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Delivery of lactase using chocolate-coated agarose carriers
Food Research International 46 (2012) 41–45
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Delivery of lactase using chocolate-coated agarose carriers Amos Nussinovitch ⁎, Nava Chapnik, Jenny Gal, Oren Froy ⁎⁎ Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel
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Article history: Received 10 June 2011 Accepted 29 November 2011 Keywords: Lactase Agarose Starch Carriers Chocolate
a b s t r a c t Dried carriers based on agarose and starch as filler entrapping the enzyme lactase were studied. After freezedrying, all carriers had a spherical shape, resembling the properties of porous cellular solids. The inclusion of starch resulted in a rough surface of the dried carriers. The dried carriers were coated with chocolate to facilitate enzyme protection and their possible inclusion within functional food products. Lactase release from non-coated and chocolate-coated carriers revealed that the conditions in buffer and stomach-simulating solution led to a first mode of release due to lactase molecules located on the external or inner surfaces of the carrier but not within the matrix itself. In addition, chocolate coating led to a second mode of lactase release greater than the initial release. Thus, chocolate coated agarose-lactase-containing carriers may be considered for utilization in functional foods for lactose intolerant people. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction In the last decades, increasing attention has been paid to beads from a range of materials and dimensions suitable for the immobilization of microorganisms, enzymes, genes, and antibodies. The appeal of this concept lies in the fact that immobilization products are easy to produce, store, and handle during industrial operation (Nussinovitch, 2010). A variety of hydrocolloids have been studied for their potential use as carriers for the controlled release of drugs. When discussing drug delivery by beads, topics related to the method of drug incorporation, size and density of the bead, extent and nature of the cross-linking, physicochemical properties of the drug, interactions between the drug and the matrix material, and concentration of the matrix material and release environment, such as the presence of enzymes, are of utmost importance (Nussinovitch, 2010). Several studies have focused on alginate-based carriers, however, revealing some difficulties. For example, the loading efficacy of the drug is too low due to its leakage into the cross-linking solution (ElKamel, Al-Gohary, & Hosny, 2003; Liu & Krishnan, 1999). Combinations of alginate with other hydrocolloids have also been reported (Ferreira Almeida & Almeida, 2004; Tapia et al., 2004). For example, alginate gel beads reinforced by chitosan, which forms a complex with alginate, erode slowly in phosphate buffer (pH 6.8) and this behavior leads to the inhibition of the initial release rate of the encapsulated drug (Murata, Maeda, Miyamoto, & Kawashima, 1993). Another report dealt with chitosan-coated alginate beads containing poly(N-isopropylacrylamide) to be used as a controlled pH/temperature-sensitive ⁎ Corresponding author. Tel.: + 972 8 948 9016; fax: + 972 8 936 3208. ⁎⁎ Corresponding author. Tel.: + 972 8 948 9746; fax: + 972 8 936 3208. E-mail addresses: [email protected] (A. Nussinovitch), [email protected] (O. Froy). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.11.016
drug delivery system with advanced encapsulation efficiency and delayed release rate (Shi, Alves, & Mano, 2006, 2007). However, less information is found on carriers based on guar gum (Toti & Aminabhavi, 2004) and even fewer studies have focused on gellan, agar, or agarose carriers (Haglund, Upadrashta, Neau, & Cutrera, 1994; Kedzierewicz, Lombry, Rios, Hoffman, & Maincent, 1999). Recently, the direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium-alginate beads and calcium-agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in the presence of hydrogen peroxide (Nahakpam, Singh, & Shah, 2008). The presence of calcium ions helped immobilize the peroxidase and mediated direct binding of the enzyme to the agarose gel. Agarose appeared to be a better immobilization matrix for the peroxidase than sodium alginate (Nahakpam et al., 2008). In addition, although the use of drying can make immobilization more complicated since it might influence the entrapped encapsulate, especially if it is sensitive to heat treatment, dried agar gel beads have served as a research model for drying (Kubota, Kobatake, Suzuki, & Hosaka, 1977; Suzuki, Onishi, Esaka, Kubota, & Hosaka, 1989) and dried gel beads in general can also be used for the separation of enzymes (Han, Park, & Ruan, 1995). Drying has also been proven beneficial for the shelf life of beads that include drugs. For example, ceftriaxone-polymethylmethhacrylate beads could be made available “off the shelf” for at least 12 months after their preparation (Alonge, Ifesanya, Okoje, Nottidge, & Fashima, 2009). Lactase, a part of the β-galactosidase family of enzymes, is a glycoside hydrolase involved in the hydrolysis of the disaccharide lactose into its constituent galactose and glucose monomers. It is present predominantly along the brush border membrane of the differentiated enterocytes lining the villi of the small intestine (Skovbjerg, Sjöström, & Norén, 1981). Lactase is essential for digestive hydrolysis of lactose in milk and its deficiency causes lactose intolerance (Järvelä, Torniainen, & Kolho, 2009). It is estimated that 75% of adults
