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Manufacture and characterisation of agarose microparticles
Journal of Food Engineering 90 (2009) 141–145
Contents lists available at ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Manufacture and characterisation of agarose microparticles A. Ellis, J.C. Jacquier * School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e
i n f o
Article history: Received 13 March 2008 Received in revised form 22 May 2008 Accepted 24 May 2008 Available online 17 June 2008 Keywords: Agarose Shearing Microgels Microparticles
a b s t r a c t The mechanical properties of agarose microparticles formed by shearing bulk gels dispersed in cold water using a high speed rotor/stator device were investigated. The influence of agarose concentration on bulk gel texture (Young’s modulus, stress and strain at failure) and microparticle size (Sauter mean diameter and size distribution) was assessed. Results obtained showed that bulk gels from 1 to 8% (w/w) showed a linear increase in Young’s modulus with concentration, while the true strain values at failure levelled at high concentrations (>3%). High speed shearing of the bulk gels yielded final particle sizes for the l% and 2% w/w microgel suspensions significantly lower than for the more concentrated gels (3–8% w/w) which are statistically identical (p < 0.005) at 103 ± 2 lm, irrespective of concentration. Rheological investigation of the microparticle suspensions, over a range of bulk gel concentrations, showed that microparticles form a solid like suspension at high volume fractions, becoming fluid like above a well defined yield stress. Overall, a simple and low-cost procedure has been developed to produce an array of agarose microparticles that can confer a range of textural functionalities to beverages from liquids to fluid gels. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Agarose, a marine based polysaccharide is the gelling component of agar and has been widely used in the food industry as a gelling or stabilising agent. Found in red seaweeds, it is an alternating copolymer of b-1,3-linked D-galactose and a-1,4-linked 3,6-anhydro-aL-galactose residues (Normand et al., 2000). The gelling properties of agarose have been extensively studied (Arnott et al., 1974) with gelling occurring at 35 °C when an infinite network of threedimensional agarose fibres is formed, while melting of the agarose network only occurs at temperatures above 85 °C. In recent years, the use of microparticles made from hydrocolloids such as agarose has been investigated for various applications in the food industry. Classically, microparticles were formed by a water-in-oil emulsion route, as described for example by Adams et al. (2004) and these spherical hydrogel beads have proven to be of enormous interest as encapsulation matrices for a wide range of food supplements such as probiotics (Krasaekoopt et al., 2003; Capela et al., 2007) and antioxidants (Boadi and Neufeld, 2001). Despite these promising functionalities, microspheres are proving difficult and expensive to manufacture due to the amount of oil phase necessary (Krasaekoopt et al., 2003). Very recently, a few Japanese patents have been disclosed relating to the use of crushed or sheared hydrocolloid microparticles in semi-solid foods and beverages. Nakade (2007) crushed agarose, forming microparticles which were then used in fruit juices to disperse fruit pulp. Adachi * Corresponding author. Tel.: +353 1 7167098; fax: +353 1 7161149. E-mail address: [email protected] (J.C. Jacquier). 0260-8774/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2008.05.036
and Binshiyoo (2007) also looked at using crushed agarose for a gel based condiment that does not flow from the surface of semi-solid food products. Another recent Japanese patent Maejima (2007) describes the encapsulation of additives in a crushed agar gel as part of a fluid gel beverage. Norton et al. (1999) developed a range of fluid gels that were manufactured by applying a shear force to cooling solutions of agarose during gelation. These microparticulate fluid gels could be used as bulking agents in food products to create a sensation of satiety due to their breakdown in the GI tract (Norton et al., 2006). While the procedure used by Norton and co-workers (Brown et al., 1990) is a very innovative and inexpensive method to produce microparticles, the process has a few limitations such as a poor control of the polymer concentration in the microparticles and difficult control of material encapsulation. In this study, gel microparticles were prepared by breaking down a quiescently cooled bulk agarose gel dispersed in water and this economical and ingenuous procedure could be of use in many areas in a developing food industry. The actual underlying science behind the manufacture and properties of the microparticles produced by shearing a gel have not been previously studied in great detail and therefore are the main focus of this work. By using a high-speed rotor–stator mixer, microparticles can be produced while controlling both the bulk agarose concentration and the microparticle to water volume ratio. The effect of bulk gel concentration and speed of the rotor on the size, shape, and polydispersity of the resulting microparticles was investigated. Finally, the effect of microparticle phase volume on the flow behaviour of the dispersion was examined.
