Microencapsulating Properties of Whey Proteins. 1. Microencapsulation of Anhydrous Milk Fat

Microencapsulating Properties of Whey Proteins. 1. Microencapsulation of Anhydrous Milk Fat

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L. YOUNG. X. SARDA, and M. ROSENBERG Department of Food Science and Technology University of California, Davis Davis 95616-8598

ABSTRACT

INTRODUCTION

Anhydrous milk fat was microencapsulated in whey protein concentrates and a whey protein isolate by spray drying. The effects of microencapsulating agent type and concentration (10 to 30% wtlwt) and of fat load (25 to 75% wt/wt) on the microencapsulation yield and efficiency were determined. Encapsulation yields of more than 90% were obtained for all evaluated systems. Microencapsulation efficiency was enhanced by increased solids concentration of wall solutions only in systems with low fat load. Microencapsulation efficiency was adversely affected by anhydrous milk fat load. Protein to lactose ratio affected the microencapsulation efficiency. Partial (50%) replacement of whey proteins by lactose resulted in the highest (95%) microencapsulation efficiency even at 75% fat load. Spherical microcapsules in which the milk fat was well isolated from the environment were obtained in all cases. The results indicate that microencapsulation of anhydrous milk fat by whey proteins is feasible and may have new applications for milk fat in food and dairy products. (Key words: anhydrous milk fat. milk fat, microencapsulation, whey proteins)

Microencapsulation is a "packaging" technique by which liquid droplets or solid particles are packed into thin films of a microencapsulating agents. The structure formed by the microencapsulating agent around the microencapsulated material (the core) is called the wall system. The wall protects the core against deterioration, limits the evaporation (or losses) of volatile core materials, and releases the core under desired conditions (1. 7. 9. 10). Microencapsulation in the food industry generally involves volatiles, flavoring materials. vitamins. essential oils and oleoresins, bacteria, enzymes. and minerals (9). Although many microencapsulation techniques have been developed, spray drying is most commonly used to microencapsulate food ingredients (25). A wall material for microencapsulation by spray drying should exhibit high solubility and possess emulsification, film-forming, and drying properties; additionally, its concentrated solutions should have low viscosity (25). Although many wall materials are available for nonfood applications, the variety of wall materials approved for food applications is limited and includes natural gums, carbohydrates, waxes, and some proteins (7, 9, 14). Efforts to develop new microencapsulating agents have been made (3); however, the success has been limited so far, and a need for new agents exists. The physicochemical properties of whey proteins have been studied extensively (13, 15, 17, 18, 19,21.22, 23, 24), and whey proteins seem to possess many of the characteristics desired for a microencapsulating agent; however, this trait has not yet been confirmed. Applications for milk fat in various food systems are limited by its susceptibility to oxidation and its handling difficulties. Microencapsulation can transform milk fat into dry and stable powder and thus may offer a solution to these problems.

Abbreviation key: AMF = anhydrous milk fat, MEE = microencapsulation efficiency, MEY = microencapsulation yield, WPC = whey protein concentrate, WPC50 = WPC containing 50% protein, WPC7S = WPC containing 75% protein, WPI = whey protein isolate.

Received July 17, 1992. Accepted June 18, 1993. 1993 J Dairy Sci 76:2868-2877

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MICROENCAPSULAnON BY WHEY PROTEINS

The objective of this research was to study the effects of wall composition and the ratio of core to wall on the microencapsulation (by spray drying) of anhydrous milk fat (AMF) by whey protein concentrates (WPC) of different compositions and by a whey protein isolate

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The WPC of 50 and 75% protein (WPSO and WP7S, respectively) were obtained from Calpro Ingredients (Corona, CA); WPI was purchased from Le Sueur Isolates (Le Sueur. MN). The composition of these materials is shown in Table 1. D-Lactose monohydrate was purchased from Sigma Chemical Co. (St Louis. MO). Anhydrous milk fat of 99.8% fat (California Cooperative Creamery. Hughson, CA) was used as core material.

