properties Free fat (Sig!)
*" ** *
(%)
17.6 29.1 !65.1 2.0 20.0 1.82 2.23
* * *
(Sig)
Reconstituted emulsion
PV at 2 months
D(v, 0.9) lm
(Sig)
(meq. O kg~1 fat) 2
(Sig)
* *** *** *
2.17 0.89 !3.03 !0.75
* * ** *
2.4 2.9 0.2 !0.2
* *** * *
*** * *
0.82 0.94 1.20
* * *
0.5 0.3 0.4
* * *
!,",#,$same as Table 5. %Lactose : WPI, 0 : 100; 33 :67 or 50 : 50.
Table 7 E!ect of fat on the emulsion! D(v, 0.9) value and powder properties Fat Emulsion Powder Powder Reconstituted Taste stability" (%) D(v, 0.9) free fat surface fat emulsion (weeks) (lm) (%) (%) D(v, 0.9) lm Sour Metallic 20.2 1.90 35.0 0.63 50.2 0.66
1.11 4.85 22.31
30.8 39.2 49.4
1.80 0.72 1.73
14 14 8
18 18 18
!Emulsion total solids 30%, containing 50 : 50 WPI : lactose. "Weeks to reach an o!-#avour score of 25 out of 100.
A "nal con"rmatory trial (Table 7) was carried out in replicate to determine the e!ect of fat level on powderfree fat, surface fat and oxidised o!-#avour stability. Although the fat, free fat and surface fat levels of the powder increased, the level of metallic o!-#avour remained the same, but a sour o!-#avour was detectable 6 weeks earlier in the highest fat (50%) powder.
4. Discussion The properties of microencapsulated fat powders such as free fat, surface fat and stability to oxidation are expected to depend on the emulsion composition and homogenising conditions. Certain changes in emulsion composition in#uenced the fat globule diameter and stability to aggregation. The current work has shown that the minimisation of the fat globule diameter, free fat, surface fat and fat oxidation in powders does not depend to the same extent on the same factors. The purpose of the whey protein was to emulsify and stabilise newly created fat/water interfaces. Since whey proteins are
globular in nature, any adsorption to an oil/water interface will result in an unfolding of the protein molecule (Macritchie, 1978), stabilising the interface but denaturing the protein. Native whey proteins are generally not visually a!ected by the presence of salts (i.e. chlorides of sodium or calcium), but denatured whey proteins are susceptible to aggregation when salts are present (Doi, 1993; McClements & Keogh, 1995). Depending on the valency of the cation, salts may reduce the electrostatic repulsion or cause cross-linking between proteinstabilised fat globules (Barbut & Foegeding, 1993). It is very likely that with 2 homogenisation passes, the surface coverage by the emulsifying protein of the globules is less than at 4 passes. At low-globule surface coverage, as achieved by 2 passes, the emulsion stability depends on electrostatic forces and is therefore sensitive to salt levels. At a higher number of homogenisation passes, surface coverage of the globules by protein may be greater, emulsion stability would then depend on steric forces and the emulsion would become much less sensitive to added salts (Masson & Jost, 1986). It was more recently shown (Rientjes & Walstra, 1993) that whey protein emulsions will only display globule cluster formation if the whey proteins are denatured prior to emulsi"cation and if calcium or other divalent salts are present but not if sodium or monovalent salt levels are high. The source of whey protein was found to be particularly important for control of initial fat globule diameter and subsequent aggregation of fat globules. This was evident from observing the behaviour of WPI stabilised emulsions (Table 2) with a 15 : 12 : 12 fat : WPI : lactose ratio where very little time-dependant aggregation was observed at low levels of salt addition, especially after 4 homogenisation passes. However, as WPI was replaced by WPC-35 as emulsi"er (Table 3), the fat globules displayed signi"cant aggregation after
662
M.K. Keogh, B.T. O+Kennedy / International Dairy Journal 9 (1999) 657}663
2 homogenisation passes. This behaviour parallels that of WPI at the higher levels of added salts. Thus, WPI (without added salts) appears to be preferable as an emulsi"er. The requirements for the minimisation of free fat and surface fat in powder manufacture are not the same as for lowering the fat globule diameter in the emulsion. In microencapsulated fat powders, hydrophilic components with glass transition temperatures or gel points above ambient conditions such as sugars or hydrocolloids should be suitable as "llers. Lactose is a suitable "ller, but, being hygroscopic, will recrystallise in high humidity conditions and cause fat globule coalescence (Saito, 1985). The free fat in the powders increased with the fat and whey protein levels and decreased