A. Ellis, J.C. Jacquier / Journal of Food Engineering 90 (2009) 141–145
2.1. Materials Gelagar CT type 1.0 from the Gracilaria rhodophyta species of red algae was obtained from B&V, Gattatico, Italy and was used as is. Distilled water was used to prepare microgel suspensions. Sodium azide (200 ppm) from Sigma, Ireland was added to all bulk gel batches as a preservative. Blue Dextran (2,000,000 average molecular weight, from Sigma, Ireland) was used to determine the microgel phase volume. 2.2. Methods 2.2.1. Bulk gel concentration The bulk gels were prepared by dispersing the powered agarose in distilled water while stirring, before being placed in an autoclave (MP 24 Control, by Rodwell Scientific Instruments) for 15 min at 121 °C to ensure complete dissolution of the polysaccharide. Dry weight measurements were carried out at 105 °C in order to establish final bulk gel concentration. A known amount of bulk gel was placed in pre-weighed vessels and dried to constant mass. Triplicates of each sample were prepared. 2.2.2. Deformation tests Compression test experiments were carried out on an Instron 5544 (High Wycombe, UK) fitted with a 500 N load cell. The cylindrical samples (15 mm diameter, 15 mm length) were compressed at a deformation rate of 50 mm/min. The samples were lubricated with oil to avoid shearing due to friction between the sample and the load plates and at least 15 replicates were tested for each bulk gel concentration. Assuming incompressibility of the agar sample (Normand et al., 2000), the true stress and true or Hencky strain were estimated according to the following equations:
true stress
r¼
FH
pr2 H0
true strain e ¼ ln
H H0
where F is the applied load, H0 is the specimen height and H is the compression displacement. The Young’s Modulus (E) was calculated in the initial linear region of the stress–strain curves. 2.2.3. Generation of microparticles The microparticle suspensions were prepared by placing the bulk gel in distilled water (1:2 gel:water by volume) and blending initially using a standard food blender. A Silverson high speed rotor–stator mixer (model L4RT, Silverson Machines Ltd. Chesham, England) equipped with a fine emulsor screen (800 lm mesh size) was used to further reduce the gel particle size by shearing at speeds of 500–7800 rpm. In order to prevent any melting of the gel during shearing, the temperature of the suspensions were kept below 10 °C by placing the container in iced water. 2.2.4. Particle size analysis Particle size analysis was carried out in distilled water using a Malvern Mastersizer S (Malvern Instruments Ltd., Morchester, UK) fitted with a 300 RF range lens (0.05–900 lm) and a small volume sample dispersion unit. The Fraunhofer optical model was used as particle size was large enough for this model to give a good approximation of particle distribution ISO13320, 1999.
2.2.5. Rheological measurements Steady stress sweep tests were carried out on the microparticle suspensions using a stress controlled rheometer (model SR-2000, Rheometric Scientific, Piscataway, NJ, USA) with couette geometry (bob diameter 29.5 mm and internal diameter of cup 32 mm) at 25 °C. 2.2.6. Particle phase volume Particle phase volume (/) of all samples was established using blue dextran according to Tester and Morrison (1990). As agarose gel does not swell in excess water (Frith et al., 1999) it can therefore be assumed that the concentration of the gel within the microparticle is that of the initial bulk gel. 3. Results and discussion 3.1. Structural properties of bulk agarose gel Compression tests were carried out on agarose gels, ranging in concentration from 1–8% w/w. The evolution of Young’s modulus (E) as a function of increasing bulk gel concentration is shown in Fig. 1 together with the evolution of the stress at failure in the same concentration range. Both these parameters seem to increase linearly with the bulk agar concentration (linear regression coefficients r2 P 0.99 in both cases), which tends to indicate a proportional strengthening of the gel network with the amount of polymer strands in the medium. On the other hand, the effect of concentration on strain at the failure point (Fig. 2) shows a levelling from 4% w/w onwards. The initial increase in strain at failure for the 1–3% w/w gels, seem to indicate shorter network connections at these low concentrations. From 4% w/w onwards, the values of the strain at failure plateau at 0.35, consistent with the results found by Normand et al. (2000). Overall, these compression results would seem to indicate that at low concentration the agar network develops first at short distances, and that the increase in concentration up to 4% w/w increases the occurrence of long distance gel points. Above 4% w/ w, the network is fully established and all additional agar chains seem to only reinforce the existing junction fibres (Guenet, 1992). 3.2. Manufacture of agarose microparticles The mechanism by which the microgel suspension is formed hinges on the suction of the bulk gel pieces and water into the rotor head. Centrifugal forces push the components towards the edge
1200 6000
1000 800
4000 600 400
2000
Failure Stress σ (kPa )
2. Experimental
Young's Modulus E (kPa)
142
200 0
0 0
1
2
3
4
5
6
7
8
Agarose concentration (% w/w) Fig. 1. Evolution of Young’s modulus ( ) and true stress at failure ( ) with agar concentration (% w/w). The dashed lines are linear regressions of the experimental points.