using a Mini-Lab high pressure homogenizer (type 8.30H; APV Rannie, St. Paul, MN) operated at SO MPa. The emulsion constituents were heated to SO·C prior to the emulsification. and this temperature was maintained throughout the emulsification process. Spray Drying. The emulsions were spray dried using an APV Anhydro Laboratory Spray Dryer (APV Anhydro AlS, S~borg, Denmark). The dryer had an evaporation rate of 7.5 kgIh and a chamber diameter of 1 m. The height, width. and length of the dryer were 2.6, 1.2, and 1.3 m. respectively. Drying was carried out in the concurrent mode. The emulsions (at SO·C) were atomized by the centrifugal atomizer of the dryer operated at 50,000 rpm. Inlet and outlet air temperatures were 160 and 80·C, respectively. The microcapsule powder was collected at the bottom of the dryer's cyclone and was kept in glass jars in an evacuated desiccator at 2S·C. All experiments were carried out in two replicates.

Microencapsulation by Spray Drying

Analyan

Emulsion Preparation. Wall solutions containing 10 to 30% (wtIwt) solids were prepared in deionized water. When the wall system consisted of combinations of WPI with lactose. the lactose partially replaced the WPI. The AMF was emulsified into the wall solutions at proportions of 25, SO. and 7S% (wtIwt) of wall solids. The emulsification was carried out in two stages. A coarse emulsion was prepared using an Ultra-Turraxl!ll T25-S 1 homogenizer (IKA Works, Cincinnati, OH) operated at 13,500 rpm for 30 s. The second stage consisted of four successive homogenization steps

The mean particle size of the emulsions was determined using a Malvern Mastersizer MS20 (Malvern Instruments, Malvern. England). A 2-mW He-Ne laser beam (633 nm) and a 45-mm focus lens were used. The moisture content of the microcapsules was determined gravimetrically after 12 h of vacuum drying (6S·C, 6.7 kPa). The AMP content of the dry capsules was determined according to the Rose-Gottlieb method ('Z7) and was expressed as grams of AMP per gram of dry nonfat solids, which was defined as the microencapsulated fat load. The microencapsulation yield (MEV) was defined as the ratio (expressed as percentage) of microencapsulated fat load to the fat load in the emulsion. A microencapsulation efficiency (MEE) parameter was defined as the percentage of AMP that could not be extracted from the microcapsules by petroleum ether using a method similar to that described by Sankarikutty et a1. (37). One gram of dry capsules was weighed into a glass extraction flask, and 25 ml of petroleum ether (Sigma Chemical Co.) were added. The extraction flask was placed on a Garver shaker (model 360; Garver Mfg., Union City, IN), and the extraction was carried out for IS min at

(WPI). MATERIALS AND METHODS Wall and Core Materials

TABLE 1. Composition 1 of whey protein concentrates and isolate used as wall materials.

Protein, % Ash, % Fat, % Lactose, % Moisture, %

wpp

WPCS()3

WPC754

95.4 1.84

51.8 5.2 4.0 37.1 1.9

76.5 3.5 8.0 10.0 2.0

2.68

IBased on product data provided by supplier. 2Whey protein isolate. 3Whey protein concentrate of 50% protein. 4Whey protein concentrate of 75% protein.

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YOUNG ET AL.

2S·C. The mixture was filtered (GN-6 filter; Gelman Science, Ann Arbor, MI), the solvent was evaporated over a water bath at 70'C, and the solvent-free extract was dried under vacuum (4S0C, SO mm Hg). The amount of extracted fat was then determined gravimetrically. The MEE (percentage) was defined as (MEFL - EF) x lOOIMEFL, where EF = extracted fat, and MEFL =microencapsulated fat load. The fat content of the WPI and WPC and the AMF that could be extracted by solvent from these materials were determined by the same methods; appropriate corrections were made when MEY and MEE of the microcapsulated systems were determined. In all cases, the analyses were carried out in duplicate, and the presented results are the mean values. Moisture Uptake