with lactose (Table 4). Higher lactose : protein ratios reduced the accessibility of solvent through the powder protein wall to the fat. Higher lactose : protein ratios also reduced aggregation of fat globules in the emulsion during drying which resulted in lower free fat in the powder and lower D(v, 0.9) values in reconstituted emulsions (Table 6). The use of a combination of WPI and lactose largely avoids the inclusion of salts present in WPCs, but WPI is a signi"cant cost factor commercially. Since fats can di!use through a hydrophobic protein layer, a key consideration is the prevention of fat migration on to the surface of the powder particle. The surface fat in the powders increased with the fat level but unlike free fat, it was not a!ected by increasing the WPC35 : WPI ratio, in e!ect the lactose : protein ratio (Table 5). Microencapsulated powders containing WPC-35 had elevated PV levels immediately after manufacture, indicating that that the fat present in the WPC-35 was already oxidised. However, the dairy industry has a lowsalt high-protein powder equivalent to WPI in sodium caseinate which has been shown to have microencapsulating properties superior to WPI, that is, it results in a greater reduction in surface fat both singly and synergistically with lactose (FaK ldt, 1995). The fact that only the higher fat level adversely a!ected the stability to oxidation suggests that factors other than gross composition and homogenisation conditions are involved. These factors need further investigation.
5. Conclusions Emulsions of su$ciently small fat globule diameter which were stable over time could be prepared using WPI or WPC-35 as an emulsifying agent. A high homogenisation pressure with 4 passes is required. The major components of the emulsion as well as the minor salt components a!ected globule diameter and stability. Increasing the number of homogenisation passes was shown to counterbalance the negative e!ects of the salts.
Increasing lactose and lactose : whey protein ratios reduced free fat signi"cantly but surface fat only marginally. The only signi"cant composition factor a!ecting surface fat and thereby the level of oxidation was the fat level in the emulsion. Other work (FaK ldt & Bergansta> hl, 1995) has already shown that sodium caseinate was superior to whey protein as a microencapsulating agent, but that sodium caseinate with lactose was by far the most e!ective combination.
Acknowledgements This project was funded in part under the European Regional Development Fund within the Non-Commissioned Food Research Programme. We are indebted to the Institute of Surface Chemistry, Stockholm, in particular Prof. B. Bergensta> hl, for carrying out the ESCA analyses.
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M.K. Keogh, B.T. O+Kennedy / International Dairy Journal 9 (1999) 657}663 McClements, D. J., & Keogh, M. K. (1995). Physical properties of cold-setting gels formed from heat-denatured whey protein isolate. Journal of Food Science, 69, 7}14. Rientjes, G. J., & Walstra, P. (1993). Factors a!ecting the stability of whey-based emulsions. Milchwissenschaft, 48, 63}67. Rosenberg, M., & Lee, S. L. (1993). Microstructure of whey protein/anhydrous milkfat emulsions. Food Structure, 12, 267}274. Rosenberg, M., & Young, S. L. (1993). Whey proteins as microencapsulating agents. Microencapsulation of anhydrous milk fat*structure evaluation. Food Structure, 12, 31}41. Saito, Z. (1985). Particle structure in spray-dried whole milk and in instant skim milk powder as related to lactose crystallization. Food Microstructure, 4, 333}340. Stone, H., & Sidel, J. L. (1993). Measurement. In H. Stone, Sensory evaluation practices (pp. 66}81). London, UK: Academic Press.
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Tuohy, S. (1987). Milkfat autoxidation in whole milk powder during storage. Ph.D. Thesis. National University of Ireland. Van Boekel, M. A. J. S., & Walstra, P. (1991). Colloids Surfaces, 3, 109. Walstra, P. (1986). Overview of emulsion and foam stability. In E. Dickenson, Food emulsions and foams (pp. 242}257). London, UK: Royal Society of Chemistry. Wheeler, B. (1989). ECHIPTM Course Text. ECHIP Inc., Hockessin, DE, USA. Young, S. L., Sarda, X., & Rosenberg, M. (1993a). Microencapsulating properties of whey proteins. 1. Microencapsulation of anhydrous milk fat. Journal of Dairy Science, 76, 2868}2877. Young, S. L., Sarda, X., & Rosenberg, M. (1993b). Microencapsulating properties of whey proteins. 2. Combination of whey proteins with carbohydrates. Journal of Dairy Science, 76, 2878}2885.