143
A. Ellis, J.C. Jacquier / Journal of Food Engineering 90 (2009) 141–145
38
16 14 12
34
Volume %
Failure Strain ε (%)
36
32
10 8 6
30 4 28
2 0
26 0
2
4
6
10
8
100
Agarose Concentration (% w/w)
Fig. 2. Effect of agar concentration (% w/w) on true strain at failure.
of the rotor blades and the inside stator wall. Shear forces then push the gel pieces and water at high velocity through the fine emulsor screen. The resulting microparticles are angular and jagged in shape as shown in Figs. 3a and b with the low concentration agarose microparticles (Fig. 3a) seemingly more serrated and oblong than the more concentrated ones (Fig. 3b). In order to understand the effect of rotor speed on the shearing process, the size distribution of the generated microgel particles was examined at fixed bulk gel concentration (5% w/w) on the entire speed range of the Silverson mixer (500–7800 rpm). In Fig. 4 the size distribution of the 5% w/w agarose bulk gel is shown after
1000
Sizes (μm) Fig. 4. Particle size distribution for each rotor speed (rpm) on the 5% agarose bulk gel. 500 rpm , 1000 rpm , 3000 rpm , 5000 rpm , 6000 rpm , 7800 rpm .
it had been ‘sheared’ over time at a range of speeds. For each given speed, shearing of the gel was ceased when repeated shearing showed no change in particle size. Therefore it should be noted that particle size was dependent on the speed at which the mixture was being broken down rather than the length of time it was under this stress for. At speeds of up to 3000 rpm there is a broad distribution of sizes, with a greater volume of large particles (above 100 lm) and a small second shoulder representing particles of smaller sizes than the rest of the mixture. Within this low speed range, the shearing process seems to reduce the size of the larger particles while the small second shoulder seems unaffected. As speed increases up to 6000 rpm the particle size becomes smaller with the second shoulder becoming part of a narrowing size distribution. At 7800 rpm, speed has caused the size distribution to narrow significantly with the particle size being more uniform than was seen at the lower speeds. Polydispersity of the samples, expressed as the ratio of the De Brouckere to the Sauter Diameters (D[4, 3]/D[3, 2]), reflects this narrowing of the size distribution as it decreases from 2.153 at 500 rpm to 1.322 at 7800 rpm. This high speed of 7800 rpm, the highest speed that could be obtained from the Silverson, will therefore be used for all bulk gel concentrations. In order to determine an effective shearing time, samples of the microparticle dispersions were taken at varying time intervals over the shearing period (until no further change in particle size was recorded after repeated shearing). Fig. 5 shows the decrease in size (D[3, 2]) of the microparticles with time. The microparticle size seems to decrease in size following a pseudo-first order kinetic 200 180
D[3,2] (μm)
160 140 120 100 80 60 40 0
2
4
6
8
10
12
14
16
18
Time (min) Fig. 3. Light microscopy of microparticles.
Fig. 5. Effect of shearing time on Sauter diameter (D[3, 2] (lm). 1 (% w/w) – d, 2 (% w/w) – s, 4 (% w/w) – ., 8 (%w/w) – 4.