In order to study the effects of lactose on MEE as a function of moisture uptake, samples of microcapsules in which WPI or a combination of 1:1 (wtlwt) WPI and lactose served as wall systems were incubated at different relative humidities. The MEE was determined prior to and after humidification using the method described. About 3 g of powder were weighed (in triplicate) into glass weighing dishes. The powder-containing dishes were dried for 12 h at 6SoC and 6.7 kPa, cooled to 2SoC, and then placed into desiccators containing saturated salt solutions at relative humidities ranging between 11.3 and 64.3 at 2SoC. No fat losses could be detected when the fat contents of the microcapsules before and after the drying stage were compared. The relative humidity of the solutions were determined at 2SoC using a Fisher electronic hygrometer (Fisher Scientific, Pittsburgh, PA). The samples were incubated until no change in weight was detected. Electron Microscopy

The inner and outer structures of the microcapsules were studied by scanning electron microscopy. Specimen preparation procedures for the examination of the outer topography of the microcapsules were those described by Rosenberg et al. (31,33) and Rosenberg and Young (34). Examination of the inner structure of microcapsules required a fracturing step. In Journal of Dairy Science Vol. 76, No. 10, 1993

our study, fracturing was done by moving a razor blade perpendicularly through a layer of microcapsules attached by doubled-sided adhesive tape (fed Pella, Redding, CA) to a specimen holder. In all cases, the specimens were coated with gold using a Polaron sputter coater (model E-SOOSO; Bio-Rad, San Jose, CA) and studied microscopically using a scanning electron microscope (lSI DS-130; International Scientific Instruments, Pleasanton, CA). Statistical Analysis

An ANOVA was performed on mean MEY to test the effect of wall solution composition. RESULTS AND DISCUSSION Emulsification Conditions

The fIrst stage in the microencapsulation process is the formation of a fIne (and stable) emulsion of the core material in wall solution. Figure 1 shows representative particle size distributions of AMF emulsifIed in WPI and WPC. For all three wall materials, and regardless of wall solution solids concentration or AMF load, unimodal particle size distributions, similar to those presented in Figure 1, were obtained. The mean particle size (of AMP) in these emulsions ranged between .3 to .6 ILm. No associated adverse effects could be detected as a function of wall material composition. As reported elsewhere (16), electron microscopic analysis of such emulsions revealed the presence of adsorbed protein films surrounding each of the AMF droplets. Emulsion viscosity and particle size distribution have signifIcant effects on microencapsulation by spray drying (28, 32). High viscosities interfere with the atomization process and lead to the formation of large, elongated droplets that adversely affect the drying rate (32). Homogenization of AMF emulsions stabilized by whey proteins and with a high fat load at pressure higher than SO MPa or passing such emulsions more than four successive passes through the homogenizer resulted in the formation of clusters consisting of protein-coated AMF droplets and in a significant increase in viscosity (16, 34, 39). In order to attain small particle size (of the AMP) while preventing clustering, four succes-

MICROENCAPSULATION BY WHEY PROTEINS

sive homogenization steps at 50 MPa were selected for preparing the emulsions prior to spray drying. Retention of AMF

The MEY of more than 90% were found for all solids concentrations and fat loads evaluated in this study (Table 2). At an AMP load of 25%, the MEY ranged between 92.5 and 99.6%. The highest MEY was obtained with 30% WPC75 as wall material, and the lowest MEY was obtained with a wall solution consisting of 10% WPI. At an AMF load of 50%, the MEY ranged between 91.3 and 98.5%. The highest and lowest MEY were obtained with

2871

wall solutions consisting of 20% WPC50 or 20% WPC75, respectively. The emulsions, consisting of 30% WPI or WPC50, each with an AMP load of 75%, were too viscous and could not be dried. At the highest AMF load. the MEY ranged between 95.6 and 98.4%, and no significant differences between the different systems were detected (P > .05). The retention of the core material during microencapsulation by spray drying is affected (among other things) by the properties and composition of the emulsion and by the drying conditions (25, 32). An ideal microencapsulation process should result in no losses of the encapsulated core during the process. The effeets of process conditions and of composi-

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Figure 1. Representative spectra of particle size distribution of emulsions consisting of 25 or 50% (wtIwt) anhydrous milk fat (for A and C and for B and D. respectively) emulsified in different wall solutions. A and B) 10 and 20% (wtIwt) whey protein isolate. respectively; C and D) 10 and 20% (wtIwt) whey protein concentrate (50% protein). respectively. Journal of Dairy Science Vol. 76. No. 10. 1993