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worldwide show some decrease in lactase activity during adulthood (Hertzler, Huynh, & Savaiano, 1996). The prevalence of lactose intolerance warrants proper treatment. Lactose intolerance is treated by a few approaches: dietary changes, adaptation treatment sometimes combined with calcium and vitamin D supplements, and treatment with caplets, tablets, drops or soft gels of lactase that are available to take with milk-containing foods. As dried agar gels are more advantageous, the objectives of this study were to formulate and characterize dried, rather than soft carriers based on agarose that contain starch as filler and the enzyme lactase. In particular, we studied the physical properties of chocolate coated carriers, examined their stability in a continuous simulated gastrointestinal fluid and analyzed the profiles of lactase release from the enzyme-fillerhydrocolloid carrier. 2. Materials and methods 2.1. Carrier preparation Agarose powder (3%, w/w) was added to doubly distilled water and stirred by hand using a glass stirring rod to achieve agarose dispersion. The solution was heated rapidly with constant hand stirring until it reached ~90 °C and remained at this temperature for 10 min. When boiling started, it was continued for exactly 1.3 min. The solution was then removed from the heat source and the foam was broken by swirling and stirring for a few minutes. After cooling the solution to 50 °C, 10% (w/w) starch was added as filler. To produce spherical beads, the solution containing the agarose and filler was dropped into doubly-distilled water through a paraffin oil layer (~5 mm) (Frutarom Ltd., Haifa, Israel). 2.2. Bead drying, entrapment, and coating The agarose beads with or without the enzyme were freeze-dried. Freeze-dried beads were obtained by storing gel beads (24 h) at −80 °C before freeze-drying (48 h) in a pilot-plant unit (Model 15 RSRC-X, Repp Industries, Inc., Gardliner, NY, USA), operating at 33.3 Pa (150 mbar) and −45 °C. To determine the moisture content in the beads, beads were further dried in a vacuum oven for 24 h at 105 °C and 104 Pa. After drying, 30 U lactase solution in 100 mM phosphate buffer pH 7.3 was added to each bead for 2 h at 4 °C. After drying at ambient temperature, the dried agarose beads were immersed ~ 50 s in melted chocolate (Elite, Upper Nazareth, Israel), which was first heated to 45 °C and then cooled to ~30 °C. Excess of chocolate was removed and the coating was allowed to solidify at room temperature. 2.3. Measurements of carrier diameter and weight Carrier diameter (±0.03 mm) was measured with a digital caliper (Mitutoyo, Tokyo, Japan). Carrier weight (±0.001 g) before and after enzyme inclusion and chocolate coating, was measured with semimicrobalance 262SMA-FR SCS (Precisa Instruments, Bern, Switzerland).
versus engineering strain relationships using the following equations: σ = F/A0 and εE = ΔD/D0, where σ is the stress (Pa), F is the force at a given time, A0 is the initial cross-sectional area of the carrier, εE is the engineering strain (dimensionless), ΔD is the absolute deformation caused by the compression test (length units), and D0 is the diameter of the carrier at time zero (length units). Young's modulus was calculated from the initial linear portion of the stress versus engineering strain curve. The Young's modulus value, i.e., the slope of the curve, is a parameter reflecting the toughness of the compressed carrier. Compression tests for the different carriers were carried out in five replicates. 2.5. Stability in simulated gastrointestinal fluid The simulated gastrointestinal fluid was based on a previous report of Hack and Selenka (1996) with some modifications. Simulated gastric fluid was prepared by dissolving 10 mg of pepsin from porcine stomach mucosa (Sigma, Rehovot, Israel) in 112 mL of doublydistilled water and 350 mg of mucine from porcine stomach (Sigma, Rehovot, Israel). Then, 3.5 mL of 3 N NaCl solution and 2 mL of 1.2 N KCl solution were added to the simulated gastric fluid. The pH value was adjusted and kept at pH 1.2 by adding an appropriate volume of 1 N HCl (Sigma, Rehovot, Israel). pH values (±0.03) were checked with an Extech Heavy Duty pH/mV Temperature Meter (Extech Instruments Co., Waltham, MA). A laboratory bottle (250 mL) filled with 120 mL of simulated gastric fluid was placed in a TEP-3 water bath using a magnetic stirrer (50 rpm). The bath, filled with preheated water (37 ± 0.5 °C), was controlled by an electric heating element. The enzyme-agarose carriers were immersed in this simulated gastric fluid for 2 h at 37 °C with shaking (75 rpm). Then, the simulated gastric fluid was titrated by adding an appropriate amount of sodium bicarbonate (Frutarom Ltd., Haifa, Israel). The pH value was adjusted and kept at pH 6.8. Trypsin (10 mg) from porcine pancreas (Sigma, Rehovot, Israel), 350 mg of pancreatin from porcine pancreas (Sigma, Rehovot, Israel), and 350 mg of dried bovine bile (Sigma, Rehovot, Israel) were added to obtain simulated intestinal fluid. Carriers were immersed in the simulated intestinal fluid for 6 h at 37 °C with shaking (75 rpm). Time of immersion of the agarose–lactase carriers in the simulated fluids was in accordance to the model developed by Hack and Selenka (1996) and to the literature referring to the passage time of solids through the stomach and small intestine (Washington, Washington, & Wilson, 2003).
2.6. Lactase activity measurements Lactase activity was performed according to the manufacturer's instructions (Sigma, Rehovot, Israel). Lactase activity of retained enzyme was performed by addition of agarose at 1:12 ratio and grinding by mortar and pestle. Bead powder was dissolved in 100 mM phosphate buffer pH 7.3, centrifuged at 8000 rpm for 15 min at 4 °C and the supernatant was tested for lactase activity. Lactase activity versus time relationship was linearized via the equation:
2.4. Compression tests Compression tests were carried out to evaluate properties, such as strength, fragility, and toughness. Carriers of each composition were compressed between lubricated parallel plates to a deformation of 90% at a constant deformation rate of 10 mm/min with an Instron Universal Testing Machine (UTM), Model 5544 (Canton, MA, USA), connected to an IBM-compatible personal computer using a card. Data acquisition and conversion of the Instron's continuous voltage versus time output into digitized force versus time relationships was performed by Merlin software from Instron Corporation (Canton, MA, USA). Finally, the force versus time data was converted to stress
y¼
abt 1 þ bt
where y = lactase activity, a and b are constants and t is the elapsed time. The applicability of this equation was tested through the fit of its linear form.