A. Ellis, J.C. Jacquier / Journal of Food Engineering 90 (2009) 141–145
model, where most of the size decrease happens in the first four minutes while no significant change occurs after 16 min (p < 0.05) of shearing for all bulk gel concentrations. There is a significant difference between the 1% w/w and 2% w/w gels while the 4% w/w and 8% w/w have final D[3, 2] (lm) values that are overlapping. Above 2% w/w, all bulk gels seem to decrease in size at the same rate (k = 0.35 ± 0.05 min1) while the 1% w/w gel has a higher k value of 0.52 min1 and no significant change in size was detected for this low concentration after 8 min (p < 0.05). The evolution of final particle size with bulk agarose concentration is shown in Fig. 6 and reveals that the particle sizes obtained for the l% and 2% w/w microgel suspensions are significantly lower than for the more concentrated gels (3–8% w/w) which are statistically identical (p < 0.005) at 103 ± 2 lm, irrespective of concentration. It is noteworthy to mention here that these final sizes are considerably smaller than the mesh size of the Silverson grid (800 lm) so that the physical extrusion of the gel through the screen is not the main force behind the generation of small particles. However, the plateau in yield strain with bulk gel concentration seen in Fig. 2 is very similar to the observed plateau in particle size seen in Fig. 6. It seems therefore that the ability of the gels to withstand large deformation strain before breaking down into smaller particles may explain the final D[3, 2] values obtained. As mentioned previously, the bulk gels of higher concentrations could withstand larger strain before fracture than the 1% and 2% w/w gels. It could therefore be rationalised that, as the gel is being forced through the emulsor screen at high speed, it is subjected to tremendous shearing strain before breaking down in smaller particles. As the higher concentration gels can withstand higher strain before failure, there final size will be larger than the lower concentration gels. 3.3. Rheological properties of the microparticles The influence of the microparticle volume fraction on the dispersion viscosity was also investigated. Samples were prepared over a range of phase volumes (/) for each bulk gel concentration. In Fig. 7 the effect of shear stress on a 6% w/w bulk gel concentration is shown. At dispersion volume fractions up to 30%, the flow behaviour of the microparticles in solution is essentially Newtonian with low viscosity (0.010 Pa s). The flow behaviour of the microgel dispersions changes dramatically as the volume fraction increases towards the maximum packing fraction /m. The flow curve at 37% shows a very high viscosity at rest (50 Pa s at 0.2 Pa stress) indicative of a solid-like structure that is easily disrupted with increasing stress. As the applied stress increases above an apparent yield stress, the viscosity of the dispersion plummets nearly four orders of magnitude to reach 0.017 Pa s at stresses above 30 Pa. This final low viscosity is similar to the more dilute
10000 1000 100
η[Pa.s]
144
1 0.1 0.01 0.001 0.1
1
10
100
σ [Pa] Fig. 7. Flow curves for 6% w/w microparticle suspensions with different microparticle volume fractions (/) where d 28.5%, s 37%, . 54%.
and liquid-like dispersions. This apparent breakdown in the dispersion viscosity is replicated at higher volume fractions up to 54%, where the initial viscosity at rest reaches 3000 Pa s. At this very high dispersion volume fraction, probably above the maximum packing fraction /m, the apparent yield stress is quite high (above 20 Pa) and the sample is solid like, resembling a gel. Although shear thinning follows at higher shear stress values, making the suspension quite fluid, this dispersion never reaches the low viscosity values obtained at lower dispersion volume fractions, probably because of our instrument limitations. The relationships between the minimum viscosity of the dispersions at different bulk gel concentrations and their particle phase volume are shown in Fig. 8 as well as their fit to the Krieger– Dougherty model (Krieger and Dougherty, 1959)
g ¼ gf 1
/ /m
½g/m
where gf is the suspending fluid viscosity, / is the true volume fraction of the solid particles, /m is the maximum packing fraction and [g] the intrinsic viscosity. The dispersions made from highly concentrated bulk gels (4–8% w/w) seem to follow the same evolution of viscosity with phase volume, with a calculated maximum packing fraction /m of 46.4%, while the dispersions made from less concentrated bulk gels (3% down to 1% w/w agarose) show distinct patterns with decreasing values of maximum packing fraction (40.8%, 34.8% and 30.7% for the 3%, 2% and 1% w/w gels, respec-
10
120
1
η (Pa.s)
100
D[3,2] μm
10
80
0.1
60 0.01 40 20
0.001 0
10
20
30
40
50
Φ (%v/v)
0 0
2
4
6
8
Agar concentration (% w/w) Fig. 6. Effect of agar concentration on Sauter diameter (D[3, 2] (lm).