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YOUNG ET AL.

tional aspects on core retention during microencapsulation by spray drying have been discussed in the literature, primarily as they apply to volatiles or essential oils (2, 4, 5, 25, 26, 28, 29, 30, 32). High drying rates that lead to rapid crust formation around the drying particles also lead to high core retention. Losses seem to occur mainly at stages prior to the formation of the dry crust. Anhydrous milk fat is not a volatile material; however, some losses did occur during spray drying. Possibly, AMF droplets that were present at the surface of emulsion droplets leaving the atomizer, or those that migrated to the surface prior to the formation of dry crust around the drying particles, were swept off the particle surface. Similar explanations regarding the retention of other core materials were suggested by others (11, 12,32) and were attributed in part to the effects of internal mixing that exists in the drying droplets at stages prior to crust formation. No indication for the effect of wall solution solids concentration was detected in this study. This

TABLE 2. Effects of wall material type, wall solution concentration, and anhydrous milk fat load on microencapsulation yield. Microencapsulation yield l Wall system

2S% Fat2

SO% Fat

10%3 WPCSO" 20% WPCSO 30% WPCSO 10% WPC7Ss 20% WPC7S 30% WPC7S 10% WPI6 20% WPI 30% WPI SEM

96.lb 96.81b 96.6"b 97.31b 96.S"b 99.6"

98.7" 91.3c 97.4"b 98.6"b 98.9S" 98.31b 97.3 ab 9S.I b 96.8Ib 1.12

7S% Fat

(%)

92.Sc 99.Qlb 99.2"b .97

98.4" 97.5" 97.4" 97.3" 96.8"

9S.6" 9S.6" 1.12

l,b,CMeans in the same column with like letters are not significantly different (J' > .OS).

lThe ratio (percentage) of microencapsulated fat load to the fat load in the emulsion, 2Percentage (wtIwt) of wall solids. Each data point represents the mean of observations on four preparations. 3Percentage (wtIwt) of wall solids in wall solution. 4Whey protein concentrate with SO% protein. SWhey protein concentrate with 7S% protein. 6Whey protein isolate. Iournal of Dairy Science Vol. 76, No. 10, 1993

result can probably be explained by the low viscosity of the emulsions even at high wall solids concentration (38) and by the nonvolatile nature of AMF. Microstructural analyses of microcapsules containing AMF, and based on whey protein (our unpublished data, 1992), revealed structural details indicating stripping of core droplets from the outer surface of microcapsules and thus supporting the explanation just presented. Various attempts to prepare dairy powders with high fat content resulted in difficulties in recovering the powder from the spray dryer and sticky powder; these problems were attributed to the formation of a fat layer on the outer surfaces of the powder particles (8, 38). The powders prepared in this research were easily recovered by the dryer cyclone, and no stickiness could be detected even at a fat load of 75%. Mlcro.tructur.

Electron microscopy revealed that the spherical microcapsules were free of visible cracks or pores, and almost no surface dents could be detected when WPI served as the wall material (Figure 2A). The limited surface indentation is in contrast to significant dent formation found to be characteristic of various spray-dried, milk-derived powders (6, 20) or other spray-dried microcapsules (31, 33). The inner structure of the microcapsules (Figure 2B) is similar to that reported for other spray-dried microcapsules (31, 33). The microscopic analysis revealed that the AMF is organized in small droplets (100 to 600 nm) embedded in the protein matrix of the wall. No visible pores or cracks exposing the core droplets to the environment could be detected. MEE

The MEE defined here represents the proportion of AMF that was not available to the extracting solvent (petroleum ether) under the test conditions. In a preliminary test, when the proportion of the AMF available to the solvent was studied as a function of time, longer extraction times resulted in significantly higher amounts of extracted AMF (Figure 3). In order to compare the MEE of the different