Milk fat microencapsulation using whey proteins M. Kieran Keogh*, Brendan T. O'Kennedy Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Republic of Ireland Received 28 March 1998; accepted 18 August 1999
Abstract The e!ects of homogenisation conditions (pressure and number of passes) and emulsion composition (fat, whey protein, lactose, salts and water) on the properties of emulsions and microencapsulated milk fat powders were measured. The emulsions were assessed by fat globule diameter and size stability over time. The properties of the powders measured were particle diameter, reconstituted emulsion fat globule diameter, free fat, surface fat and fat oxidation. As expected, increasing homogenisation pressure reduced the fat globule diameter and increasing the number of homogenisation passes reduced the diameter of the largest globules. Increasing fat and salts reduced fat globule diameter stability after 2 homogenisation passes but the reduction in stability was less after 4 passes. Increasing the lactose : whey protein concentrate ratio reduced free fat and fat globule aggregation after powder reconstitution, but not the surface fat. The higher level of fat increased the surface fat on powder particles and the level of oxidation during storage. ( 1999 Elsevier Science Ltd. All rights reserved. Keywords: Microencapsulation; Homogenisation; Composition; Oxidation
1. Introduction Microencapsulation involves dispersing and "xing sub-micron-sized particles in a solid particle matrix. Techniques suitable for the microencapsulation of fat, such as high-pressure homogenisation for dispersing fat globules and spray drying for "xation of the globules, are readily available in the dairy industry. The solid matrix or wall material of the powder particles should protect the microparticles or core material against deterioration (Rosenberg & Lee, 1993; Rosenberg & Young, 1993; Young, Sarda, & Rosenberg, 1993a, b). Any microparticle can be encapsulated, but when the core material is fat, part of the wall material must have emulsifying properties and be capable of dehydration. The milk protein products sodium caseinate and whey protein concentrate have excellent emulsifying and dehydration properties. While sodium caseinate has a relatively uniform composition, commercial whey protein concentrates contain 35}75% or more protein and whey protein isolates have at least 90% protein. A critical parameter of the powder is the extent of fat on the surface on the particles, which can be measured by
* Corresponding author. E-mail address: [email protected] (M.K. Keogh)
electron spectroscopy for chemical analysis (ESCA) (FaK ldt, Bergensta> hl & Carlsson, 1993). It has been shown that surface fat does not correlate well to solvent extractable free fat, since the latter also includes some nearsurface fat (Buchheim, 1982). Surface fat but not free fat by solvent extraction (Buma, 1971) was shown to be related to the oxidative stability of cholesterol in fat powders (Granelli, Fa> ldt, Appelqvist & Bergensta> hl, 1996). Some authors do not seem to distinguish clearly between free fat and surface fat, but associate the latter with the free fat which can be removed from the surface of the powder particles during drying. It was suggested (van Boekel & Walstra, 1991; see also FaK ldt & Bergansta> hl, 1995) that the extent of surface fat resulted from emulsion instability leading to coalescence. Coalescence instability in emulsions is assumed to increase the di!usion of fat onto the powder surface during drying (FaK ldt & Bergansta> hl, 1995). It should be noted that fat globule aggregation and increase in emulsion viscosity usually precede fat coalescence (Walstra, 1986) leading to higher free fat and surface fat in powders. Emulsion fat globule diameter and size stability over time, therefore, seem to be of fundamental importance for successful microencapsulation. The advantages of microencapsulation are that milk fat can be converted to a stable powder form and better powder #ow properties may be obtained if the extent of
0958-6946/99/$ - see front matter ( 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 1 3 7 - 5
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fat on the surface of the dried particles can be minimised. Low levels of fat on the surface could result in the production of powders with higher fat content using conventional dryers and of fat powders that are more stable to oxidation. The aim of this research was to determine the e!ects of homogenisation factors and composition (fat, whey protein, lactose and salts) on the formation and stability of emulsions and encapsulated fat powder particles and to relate these to the properties of the powder.