t 1 t ¼ þ y ab a
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or in its abbreviated form t ¼ k1 þ k2 t: y Such an empirical consideration was previously performed for vibratory compaction of non-food powders (Sone, 1972) and was also mentioned in Peleg and Bagely (1983). 2.7. Porosity determination Carrier porosity was calculated using the following equation: P = 1 − (ρB / ρS), where P is the porosity value (dimensionless), ρB is the bulk density (mass/volume) of the carrier, and ρS is the solid density (mass/volume) of the carrier. Bulk density was obtained from the ratio of mass to volume of the dried carrier. Solid density was evaluated by micro-pycnometer (Quantachrome, Syosset, NY, USA). 2.8. Scanning electron microscopy (SEM) To study the structural changes within and on the surface of the carriers as a result of filler inclusion and drying, SEM micrographs were taken using a JEOL JSM 35C SEM (Tokyo, Japan). The carriers were attached to metal stubs and gold-coated (150–200 Å) in a Polaron 5150 sputter coater (Polaron Equipment Ltd., Holywall Industrial Estate Watford, Hertfordshire, England). 2.9. Statistical analysis Statistical analyses were conducted using JMP software (SAS Institute, 1995), including ANOVA and the Tukey–Kramer Honestly Significant Difference test for comparisons of means. P b 0.05 was considered significant. 3. Results and discussion 3.1. Weight and structure of carriers The agarose carriers designed for lactase immobilization and release weighed from 4.5 to 6.3 mg. The diameters of the freezedried agarose carriers ranged from 3.6 to 3.9 mm. As expected from freeze-drying, the changes in the carrier shape were small (Fellows, 2000; Fennema, 1975), i.e., all carriers remained spherical. Similar shape was described when freeze-dehydration was used to dry alginate beads that were employed to entrap probiotic bacteria for survival purposes in frozen, fermented dairy desserts. Based on SEM micrographs, those beads were also illustrated merely as “spherical” (Shah & Ravula, 2000). The inclusion of starch as filler resulted in a rough surface, i.e., the carriers contained dents and holes on their external surface area (Fig. 1A). It is interesting to note that also freeze-dried alginate beads after immobilization of Acinetobacter johnsonii (i.e. inclusion of entrapped bacteria) were described to also have some grooves on their external surface (Muyima & Cloete, 1995). The diameter of the dents varied from a few microns to ~ 0.1 mm. The size and distribution of the pores formed within the carrier were not homogeneous, as can be seen under electron microscope of the halved freeze-dried agrose–lactase carriers (Fig. 1B). The filler particles (starch granules) appear to occupy some of the pores, as well as are embedded within the carrier wall and surface (Fig. 1B, C). It is well known that freeze-dried beads can be reinforced by inclusion of starch in their formulation before the beads are manufactured and dried. Such starch granules serve not only as filler but also as a carbon source (Tal, van Rijn, & Nussinovitch, 1999). Furthermore, filler inclusion in the dried product reduced its collapse and roundness distortion during the drying process. Thus, the dried bead's components and the method by which it is formed determine its weight,
Fig. 1. Lactase–agarose carriers. A. An intact agarose–starch–lactase carrier displaying its spherical shape and large surface pores. Bar is 1 mm. B. Halved freeze-dried agarose–starch–lactase carrier. Bar is 1 mm. C. Starch granules within the inner structure of the carrier. Bar is 500 μm.
volume, shape and surface topography. Taking these parameters into consideration provides the researcher a useful tool for creating tailormade dried beads for a predetermined operation (Zohar-Perez, Chet, & Nussinovitch, 2004). Although the interior of the spherical carriers (dried beads) was porous, their outer surface appeared to be less porous. The calculated porosity values of the freeze-dried carriers ranged from 85.6 to 90.6%. Freeze-dried products are generally characterized by the least shrinkage and structural changes, and by the creation of pores in areas of former ice crystals. The porous characteristic of the formed bead is
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of utmost importance to enable its coating for example by molten chocolate, in order to further permit bead inclusion in chocolate bars or other chocolate-based products for production as functional foods. Chocolate-coated beads weighed 32.1 ± 1.2 mg. 3.2. Mechanical properties of the carriers As the chocolate-coated lactase–agar carriers can be consumed as is or as part of a confection, it is important to assess its mechanical properties which are related to its sensory qualities. In addition, the mechanical properties of the carriers are also affected by the motility of the gastrointestinal tract. Therefore, the mechanical properties of the carriers were evaluated by compression tests. Fig. 2 represents typical stress–strain relationships for freeze-dried agarose carriers with the inclusion of the filler before coating. It is clear that up to an engineering strain of ~0.4–0.5, an approximately linear stress–strain relationship exists, and the Young's modulus can, therefore, be easily calculated (Fig. 2). The values of Young's modulus for the freeze-dried agarose–lactase carriers were 0.69 ± 0.26 kPa and the R2 values ranged from 0.985 to 0.994. Similar results (0.70 ± 0.09) were detected for the chocolatecoated carriers. The shape of the compressed carrier resembles a typical sigmoid shape of a cellular solid (without a shoulder). i.e., upon mastication it assumes a character or resembles a structure of a crunchy product. While a freeze-dried agarose carrier (i.e., a cellular solid) is being compressed, the cell walls are bent, giving linear elasticity if the cell wall material is elastic. Upon reaching a critical stress, cells begin to crumple and ultimately, at high strains, collapse. It is sufficient to allow opposing cell walls to touch or their broken fragments to pack together. Further deformation compresses the cell wall material itself. This gives the final, steeply rising portion of the stress–strain curve, termed densification. The noise generated by the touch of the opposing cell walls and the pack of the broken fragments is partially responsible for the crunchiness sensation (Nussinovitch, 1997, 2003). Furthermore, the coating with chocolate reduces the absorbance of moisture of the dry product and ensures the preservation of crunchiness of the product for longer periods of time, similarly to puffed rice embedded within chocolate bars or confections whose pores are filled with chocolate (Bower & Whitten, 2000). 3.3. Lactase release from carriers To evaluate the potential of the agarose beads to serve as enzyme carriers, it was necessary to study their stability and enzyme release in solutions which mimic gastrointestinal fluids. Therefore, the ability of the carriers containing lactase to withstand enzyme-containing stomach and small intestine environments was next tested. The weight of the non-coated carriers incubated in buffer or stomach- and intestine-simulating solutions did not decrease significantly (Table 1). In contrast, the weight of the chocolate-coated carriers decreased by ~65%, suggesting that the chocolate coat dissolved quite easily at 37 °C (Le Reverend, Smart, Fryer, & Bakalis, 2011). As most of the weight
Fig. 2. Stress–strain relationships of compressed agarose–starch–lactase dried carriers.