Fig. 8. The evolution of the solution viscosity at high shear as a function of the microparticle phase volume for different bulk gel concentrations ( 1%, 2%, 3%, 4–8%).
A. Ellis, J.C. Jacquier / Journal of Food Engineering 90 (2009) 141–145
tively). Overall these maximum packing fraction values are quite low (theoretically, /m should be close to 60% for monodisperse spherical dispersions), all the more so if the polydispersity of the present dispersions is taken into account. The angularity of the broken gels might explain these low /m values, resulting in interlocking particle aggregates preventing flow at intermediate shear values. This behaviour would be increased with the angularity of the dispersion particles, increasing from low to highly concentrated gels as seen in Fig. 3 and discussed previously. But, this behaviour is quite complex, and more investigation is needed to fully explain this phenomenon. 4. Conclusion The overall diameter of the microparticles following shearing was approximately 100 lm for the more concentrated (4–8% w/ w) bulk gel concentrations, with smaller sizes found for the 1% and 2% w/w bulk gels. The small size of these particles means that they could be incorporated into a food product as a bulking agent and texture modifiers for a wide range of beverages. The results from the mechanical testing of the bulk gel indicate that their soft texture and high elastic nature will not impact negatively on the mouth feel of the product. Initial testing of the rheological properties of the microgels has shown that even at high solid content (<30% v/v), the viscosity is low and should have little effect on the overall flowability of the final food product. At higher volume fractions (>35%), the dispersion becomes solid-like at rest, with the characteristics of a fluid gel. Overall, a simple and lowcost procedure has been developed to produce an array of agarose microparticles that have a range of functionalities and could be of use in many areas throughout the food industry, for example as fat replacers or as micro-encapsulation vehicles for functional ingredient protection and delivery. Acknowledgements Authors would like to acknowledge funding of this FIRM project by the Department of Agriculture and Food under the National Development Plan 2000–2006.
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References Adachi, H., Binshiyoo, K.K., 2007. Gel condiment using agar. Japanese Patent Office, JP 2007014323A. Adams, S., Frith, W.J., Stokes, J.R., 2004. Influence of particle modulus on the rheological properties of agar microgel suspensions. Journal of Rheology 48 (6), 1195–1213. Arnott, S.A., Fulmer, A., Scott, W.E., 1974. The agarose double helix and its function in agarose gel structure. Journal of Molecular Biology 90, 269–284. Boadi, D.K., Neufeld, R.J., 2001. Encapsulation of tannase for the hydrolysis of tea tannins. Enzyme and Microbial Technology 28, 590–595. Brown, C.R.T., Cutler, A.N., Norton, I.T., 1990. Inventors. Liquid Based Composition Comprising Gelling Polysaccharide Capable of Forming a Reversible Gel and a Method Preparing Such a Composition, No. EP0355908. Capela, P., Hay, T.K.C., Shah, N.P., 2007. Effect of homogenisation on the bead size and survival of encapsulated probiotic bacteria. Food Research International 40, 1261–1269. Frith, W.J., Lips A., Norton, I.T., 1999. Steady shear flow properties of spherical microgels. In: Mashelkar, R.A., Kulkarni, B.D., Naik, V.M. (Ed.), Structure and Dynamics of Materials in the Mesoscale Domain: Proceedings of the Royal Society, Unilever Indo-UK Forum Material Science Engineering, India, 1997, Imperial College Press, pp. 207–218. Guenet, J.M., 1992. Thermoreversible Gelation of Polymers and Biopolymers. Springer, Berlin. pp. 80–87. ISO13320, 1999. Particle Size Analysis – Laser Diffraction Methods. Part 1: General Principles. Krasaekoopt, W., Bhandari, B., Deeth, H., 2003. Evaluation of encapsulation techniques of probiotics for yoghurt. International Dairy Journal 13, 3–13. Krieger, I.M., Dougherty, T.J., 1959. A mechanism for non-Newtonian flow in suspensions of rigid spheres. Transactions of the Society of Rheology 53 (21), 8259–8264. Maejima, T., 2007. Gelidium jelly drink. Japanese Patent Application, JP 2006288305A. Nakade, K., 2007. Method for producing preparation. Japanese Patent Application, JP 2006254746A. Normand, V., Lootens, D.L., Amici, E., Plucknett, K.P., Aymard, P., 2000. New insight into agarose gel mechanical properties. Biomacromolecules 1, 730–738. Norton, I.T., Jarvis, D.A., Foster, T.J., 1999. A molecular model for the formation and properties of fluid gel. International Journal of Biological Macromolecules 26, 255–261. Norton, I.T., Frith, W.J., Ablett, S., 2006. Fluid gels, mixed fluid gels and satiety. Food Hydrocolloids 20, 229–239. Tester, R.F., Morrison, W.R., 1990. Swelling and gelatinization of cereal starches. I. Effects of amylopectin, amylose and lipids. Cereal Chemistry 67 (6), 551–557.