2. Materials and methods 2.1. Ingredients Whey protein isolate (WPI) was obtained from Davisco International, (Le Sueur, MN, USA), and a whey protein concentrate (WPC-35) from Carbery Milk Products, Ballineen, Co. Cork, Ireland. Anhydrous milk fat (AMF) was obtained from Dairygold, Mallow, Co. Cork, Ireland. This AMF was produced during summer, was less than 1 month old and was within the IDF speci"cations (IDF, 1977). Lactose was obtained from Avonmore Foods plc, Ballyragget, Co. Kilkenny, Ireland. The chloride salts of sodium, potassium, calcium and magnesium used were Analar grade (BDH, Poole, Dorset, UK). All water used throughout was deionised. 2.2. Preparation of the emulsions Salts, lactose and whey proteins were sequentially added to deionised water using a Silverson mixer (Model AXR, Silverson Machines Ltd., Chesham, UK) for 5 min, adjusted to give a "nal pH of 7.0$0.1 and centrifuged at 503C at 2000 g]10 min to facilitate de-foaming. When large batches were prepared for pilot-scale drying, the aqueous phase was cooled and kept at 43C overnight to allow the foam to collapse. The aqueous phases contained di!erent levels and ratios of WPI, WPC-35, lactose and salts at total solids concentrations ranging 24}27%. Milk fat, preheated to dissolve all crystals, was mixed into the aqueous system using a Silverson mixer at the lowest speed to disperse the oil phase to give total emulsion solids in the 30}47% range. The coarse emulsion was then homogenised at 45 MPa for 2 or 4 passes at 503C using a Gaulin Mini-Lab homogeniser (APV, Silkeborg, Denmark, 60 kg h~1) before spray drying. During multiple-pass homogenisation, the temperature of the emulsions increased from 50 to about 583C after the fourth pass. 2.3. Spray drying The emulsions were spray dried in a pilot-scale Anhydro &Lab dryer' (Anhydro, Copenhagen, Denmark) us-
ing two #uid nozzle atomisation at a throughput rate of 40 kg h~1. The inlet air temperature was 1603C and the outlet temperature was 803C. 2.4. Fat globule diameter The fat globule diameter of the emulsions was measured using a Malvern Mastersizer X (Malvern Instruments, Malvern, UK) using an MSX15 small volume sample presentation unit. The instrument uses an approximation of the Mie-scattering thoery, which utilises the refractive index of the dispersed phase and its absorption. A relative refractive index n /n "1.095 and an 0*- 8!5%3 absorption value of 0.1 were used in the calculations. A 2 mW He}Ne laser beam (633 nm) and a 300 RF lens (size range 0.05}879 lm) were used for the measurements. The results were measured as the volume weighted diameter of the lower decile of the number of the fat globules D(v, 0.1), the volume weighted median diameter D(v, 0.5) and the volume weighted diameter D(v, 0.9) of the upper decile of the number of fat globules. Only the D(v, 0.9) value is reported here because it was found to be the most sensitive indicator of homogenisation e$ciency and emulsion stability. One sample of each diluted emulsion was repeatedly measured until an equilibrium value was reached. For the determination of the reconstituted powder fat globule size, the powders were suspended in deionised water at 603C at a rate of 10 g/100 g. 2.5. Storage of powders and assessment of oxidative stability The microencapsulated fat powders were stored in vacuum-treated aluminium foil sachets at 163C. The peroxide value (PV) was measured by the IDF sanctioned method (IDF, 1991). For taste assessment (Stone & Sidel, 1993), powders were suspended in deionised water at a rate of 8 g/100 g using a domestic blender (BraK un, FrankfuK rt/M, Germany), allowed to de-aerate and served to a panel of minimum 7 members at room temperature. 2.6. Analysis of free and surface fat The free fat content of the powders was determined by extraction with CCl (A/S Niro Atomizer, 1978). 4 Measurements of the surface composition of the powders were carried out by electron spectroscopy for chemical analysis (ESCA) (FaK ldt et al., 1993) at the Institute for Surface Chemistry, Stockholm, Sweden. In this method, samples are placed under very high vacuum (10~7 Torr) in an AXIS HS photoelectron spectrometer (Kratos Analytical, Manchester, UK), where they are irradiated with X-ray photons of a well-de"ned energy. This causes a complete transfer of the photon's energy to an atomic or molecular orbital electron. Where the electron binding energy is lower than the photon energy, the electron is
M.K. Keogh, B.T. O+Kennedy / International Dairy Journal 9 (1999) 657}663
emitted from the atom with a kinetic energy equal to the di!erence between the photon energy and the binding energy minus the spectrometer work function. Since the total binding energy is characteristic for each element and orbital, an analysis of the emitted photoelectrons allows an identi"cation of the elements of the nearsurface region (&10 nm depth). Each result is a mean of three analyses.