Table 1 % bead weight after incubation in buffer and stomach and intestine solutions.
Buffer solution Stomach solution Intestine solution
Non-coated bead
Chocolate-coated bead
100.72 ± 0.84 99.68 ± 0.10 90.30 ± 1.86
37.13 ± 1.03⁎ 33.77 ± 1.34⁎ 37.68 ± 1.46⁎
⁎ P b 0.05 (Student's t-test).
of the chocolate-coated carriers was due to the coating, this result is desirable. Thus, most of the chocolate-mediated protection against oxidation or moisture absorption terminated upon carrier consumption. The leftover chocolate within inner pores has beneficial influence on enzyme release (see below). After having established that lactase can be entrapped within agarose carriers, release of lactase from the carrier and its biological activity were measured. Chocolate-coated and non-coated lactase-containing carriers were incubated in stomach- and small intestine-simulating solutions (Gal & Nussinovitch, 2007) for 2 h and 6 h, respectively. Released lactase after incubation of chocolate-coated and non-coated lactase-containing carriers in buffer and in stomach- and small intestine-simulating solutions was determined by measuring lactase activity. Non-coated carriers released 32.4% of lactase activity after incubation with buffer and 22.5% and 0.22% after incubation with stomachor small intestine-simulating solutions, respectively. This indicated that the conditions in buffer and stomach-simulating solution led to a first mode of release due to lactase molecules located on the external or inner surfaces of the carrier but not within the matrix itself. However, chocolate-coated carriers exhibited similar lactase activity after incubation in all solutions (Fig. 3). We then measured lactase activity that remained in the carriers due to location inside carrier walls by grinding the beads in the presence of agarose. This was performed on carriers after incubation in buffer, stomach- and small intestine-simulating solutions. Similarly to the results achieved with the incubation in the different solutions, chocolate-coated carriers released higher levels of lactase activity after grinding. These results demonstrate the beneficial effect of the chocolate coating. This two-mode release is reminiscent of a recent publication showing release of human defensin from alginate carriers (Froy, Chapnik, & Nussinovitch, 2010). It is plausible that the leftover chocolate reached inner positions within the carrier and blocked the passage of some proteolytic enzymes, protecting lactase molecules inside the carriers. We then performed kinetic measurements of lactase release and found that chocolate-coated carriers released lactase more readily and reached maximal release after 30 min, whereas non-coated carriers reached release plateau after 1 h (Fig. 4). We tested the curves' linearized form (Fig. 4B) and found that in both cases the regression coefficients were very high, i.e., R 2 = 0.9868 for the chocolate-
Fig. 3. Percent lactase activity of non-coated and chocolate-coated carriers. Activity was compared to the activity initially incorporated into the carriers.
A. Nussinovitch et al. / Food Research International 46 (2012) 41–45
Fig. 4. Kinetics of lactase release. A. Kinetics of lactase release from non-coated and chocolate-coated carriers. Lactase activity was measured at different time-points after immersion of carriers in lactase buffer. B. Linearized form of A, a mathematical conversion and the regression lines.
coated and 0.995 for the non-coated carriers. Thus, the rate and quantity of the release is faster for the non-coated carrier. In general, this form of data presentation is very convenient for system comparison, since it only involves two experimental constants. The magnitude of these constants especially the k2 constant must depend on the conditions by which the experiments were conducted. These results support the surmise that the chocolate reached inner positions within the carrier and attenuated lactase release, leading to a two-mode release. 4. Conclusions Our results show that dried agarose beads can be utilized for the entrapment of enzymes, such as lactase. Lactase retains its activity when released from the carrier. In addition, chocolate coating aids in protecting the entrapped enzyme and supports the release of intact enzyme in a two-mode pattern. Acknowledgements We thank the Kennedy Leigh Fund for their support. References Alonge, T. O., Ifesanya, A. O., Okoje, V. N., Nottidge, T. E., & Fashima, N. A. (2009). An evaluation of the shelf life of ceftriaxone-polymethylmethhacrylate antibiotic beads. European Journal of Orthopaedic Surgery and Traumatology, 19, 571–575. Bower, J. A., & Whitten, R. (2000). Sensory characteristics and consumer liking for cereal bar snack foods. Journal of Sensory Studies, 15, 327–345.