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MICROENCAPSULATION BY WHEY PROTEINS

systems, extraction time of IS min was used in all cases. Because of this and the high solubility of AMP in the solvent, the amount of AMP extracted by the solvent is suggested to represent not only the proportion of AMF present on the microcapsule surfaces but also a fraction of the microencapsulated AMP extracted from the interior parts of the capsules by a leaching process. The effects of wall solution solids concentration and fat load on MEE are presented in Figure 4 for WPI, WPCSO, and WPC7S. Increasing the WPI concentration resulted in higher MEE in all cases. However, at an AMP load of 7S%, the effect of wall solids was very slight between the systems containing 10 and 20% wall solids. For the WPCSO systems, MEE was increased as a function of wall solids concentration at AMF load of 25% and was only slightly affected at higher AMP loads. However, the MEE of the WPCSO systems were higher than those of the WPI systems, regardless of AMP load. The MEE of the WPC7S systems was affected by the wall

solids concentration in a way that was similar to that discussed for WPCSO; however, the effect of wall solids concentration at SO and 75% AMF load was more profound than that for the WPCSO systems. For any microcapsule composition, the MEE of the WPCSQ-based systems was higher than that of the WPC7Sbased systems. Increasing the AMF load resulted in a decrease in MEE, regardless of the wall material used; however, the WPIbased systems were more sensitive to the AMF load than were the WPC-based systems and showed a more profound decrease in MEE as a function of AMF load. In order to extract AMF from the interior parts of the microcapsules, the solvent should diffuse through the wall matrices, reach the AMF droplets embedded in the wall, dissolve part of the AMF, and the AMF-solvent solution should then diffuse back through the wall. In this research, WPCSO contained the highest (36%) lactose content among the evaluated wall materials. In light of the high MEE values obtained with this wall material, different com-

Figwe 2. Representative micrographs revealing the A) outer structure and B) the inner structure of whey proteinbased microcapsules containing anhydrous milk fat. Fat content: 73% (wtIwt). OS Outer surface. IS inner surface, PO fat droplets, W capsule wall. Scale bar 17 I'm (A) and 550 nm (B).

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Journal of Dairy Science Vol. 76, No. 10, 1993

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YOUNG ET AL.

binations of WPI and lactose as the wall system were tested (Figure 5), keeping the total solids of the wall solution at 20% and the AMF load at 75%. Partially replacing the WPI by lactose gradually increased the MEE. At a ratio of 1:1 WPI to lactose (wtIwt), the MEE was 95%. A higher replacement ratio did not result in any further increase in MEE. The MEY of these systems ranged between 98 and 99.5%. After spray drying, the lactose is in an amorphous state (35, 36). In order to understand the effects of the physical state of the lactose on the MEE, the effect of moisture uptake on MEE was studied and is presented in Figure 6. No changes in MEE occurred for the 1: 1 WPI to lactose system after incubation at relative humidities of up to 36.2%. However, the MEE dropped from 95% (for the dry sample after spray drying) to 63% after incubation at relative humidity of 64.3%. The MEE of the WPI-based system was not affected by the moisture uptake at any of the relative humidities studied. Examination of the sample incubated at 64.3% relative humidity by electron microscopy (micrographs not provided) revealed the presence of large lactose crystals on the microcapsule surfaces. The structural results are in agreement with those reported by Saltmarch and Labuza (35, 36).

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Figure 3. Anhydrous milk fat (AMF) extracted from whey protein-based microcapsules as a function of extraction time. Each data point represents the mean of observations on four preparations. Bars represent standard error of the means. Journal of Dairy Science Vol. 76, No. 10, 1993

Figure 4. Effects of wall solution solids concentration and anhydrous milk fat load on the microencapsulation efficiency. Wall material type: A) whey protein isolate, B) whey protein concentrate (50% protein), and C) whey protein concentrate (75% protein). Wall solution solids concentration: 10% (wtIwt; .)' 20% (wtIwt; A), and 30% (wtIwt; .). Each data point represents the mean of observations on four preparations. Bars represent standard errors of the means.

2875

MICROENCAPSULATION BY WHEY PROTEINS 100

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Figure S. Encapsulation efficiency as affected by partial replacement of whey protein isolate (WPI) by lactose. In all cases, wall solution solids concentration was 20% (wtIwt), and anhydrous milk fat load was 7S% (Wl/wt). Each data point represents the mean of observations on four preparations. Bars represent standard errors of the means.