659
Table 1 Composition of whey protein isolate and concentrate used Product
WPI WPC-35
Composition (%)
pH of a 10% soln
Protein
Na
K
Ca
Mg
89.9 35.4
0.59 0.62
0.11 1.87
0.13 0.71
0.02 *
7.3 6.6
2.7. Data analysis Experiments where 1}2 variables were studied were carried out in replicate while experiments with 3 variables presented in Tables 5 and 6 below were statistically designed. The results presented in Table 5 were derived from a linear design of 3 variables]2 levels]2 replicates. A quadratic design was used for the results presented in Table 6. The ECHIPTM statistical package used (Wheeler, 1989) recommended 15 trials and 5 replicates for these 3-variable designs. The replicate standard deviation (Rep SD) given in the Tables, is a measure of the replicate error, while residual standard deviation (Res SD) is a measure of the model error. The F-test compares the model error to replicate error as (Res SD)2/(Rep SD)2. The replicate SD should not be more than roughly twice the residual SD for "ve degrees of freedom in the F-test for a quadratic design. In order to be signi"cant, the e!ect of a variable must be roughly twice the residual SD for "ve degrees of freedom in a quadratic design.
3. Results Table 1 shows the composition of the WPI and WPC35 used. The WPI contained 89.9 g protein and 2.7 g lactose versus 35.4 g and 50.4 g lactose/100 g in the WPC-35. Table 2 shows the e!ects of added salts and 2 or 4 homogenisation passes on the fat globule diameter in emulsions stabilised by WPI, after storage for 2 and 22 min. The WPI contained the same level of salts/g protein as WPC-35 when 1.90% salts were added. Using 2 passes only, the fat globule diameter D(v, 0.9) value measured after 2 min increased from 0.76 to 1.72 lm as the salt level increased to 0.63%, but fat globule diameter also increased with time at salt levels of 0.31% and above. At 1.90% salts addition, the emulsions were unstable and had much larger droplet size distributions which were bimodal. When 4 homogenisation passes were used, the D(v, 0.9) values did not increase with time at salt levels up to 0.63%, but the emulsions were unstable at the 1.90% level of salt addition. Table 3 shows an analogous situation where WPI/WPC-35 mixtures were used in proportions ranging from 100 : 0% to 0 : 100%. A D(v, 0.9) value of 1.0 lm was exceeded after 2 passes and the emulsion became unstable over time when 50 or 100% of the WPI was replaced by WPC-35. The D(v, 0.9)
Table 2 E!ect of added salts and 2 or 4 homogenisation passes on the fat globule diameter D(v, 0.9) in emulsions! stabilised by WPI, after standing for 2 and 22 min Added salts (%)
Fat globule diameter D(v, 0.9) lm 2 passes
0.00 0.10 0.20 0.31 0.63 1.90
4 passes
2 min
22 min
2 min
22 min
0.76 0.98 0.91 1.33 1.72 Unstable
0.80 1.04 0.99 1.61 2.42 n.m."
0.54 0.74 0.64 0.78 0.62 Unstable
0.54 n.m." n.m." 0.84 0.66 n.m."
! Containing 15% fat, 12% WPI, 12% lactose. " n.m."not measured.
Table 3 E!ect of the WPI : WPC-35 ratio and 2 or 4 homogenisation passes on the fat globule diameter D(v, 0.9) in emulsions!, after standing for 2 and 22 min WPI : WPC-35 ratio
Fat globule diameter D(v, 0.9) lm 2 passes
100 : 0 85 : 15 75 : 25 50 : 50 0 : 100
4 passes
2 min
22 min
2 min
22 min
0.76 0.81 0.83 1.10 Unstable
0.80 0.85 0.83 1.21 Unstable
0.54 0.60 0.63 0.71 0.76
0.54 0.61 0.65 0.73 0.78
! Containing 15% fat, 12% WPI : WPC-35, 12% lactose.