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Food Research International journal homepage: www.elsevier.com/locate/foodres
Delivery of lactase using chocolate-coated agarose carriers Amos Nussinovitch ⁎, Nava Chapnik, Jenny Gal, Oren Froy ⁎⁎ Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel
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Article history: Received 10 June 2011 Accepted 29 November 2011 Keywords: Lactase Agarose Starch Carriers Chocolate
a b s t r a c t Dried carriers based on agarose and starch as filler entrapping the enzyme lactase were studied. After freezedrying, all carriers had a spherical shape, resembling the properties of porous cellular solids. The inclusion of starch resulted in a rough surface of the dried carriers. The dried carriers were coated with chocolate to facilitate enzyme protection and their possible inclusion within functional food products. Lactase release from non-coated and chocolate-coated carriers revealed that the conditions in buffer and stomach-simulating solution led to a first mode of release due to lactase molecules located on the external or inner surfaces of the carrier but not within the matrix itself. In addition, chocolate coating led to a second mode of lactase release greater than the initial release. Thus, chocolate coated agarose-lactase-containing carriers may be considered for utilization in functional foods for lactose intolerant people. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction In the last decades, increasing attention has been paid to beads from a range of materials and dimensions suitable for the immobilization of microorganisms, enzymes, genes, and antibodies. The appeal of this concept lies in the fact that immobilization products are easy to produce, store, and handle during industrial operation (Nussinovitch, 2010). A variety of hydrocolloids have been studied for their potential use as carriers for the controlled release of drugs. When discussing drug delivery by beads, topics related to the method of drug incorporation, size and density of the bead, extent and nature of the cross-linking, physicochemical properties of the drug, interactions between the drug and the matrix material, and concentration of the matrix material and release environment, such as the presence of enzymes, are of utmost importance (Nussinovitch, 2010). Several studies have focused on alginate-based carriers, however, revealing some difficulties. For example, the loading efficacy of the drug is too low due to its leakage into the cross-linking solution (ElKamel, Al-Gohary, & Hosny, 2003; Liu & Krishnan, 1999). Combinations of alginate with other hydrocolloids have also been reported (Ferreira Almeida & Almeida, 2004; Tapia et al., 2004). For example, alginate gel beads reinforced by chitosan, which forms a complex with alginate, erode slowly in phosphate buffer (pH 6.8) and this behavior leads to the inhibition of the initial release rate of the encapsulated drug (Murata, Maeda, Miyamoto, & Kawashima, 1993). Another report dealt with chitosan-coated alginate beads containing poly(N-isopropylacrylamide) to be used as a controlled pH/temperature-sensitive ⁎ Corresponding author. Tel.: + 972 8 948 9016; fax: + 972 8 936 3208. ⁎⁎ Corresponding author. Tel.: + 972 8 948 9746; fax: + 972 8 936 3208. E-mail addresses: [email protected] (A. Nussinovitch), [email protected] (O. Froy). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.11.016
drug delivery system with advanced encapsulation efficiency and delayed release rate (Shi, Alves, & Mano, 2006, 2007). However, less information is found on carriers based on guar gum (Toti & Aminabhavi, 2004) and even fewer studies have focused on gellan, agar, or agarose carriers (Haglund, Upadrashta, Neau, & Cutrera, 1994; Kedzierewicz, Lombry, Rios, Hoffman, & Maincent, 1999). Recently, the direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium-alginate beads and calcium-agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in the presence of hydrogen peroxide (Nahakpam, Singh, & Shah, 2008). The presence of calcium ions helped immobilize the peroxidase and mediated direct binding of the enzyme to the agarose gel. Agarose appeared to be a better immobilization matrix for the peroxidase than sodium alginate (Nahakpam et al., 2008). In addition, although the use of drying can make immobilization more complicated since it might influence the entrapped encapsulate, especially if it is sensitive to heat treatment, dried agar gel beads have served as a research model for drying (Kubota, Kobatake, Suzuki, & Hosaka, 1977; Suzuki, Onishi, Esaka, Kubota, & Hosaka, 1989) and dried gel beads in general can also be used for the separation of enzymes (Han, Park, & Ruan, 1995). Drying has also been proven beneficial for the shelf life of beads that include drugs. For example, ceftriaxone-polymethylmethhacrylate beads could be made available “off the shelf” for at least 12 months after their preparation (Alonge, Ifesanya, Okoje, Nottidge, & Fashima, 2009). Lactase, a part of the β-galactosidase family of enzymes, is a glycoside hydrolase involved in the hydrolysis of the disaccharide lactose into its constituent galactose and glucose monomers. It is present predominantly along the brush border membrane of the differentiated enterocytes lining the villi of the small intestine (Skovbjerg, Sjöström, & Norén, 1981). Lactase is essential for digestive hydrolysis of lactose in milk and its deficiency causes lactose intolerance (Järvelä, Torniainen, & Kolho, 2009). It is estimated that 75% of adults