Lactose has no surface active properties and therefore does not participate in the formation of the stabilizing films at the oil-water interfaces during the emulsification stage. Also, no significant changes in the amount of fat present on the capsule surfaces could be expected as a result of water uptake at 25·C. The results suggest that lactose in its amorphous state acts as a hydrophilic filler or sealant that significantly limits diffusion of the solvent through the wall toward the microencapsulated AMF droplets and thus leads to high MEE values. Once crystallization of lactose occurs, the solvent diffusion is less limited, and, hence, MEE decreases. The results also support the aforementioned assumption that the amount of AMF extracted by the solvent represents in this study not only the proportion of AMF that was present on the capsule surfaces but also AMF that was extracted from interior parts of the microcapsules. The results of the 1: 1 WPI to lactose system may suggest that about 5% of the AMF microencapsulated in this wall system can be considered as surface oil. Based on these considerations, the superiority of WPC50 over the other two wall materials can be attributed to its relatively high lactose content. The WPC75 contained only

10% lactose, which can explain its inferiority to WPC50. However, the differences in MEE obtained with the three wall materials reflect the influence of more than just their lactose content. The WPI did not contain any milk fat, but the two WPC contained significant amounts of lipids (4 and 8% for WPC50 and WPC75 , respectively). The hydrophobicity of the WPC75-based wall is probably higher than that of the wall consisting of WPI. This result, along with the relatively low lactose content, which could not effectively limit the diffusion of the solvent, can explain the lower MEE that was obtained with WPC75 than with WPI. Increased wall solution solids concentration resulted in an increase in MEE, which can be linked with the effect of wall solids concentration on the formation of surface core prior to the formation of crust around the drying droplets. Higher wall solids were probably associated with less AMF migration to the surface at early stages of the drying, thus affecting the amount of AMF that was available to the solvent as surface fat. Additionally, the increase in MEE as a function of wall solution solids concentration can be attributed to the formation of thicker wall matrices around the AMF droplets that resulted in increasing the

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Figure 6. Effect of moisture uptake (at 2S'C) on the microencapsulation efficiency. Wall solutions consisted of whey protein isolate (solid bars) or 1: 1 (wtIwt) whey protein isolate and lactose (striped bars). In all cases, wall solution solids concentration was 20% (wtIwt) and anhydrous milk fat load was 75% (wtIwt). Each data point represents the mean of observations on four preparations. Bars represent standard errors of the means. Journal of Dairy Science Vol. 76, No. 10, 1993

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YOUNG ET AL.

diffusional path, which in tum resulted in less AMP extracted by the solvent (at a constant contact time). However, for a given wall material, when the fat load was increased, the total surface of oil-water interface per unit mass of wall material increased, and the layers of wall material between the encapsulated fat droplets became thinner. This change enhanced the accessibility of the AMP to the solvent, and, hence, lower MEE values were obtained. The results suggest that a combination of WPI with lactose represents a wall system superior to WPC. CONCLUSIONS

Whey proteins can be considered as potential microencapsulating agents for AMP. The microcapsules produced by spray drying are spherical and are characterized by smooth surfaces free of visible cracks or pores. The AMP is embedded in the wall system in the form of small droplets. High MEY is feasible even at high AMP loads. The results of this study suggest that diffusion of an apolar substance through a whey protein-based wall is possible and can be limited by incorporating lactose as a wall constituent. Although AMP is not expected to migrate through the capsule's wall during storage, migration may occur when apolar materials of relatively low molecular mass (Le., aroma compounds) are microencapsulated in whey proteins. In such cases, the effect of lactose in restricting the diffusion of apolar substances through the wall may offer a solution. The structural details of the microcapsules suggest that the AMP is well isolated from the environment; however, the chemical stability of the encapsulated system should be evaluated by stability tests. Chemical stability and the microencapsulation of materials other than AMP are subjects of ongoing research in this lab. ACKNOWLEDGMENTS

The California Dairy Poods Research Center funded this research. REFERENCES I Andres, C. 1981. Microencapsulation extends shelf life of marginally stable ingredients. Food Process. (OIic.) 42:40. Journal of Dairy Science Vol. 76, No. 10, 1993

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Journal of Dairy Science Vol. 76, No. 10, 1993