value remained stable over time after 4 passes for all proportions of WPC-35 up to 100%. This contrasts with the behaviour of WPI-stabilised emulsions in the presence of 1.90% added salts, where emulsion instability was observed. Table 4 shows the e!ect of the composition of emulsions, after 2 and 4 homogenisation passes, on the fat globule diameter after 2 and 22 min. With an emulsion containing fat, WPC-35 and lactose levels of 11, 12 and 12%, the D(v, 0.9) value remained below 1.6 lm for 22 min after 2 passes and below 0.6 lm after 4 passes. When
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Table 4 E!ect of the composition of emulsions after 2 and 4 homogenisation passes on the fat globule diameter after 2 and 22 min Fat globule diameter D(v, 0.9) lm
Composition (%) Fat
11 15 11
WPC-35
12 12 24
Lactose
12 12 0
2 passes
4 passes
2 min
22 min
2 min
22 min
1.50 3.34 1.26
1.58 2.46 11.65
0.55 0.76 0.61
0.57 0.78 14.53
Table 5 E!ect of fat, whey protein source and number of homogenisation passes on the emulsion D(v, 0.9), powder free fat and surface fat values Term
Emulsion D(v, 0.9)
Powder Free fat
(lm)
Surface fat
(Sig!) (%)
Linear Constant# 0.77 Fat (%)$ !0.12 *" Whey protein !0.10 * powder% Homogenis- !0.25 ** ation passes& Res SD' 0.10 * Rep SD) 0.10 *
(Sig)
(%)
(Sig)
2.6 13.2 *** !10.2 ***
46.1 8.1 * !4.1 *
!1.5 *
!2.9 *
1.5 * 1.6 *
5.0 * 7.0 *
! Sig denotes signi"cance; *"P40.05; **"P40.01; ***"P4 0.001. " Not signi"cant. #Constant is the response value of the model at the centred value of all the variables. $ Fat (%); 26 or 40 in powder. % Whey protein powder; 50 : 50 WPC-35 : WPI, or WPC-35 only. & Homogenisation passes; 2 or 4. ' Res SD is a measure of the model error. ) Rep SD is a measure of the replication error.
the fat level was increased to 15%, the D(v, 0.9) values were higher in all cases, about 3.0 lm after 2 passes but below 0.8 lm after 4 passes. However, when lactose addition was omitted, signi"cant fat globule aggregation/coalescence occurred after 22 min after both 2 and 4 passes. Three series of microencapsulated milk fat powders were made to test the e!ects of emulsion composition and number of homogenisation passes on the resulting milk powder properties. The results are shown in Tables 5}7. Tables 5 and 6 show the e!ects on the response variables as each input variable changes from its low to its high value. The constant corresponds to the model response at the centred value of all the input variables. Because of this, the linear or main e!ect ranges from half the e!ect
value below the constant to half the e!ect value above the constant. The polynomial coe$cients of the models were determined but are not presented. In the "rst experiment (Table 5), milk fat and two whey protein powders (50 : 50 WPC-35 : WPI and WPC-35 only) were used to make 10% fat emulsions (25 and 38.5% solids) which were homogenised at 45 MPa using 2 and 4 passes. The emulsion fat globule diameter D(v, 0.9) value was not signi"cantly a!ected by the emulsion composition in this case, but was signi"cantly reduced by the higher number of homogenisation passes. After drying the emulsions (26 and 40% fat powders), the surface fat values were high, ranging from 38.4 to 52.7% and agreeing with values found for whey protein emulsion powders (FaK ldt, 1995). The free fat and surface fat values of the higher (40%) fat powders were higher by 13.2 and 8.1%, respectively. Increasing the level of WPC-35 from 50 to 100% (thus increasing the lactose level and reducing the whey protein level) reduced the free fat signi"cantly