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worldwide show some decrease in lactase activity during adulthood (Hertzler, Huynh, & Savaiano, 1996). The prevalence of lactose intolerance warrants proper treatment. Lactose intolerance is treated by a few approaches: dietary changes, adaptation treatment sometimes combined with calcium and vitamin D supplements, and treatment with caplets, tablets, drops or soft gels of lactase that are available to take with milk-containing foods. As dried agar gels are more advantageous, the objectives of this study were to formulate and characterize dried, rather than soft carriers based on agarose that contain starch as filler and the enzyme lactase. In particular, we studied the physical properties of chocolate coated carriers, examined their stability in a continuous simulated gastrointestinal fluid and analyzed the profiles of lactase release from the enzyme-fillerhydrocolloid carrier. 2. Materials and methods 2.1. Carrier preparation Agarose powder (3%, w/w) was added to doubly distilled water and stirred by hand using a glass stirring rod to achieve agarose dispersion. The solution was heated rapidly with constant hand stirring until it reached ~90 °C and remained at this temperature for 10 min. When boiling started, it was continued for exactly 1.3 min. The solution was then removed from the heat source and the foam was broken by swirling and stirring for a few minutes. After cooling the solution to 50 °C, 10% (w/w) starch was added as filler. To produce spherical beads, the solution containing the agarose and filler was dropped into doubly-distilled water through a paraffin oil layer (~5 mm) (Frutarom Ltd., Haifa, Israel). 2.2. Bead drying, entrapment, and coating The agarose beads with or without the enzyme were freeze-dried. Freeze-dried beads were obtained by storing gel beads (24 h) at −80 °C before freeze-drying (48 h) in a pilot-plant unit (Model 15 RSRC-X, Repp Industries, Inc., Gardliner, NY, USA), operating at 33.3 Pa (150 mbar) and −45 °C. To determine the moisture content in the beads, beads were further dried in a vacuum oven for 24 h at 105 °C and 104 Pa. After drying, 30 U lactase solution in 100 mM phosphate buffer pH 7.3 was added to each bead for 2 h at 4 °C. After drying at ambient temperature, the dried agarose beads were immersed ~ 50 s in melted chocolate (Elite, Upper Nazareth, Israel), which was first heated to 45 °C and then cooled to ~30 °C. Excess of chocolate was removed and the coating was allowed to solidify at room temperature. 2.3. Measurements of carrier diameter and weight Carrier diameter (±0.03 mm) was measured with a digital caliper (Mitutoyo, Tokyo, Japan). Carrier weight (±0.001 g) before and after enzyme inclusion and chocolate coating, was measured with semimicrobalance 262SMA-FR SCS (Precisa Instruments, Bern, Switzerland).
versus engineering strain relationships using the following equations: σ = F/A0 and εE = ΔD/D0, where σ is the stress (Pa), F is the force at a given time, A0 is the initial cross-sectional area of the carrier, εE is the engineering strain (dimensionless), ΔD is the absolute deformation caused by the compression test (length units), and D0 is the diameter of the carrier at time zero (length units). Young's modulus was calculated from the initial linear portion of the stress versus engineering strain curve. The Young's modulus value, i.e., the slope of the curve, is a parameter reflecting the toughness of the compressed carrier. Compression tests for the different carriers were carried out in five replicates. 2.5. Stability in simulated gastrointestinal fluid The simulated gastrointestinal fluid was based on a previous report of Hack and Selenka (1996) with some modifications. Simulated gastric fluid was prepared by dissolving 10 mg of pepsin from porcine stomach mucosa (Sigma, Rehovot, Israel) in 112 mL of doublydistilled water and 350 mg of mucine from porcine stomach (Sigma, Rehovot, Israel). Then, 3.5 mL of 3 N NaCl solution and 2 mL of 1.2 N KCl solution were added to the simulated gastric fluid. The pH value was adjusted and kept at pH 1.2 by adding an appropriate volume of 1 N HCl (Sigma, Rehovot, Israel). pH values (±0.03) were checked with an Extech Heavy Duty pH/mV Temperature Meter (Extech Instruments Co., Waltham, MA). A laboratory bottle (250 mL) filled with 120 mL of simulated gastric fluid was placed in a TEP-3 water bath using a magnetic stirrer (50 rpm). The bath, filled with preheated water (37 ± 0.5 °C), was controlled by an electric heating element. The enzyme-agarose carriers were immersed in this simulated gastric fluid for 2 h at 37 °C with shaking (75 rpm). Then, the simulated gastric fluid was titrated by adding an appropriate amount of sodium bicarbonate (Frutarom Ltd., Haifa, Israel). The pH value was adjusted and kept at pH 6.8. Trypsin (10 mg) from porcine pancreas (Sigma, Rehovot, Israel), 350 mg of pancreatin from porcine pancreas (Sigma, Rehovot, Israel), and 350 mg of dried bovine bile (Sigma, Rehovot, Israel) were added to obtain simulated intestinal fluid. Carriers were immersed in the simulated intestinal fluid for 6 h at 37 °C with shaking (75 rpm). Time of immersion of the agarose–lactase carriers in the simulated fluids was in accordance to the model developed by Hack and Selenka (1996) and to the literature referring to the passage time of solids through the stomach and small intestine (Washington, Washington, & Wilson, 2003).
2.6. Lactase activity measurements Lactase activity was performed according to the manufacturer's instructions (Sigma, Rehovot, Israel). Lactase activity of retained enzyme was performed by addition of agarose at 1:12 ratio and grinding by mortar and pestle. Bead powder was dissolved in 100 mM phosphate buffer pH 7.3, centrifuged at 8000 rpm for 15 min at 4 °C and the supernatant was tested for lactase activity. Lactase activity versus time relationship was linearized via the equation:
2.4. Compression tests Compression tests were carried out to evaluate properties, such as strength, fragility, and toughness. Carriers of each composition were compressed between lubricated parallel plates to a deformation of 90% at a constant deformation rate of 10 mm/min with an Instron Universal Testing Machine (UTM), Model 5544 (Canton, MA, USA), connected to an IBM-compatible personal computer using a card. Data acquisition and conversion of the Instron's continuous voltage versus time output into digitized force versus time relationships was performed by Merlin software from Instron Corporation (Canton, MA, USA). Finally, the force versus time data was converted to stress
y¼
abt 1 þ bt
where y = lactase activity, a and b are constants and t is the elapsed time. The applicability of this equation was tested through the fit of its linear form.