by 10.2%, but not the surface fat or emulsion globule D(v, 0.9) value. The higher number of homogenisation passes reduced the D(v, 0.9) value signi"cantly by 0.25 lm but not the free fat or surface fat. When WPC-35 alone was used to make these powders, a signi"cantly higher PV (3.4 vs 1.3 meq O kg~1 fat) was found at zero time than when 2 the WPC-35/WPI blend was used. This indicated that the fat present in the WPC-35 was already oxidised. The results of this experiment using WPC-35 could not therefore be used for testing for stability to oxidation. The lactose : whey protein ratio was then varied by addition of lactose to whey protein isolate (Table 6), but in this experiment, surface fat values were not measured. Milk fat and three blends of lactose : WPI (0 : 100, 33 : 67 and 50 : 50) were used to make 10% fat emulsions which were spray dried. The emulsion solids levels were 25 and 38.5%. In this experiment, the D(v, 0.9) values of the emulsions were signi"cantly reduced by 0.55 lm by increasing the lactose : WPI ratio, unlike the WPC-35/WPI blend which contains salts (Table 5). The reconstituted emulsion globule diameter decreased signi"cantly by 3.0 lm with increasing lactose : WPI ratio, a much greater decrease than for the emulsion globule diameter before drying (Table 6). On drying, the free fat levels of the powders (26 and 40% fat) were signi"cantly higher (by 29.1%) at the higher powder fat level and lower (by 65.1%) as the lactose : WPI ratio increased, (Table 6) as shown by other workers (Young et al., 1993a). Despite this very large decrease in free fat, the result from the previous experiment (Table 5) and other work (FaK ldt, 1995) indicated that surface fat did not decrease as lactose : WPI ratio increased, but increased signi"cantly only as fat increased. This could account for the higher PV of 3.85 (2.4#0.5]2.9) in the higher fat, but not in the higher lactose : WPI ratio powders after 2 months storage. A PV of 3.5 was associated with oxidised o!-#avour in whole milk powders stored at 603C (Tuohy, 1987).
M.K. Keogh, B.T. O+Kennedy / International Dairy Journal 9 (1999) 657}663
661
Table 6 E!ect of fat and lactose : WPI ratio on the emulsion D(v, 0.9) value and powder properties Term
Emulsion D(v, 0.9) (lm)
Linear Interaction Quadratic
Constant# Fat$ Lactose : WPI% Fat x Lactose : WPI Lactose : WPI'2 Res SD Rep SD
0.30 !0.18 !0.55 0.60 0.20 0.09 0.10
Powder properties Free fat (Sig!)
*" ** *
(%)
17.6 29.1 !65.1 2.0 20.0 1.82 2.23
* * *
(Sig)
Reconstituted emulsion
PV at 2 months
D(v, 0.9) lm
(Sig)
(meq. O kg~1 fat) 2
(Sig)
* *** *** *
2.17 0.89 !3.03 !0.75
* * ** *
2.4 2.9 0.2 !0.2
* *** * *
*** * *
0.82 0.94 1.20
* * *
0.5 0.3 0.4
* * *
!,",#,$same as Table 5. %Lactose : WPI, 0 : 100; 33 :67 or 50 : 50.
Table 7 E!ect of fat on the emulsion! D(v, 0.9) value and powder properties Fat Emulsion Powder Powder Reconstituted Taste stability" (%) D(v, 0.9) free fat surface fat emulsion (weeks) (lm) (%) (%) D(v, 0.9) lm Sour Metallic 20.2 1.90 35.0 0.63 50.2 0.66
1.11 4.85 22.31
30.8 39.2 49.4
1.80 0.72 1.73
14 14 8
18 18 18
!Emulsion total solids 30%, containing 50 : 50 WPI : lactose. "Weeks to reach an o!-#avour score of 25 out of 100.
A "nal con"rmatory trial (Table 7) was carried out in replicate to determine the e!ect of fat level on powderfree fat, surface fat and oxidised o!-#avour stability. Although the fat, free fat and surface fat levels of the powder increased, the level of metallic o!-#avour remained the same, but a sour o!-#avour was detectable 6 weeks earlier in the highest fat (50%) powder.
4. Discussion The properties of microencapsulated fat powders such as free fat, surface fat and stability to oxidation are expected to depend on the emulsion composition and homogenising conditions. Certain changes in emulsion composition in#uenced the fat globule diameter and stability to aggregation. The current work has shown that the minimisation of the fat globule diameter, free fat, surface fat and fat oxidation in powders does not depend to the same extent on the same factors. The purpose of the whey protein was to emulsify and stabilise newly created fat/water interfaces. Since whey proteins are
globular in nature, any adsorption to an oil/water interface will result in an unfolding of the protein molecule (Macritchie, 1978), stabilising the interface but denaturing the protein. Native whey proteins are generally not visually a!ected by the presence of salts (i.e. chlorides of sodium or calcium), but denatured whey proteins are susceptible to aggregation when salts are present (Doi, 1993; McClements & Keogh, 1995). Depending on the valency of the cation, salts may reduce the electrostatic repulsion or cause cross-linking between proteinstabilised fat globules (Barbut & Foegeding, 1993). It is very likely that with 2 homogenisation passes, the surface coverage by the emulsifying protein of the globules is less than at 4 passes. At low-globule surface coverage, as achieved by 2 passes, the emulsion stability depends on electrostatic forces and is therefore sensitive to salt levels. At a higher number of homogenisation passes, surface coverage of the globules by protein may be greater, emulsion stability would then depend on steric forces and the emulsion would become much less sensitive to added salts (Masson & Jost, 1986). It was more recently shown (Rientjes & Walstra, 1993) that whey protein emulsions will only display globule cluster formation if the whey proteins are denatured prior to emulsi"cation and if calcium or other divalent salts are present but not if sodium or monovalent salt levels are high. The source of whey protein was found to be particularly important for control of initial fat globule diameter and subsequent aggregation of fat globules. This was evident from observing the behaviour of WPI stabilised emulsions (Table 2) with a 15 : 12 : 12 fat : WPI : lactose ratio where very little time-dependant aggregation was observed at low levels of salt addition, especially after 4 homogenisation passes. However, as WPI was replaced by WPC-35 as emulsi"er (Table 3), the fat globules displayed signi"cant aggregation after
662
M.K. Keogh, B.T. O+Kennedy / International Dairy Journal 9 (1999) 657}663
2 homogenisation passes. This behaviour parallels that of WPI at the higher levels of added salts. Thus, WPI (without added salts) appears to be preferable as an emulsi"er. The requirements for the minimisation of free fat and surface fat in powder manufacture are not the same as for lowering the fat globule diameter in the emulsion. In microencapsulated fat powders, hydrophilic components with glass transition temperatures or gel points above ambient conditions such as sugars or hydrocolloids should be suitable as "llers. Lactose is a suitable "ller, but, being hygroscopic, will recrystallise in high humidity conditions and cause fat globule coalescence (Saito, 1985). The free fat in the powders increased with the fat and whey protein levels and decreased with lactose (Table 4). Higher lactose : protein ratios reduced the accessibility of solvent through the powder protein wall to the fat. Higher lactose : protein ratios also reduced aggregation of fat globules in the emulsion during drying which resulted in lower free fat in the powder and lower D(v, 0.9) values in reconstituted emulsions (Table 6). The use of a combination of WPI and lactose largely avoids the inclusion of salts present in WPCs, but WPI is a signi"cant cost factor commercially. Since fats can di!use through a hydrophobic protein layer, a key consideration is the prevention of fat migration on to the surface of the powder particle. The surface fat in the powders increased with the fat level but unlike free fat, it was not a!ected by increasing the WPC35 : WPI ratio, in e!ect the lactose : protein ratio (Table 5). Microencapsulated powders containing WPC-35 had elevated PV levels immediately after manufacture, indicating that that the fat present in the WPC-35 was already oxidised. However, the dairy industry has a lowsalt high-protein powder equivalent to WPI in sodium caseinate which has been shown to have microencapsulating properties superior to WPI, that is, it results in a greater reduction in surface fat both singly and synergistically with lactose (FaK ldt, 1995). The fact that only the higher fat level adversely a!ected the stability to oxidation suggests that factors other than gross composition and homogenisation conditions are involved. These factors need further investigation.
5. Conclusions Emulsions of su$ciently small fat globule diameter which were stable over time could be prepared using WPI or WPC-35 as an emulsifying agent. A high homogenisation pressure with 4 passes is required. The major components of the emulsion as well as the minor salt components a!ected globule diameter and stability. Increasing the number of homogenisation passes was shown to counterbalance the negative e!ects of the salts.
Increasing lactose and lactose : whey protein ratios reduced free fat signi"cantly but surface fat only marginally. The only signi"cant composition factor a!ecting surface fat and thereby the level of oxidation was the fat level in the emulsion. Other work (FaK ldt & Bergansta> hl, 1995) has already shown that sodium caseinate was superior to whey protein as a microencapsulating agent, but that sodium caseinate with lactose was by far the most e!ective combination.
Acknowledgements This project was funded in part under the European Regional Development Fund within the Non-Commissioned Food Research Programme. We are indebted to the Institute of Surface Chemistry, Stockholm, in particular Prof. B. Bergensta> hl, for carrying out the ESCA analyses.
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