t 1 t ¼ þ y ab a
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or in its abbreviated form t ¼ k1 þ k2 t: y Such an empirical consideration was previously performed for vibratory compaction of non-food powders (Sone, 1972) and was also mentioned in Peleg and Bagely (1983). 2.7. Porosity determination Carrier porosity was calculated using the following equation: P = 1 − (ρB / ρS), where P is the porosity value (dimensionless), ρB is the bulk density (mass/volume) of the carrier, and ρS is the solid density (mass/volume) of the carrier. Bulk density was obtained from the ratio of mass to volume of the dried carrier. Solid density was evaluated by micro-pycnometer (Quantachrome, Syosset, NY, USA). 2.8. Scanning electron microscopy (SEM) To study the structural changes within and on the surface of the carriers as a result of filler inclusion and drying, SEM micrographs were taken using a JEOL JSM 35C SEM (Tokyo, Japan). The carriers were attached to metal stubs and gold-coated (150–200 Å) in a Polaron 5150 sputter coater (Polaron Equipment Ltd., Holywall Industrial Estate Watford, Hertfordshire, England). 2.9. Statistical analysis Statistical analyses were conducted using JMP software (SAS Institute, 1995), including ANOVA and the Tukey–Kramer Honestly Significant Difference test for comparisons of means. P b 0.05 was considered significant. 3. Results and discussion 3.1. Weight and structure of carriers The agarose carriers designed for lactase immobilization and release weighed from 4.5 to 6.3 mg. The diameters of the freezedried agarose carriers ranged from 3.6 to 3.9 mm. As expected from freeze-drying, the changes in the carrier shape were small (Fellows, 2000; Fennema, 1975), i.e., all carriers remained spherical. Similar shape was described when freeze-dehydration was used to dry alginate beads that were employed to entrap probiotic bacteria for survival purposes in frozen, fermented dairy desserts. Based on SEM micrographs, those beads were also illustrated merely as “spherical” (Shah & Ravula, 2000). The inclusion of starch as filler resulted in a rough surface, i.e., the carriers contained dents and holes on their external surface area (Fig. 1A). It is interesting to note that also freeze-dried alginate beads after immobilization of Acinetobacter johnsonii (i.e. inclusion of entrapped bacteria) were described to also have some grooves on their external surface (Muyima & Cloete, 1995). The diameter of the dents varied from a few microns to ~ 0.1 mm. The size and distribution of the pores formed within the carrier were not homogeneous, as can be seen under electron microscope of the halved freeze-dried agrose–lactase carriers (Fig. 1B). The filler particles (starch granules) appear to occupy some of the pores, as well as are embedded within the carrier wall and surface (Fig. 1B, C). It is well known that freeze-dried beads can be reinforced by inclusion of starch in their formulation before the beads are manufactured and dried. Such starch granules serve not only as filler but also as a carbon source (Tal, van Rijn, & Nussinovitch, 1999). Furthermore, filler inclusion in the dried product reduced its collapse and roundness distortion during the drying process. Thus, the dried bead's components and the method by which it is formed determine its weight,
Fig. 1. Lactase–agarose carriers. A. An intact agarose–starch–lactase carrier displaying its spherical shape and large surface pores. Bar is 1 mm. B. Halved freeze-dried agarose–starch–lactase carrier. Bar is 1 mm. C. Starch granules within the inner structure of the carrier. Bar is 500 μm.
volume, shape and surface topography. Taking these parameters into consideration provides the researcher a useful tool for creating tailormade dried beads for a predetermined operation (Zohar-Perez, Chet, & Nussinovitch, 2004). Although the interior of the spherical carriers (dried beads) was porous, their outer surface appeared to be less porous. The calculated porosity values of the freeze-dried carriers ranged from 85.6 to 90.6%. Freeze-dried products are generally characterized by the least shrinkage and structural changes, and by the creation of pores in areas of former ice crystals. The porous characteristic of the formed bead is
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of utmost importance to enable its coating for example by molten chocolate, in order to further permit bead inclusion in chocolate bars or other chocolate-based products for production as functional foods. Chocolate-coated beads weighed 32.1 ± 1.2 mg. 3.2. Mechanical properties of the carriers As the chocolate-coated lactase–agar carriers can be consumed as is or as part of a confection, it is important to assess its mechanical properties which are related to its sensory qualities. In addition, the mechanical properties of the carriers are also affected by the motility of the gastrointestinal tract. Therefore, the mechanical properties of the carriers were evaluated by compression tests. Fig. 2 represents typical stress–strain relationships for freeze-dried agarose carriers with the inclusion of the filler before coating. It is clear that up to an engineering strain of ~0.4–0.5, an approximately linear stress–strain relationship exists, and the Young's modulus can, therefore, be easily calculated (Fig. 2). The values of Young's modulus for the freeze-dried agarose–lactase carriers were 0.69 ± 0.26 kPa and the R2 values ranged from 0.985 to 0.994. Similar results (0.70 ± 0.09) were detected for the chocolatecoated carriers. The shape of the compressed carrier resembles a typical sigmoid shape of a cellular solid (without a shoulder). i.e., upon mastication it assumes a character or resembles a structure of a crunchy product. While a freeze-dried agarose carrier (i.e., a cellular solid) is being compressed, the cell walls are bent, giving linear elasticity if the cell wall material is elastic. Upon reaching a critical stress, cells begin to crumple and ultimately, at high strains, collapse. It is sufficient to allow opposing cell walls to touch or their broken fragments to pack together. Further deformation compresses the cell wall material itself. This gives the final, steeply rising portion of the stress–strain curve, termed densification. The noise generated by the touch of the opposing cell walls and the pack of the broken fragments is partially responsible for the crunchiness sensation (Nussinovitch, 1997, 2003). Furthermore, the coating with chocolate reduces the absorbance of moisture of the dry product and ensures the preservation of crunchiness of the product for longer periods of time, similarly to puffed rice embedded within chocolate bars or confections whose pores are filled with chocolate (Bower & Whitten, 2000). 3.3. Lactase release from carriers To evaluate the potential of the agarose beads to serve as enzyme carriers, it was necessary to study their stability and enzyme release in solutions which mimic gastrointestinal fluids. Therefore, the ability of the carriers containing lactase to withstand enzyme-containing stomach and small intestine environments was next tested. The weight of the non-coated carriers incubated in buffer or stomach- and intestine-simulating solutions did not decrease significantly (Table 1). In contrast, the weight of the chocolate-coated carriers decreased by ~65%, suggesting that the chocolate coat dissolved quite easily at 37 °C (Le Reverend, Smart, Fryer, & Bakalis, 2011). As most of the weight
Fig. 2. Stress–strain relationships of compressed agarose–starch–lactase dried carriers.
Table 1 % bead weight after incubation in buffer and stomach and intestine solutions.
Buffer solution Stomach solution Intestine solution
Non-coated bead
Chocolate-coated bead
100.72 ± 0.84 99.68 ± 0.10 90.30 ± 1.86
37.13 ± 1.03⁎ 33.77 ± 1.34⁎ 37.68 ± 1.46⁎
⁎ P b 0.05 (Student's t-test).
of the chocolate-coated carriers was due to the coating, this result is desirable. Thus, most of the chocolate-mediated protection against oxidation or moisture absorption terminated upon carrier consumption. The leftover chocolate within inner pores has beneficial influence on enzyme release (see below). After having established that lactase can be entrapped within agarose carriers, release of lactase from the carrier and its biological activity were measured. Chocolate-coated and non-coated lactase-containing carriers were incubated in stomach- and small intestine-simulating solutions (Gal & Nussinovitch, 2007) for 2 h and 6 h, respectively. Released lactase after incubation of chocolate-coated and non-coated lactase-containing carriers in buffer and in stomach- and small intestine-simulating solutions was determined by measuring lactase activity. Non-coated carriers released 32.4% of lactase activity after incubation with buffer and 22.5% and 0.22% after incubation with stomachor small intestine-simulating solutions, respectively. This indicated that the conditions in buffer and stomach-simulating solution led to a first mode of release due to lactase molecules located on the external or inner surfaces of the carrier but not within the matrix itself. However, chocolate-coated carriers exhibited similar lactase activity after incubation in all solutions (Fig. 3). We then measured lactase activity that remained in the carriers due to location inside carrier walls by grinding the beads in the presence of agarose. This was performed on carriers after incubation in buffer, stomach- and small intestine-simulating solutions. Similarly to the results achieved with the incubation in the different solutions, chocolate-coated carriers released higher levels of lactase activity after grinding. These results demonstrate the beneficial effect of the chocolate coating. This two-mode release is reminiscent of a recent publication showing release of human defensin from alginate carriers (Froy, Chapnik, & Nussinovitch, 2010). It is plausible that the leftover chocolate reached inner positions within the carrier and blocked the passage of some proteolytic enzymes, protecting lactase molecules inside the carriers. We then performed kinetic measurements of lactase release and found that chocolate-coated carriers released lactase more readily and reached maximal release after 30 min, whereas non-coated carriers reached release plateau after 1 h (Fig. 4). We tested the curves' linearized form (Fig. 4B) and found that in both cases the regression coefficients were very high, i.e., R 2 = 0.9868 for the chocolate-
Fig. 3. Percent lactase activity of non-coated and chocolate-coated carriers. Activity was compared to the activity initially incorporated into the carriers.
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Fig. 4. Kinetics of lactase release. A. Kinetics of lactase release from non-coated and chocolate-coated carriers. Lactase activity was measured at different time-points after immersion of carriers in lactase buffer. B. Linearized form of A, a mathematical conversion and the regression lines.
coated and 0.995 for the non-coated carriers. Thus, the rate and quantity of the release is faster for the non-coated carrier. In general, this form of data presentation is very convenient for system comparison, since it only involves two experimental constants. The magnitude of these constants especially the k2 constant must depend on the conditions by which the experiments were conducted. These results support the surmise that the chocolate reached inner positions within the carrier and attenuated lactase release, leading to a two-mode release. 4. Conclusions Our results show that dried agarose beads can be utilized for the entrapment of enzymes, such as lactase. Lactase retains its activity when released from the carrier. In addition, chocolate coating aids in protecting the entrapped enzyme and supports the release of intact enzyme in a two-mode pattern. Acknowledgements We thank the Kennedy Leigh Fund for their support. References Alonge, T. O., Ifesanya, A. O., Okoje, V. N., Nottidge, T. E., & Fashima, N. A. (2009). An evaluation of the shelf life of ceftriaxone-polymethylmethhacrylate antibiotic beads. European Journal of Orthopaedic Surgery and Traumatology, 19, 571–575. Bower, J. A., & Whitten, R. (2000). Sensory characteristics and consumer liking for cereal bar snack foods. Journal of Sensory Studies, 15, 327–345.
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