Contents lists available at ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Manufacture and characterisation of agarose microparticles A. Ellis, J.C. Jacquier * School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e
i n f o
Article history: Received 13 March 2008 Received in revised form 22 May 2008 Accepted 24 May 2008 Available online 17 June 2008 Keywords: Agarose Shearing Microgels Microparticles
a b s t r a c t The mechanical properties of agarose microparticles formed by shearing bulk gels dispersed in cold water using a high speed rotor/stator device were investigated. The influence of agarose concentration on bulk gel texture (Young’s modulus, stress and strain at failure) and microparticle size (Sauter mean diameter and size distribution) was assessed. Results obtained showed that bulk gels from 1 to 8% (w/w) showed a linear increase in Young’s modulus with concentration, while the true strain values at failure levelled at high concentrations (>3%). High speed shearing of the bulk gels yielded final particle sizes for the l% and 2% w/w microgel suspensions significantly lower than for the more concentrated gels (3–8% w/w) which are statistically identical (p < 0.005) at 103 ± 2 lm, irrespective of concentration. Rheological investigation of the microparticle suspensions, over a range of bulk gel concentrations, showed that microparticles form a solid like suspension at high volume fractions, becoming fluid like above a well defined yield stress. Overall, a simple and low-cost procedure has been developed to produce an array of agarose microparticles that can confer a range of textural functionalities to beverages from liquids to fluid gels. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Agarose, a marine based polysaccharide is the gelling component of agar and has been widely used in the food industry as a gelling or stabilising agent. Found in red seaweeds, it is an alternating copolymer of b-1,3-linked D-galactose and a-1,4-linked 3,6-anhydro-aL-galactose residues (Normand et al., 2000). The gelling properties of agarose have been extensively studied (Arnott et al., 1974) with gelling occurring at 35 °C when an infinite network of threedimensional agarose fibres is formed, while melting of the agarose network only occurs at temperatures above 85 °C. In recent years, the use of microparticles made from hydrocolloids such as agarose has been investigated for various applications in the food industry. Classically, microparticles were formed by a water-in-oil emulsion route, as described for example by Adams et al. (2004) and these spherical hydrogel beads have proven to be of enormous interest as encapsulation matrices for a wide range of food supplements such as probiotics (Krasaekoopt et al., 2003; Capela et al., 2007) and antioxidants (Boadi and Neufeld, 2001). Despite these promising functionalities, microspheres are proving difficult and expensive to manufacture due to the amount of oil phase necessary (Krasaekoopt et al., 2003). Very recently, a few Japanese patents have been disclosed relating to the use of crushed or sheared hydrocolloid microparticles in semi-solid foods and beverages. Nakade (2007) crushed agarose, forming microparticles which were then used in fruit juices to disperse fruit pulp. Adachi * Corresponding author. Tel.: +353 1 7167098; fax: +353 1 7161149. E-mail address: [email protected] (J.C. Jacquier). 0260-8774/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2008.05.036
and Binshiyoo (2007) also looked at using crushed agarose for a gel based condiment that does not flow from the surface of semi-solid food products. Another recent Japanese patent Maejima (2007) describes the encapsulation of additives in a crushed agar gel as part of a fluid gel beverage. Norton et al. (1999) developed a range of fluid gels that were manufactured by applying a shear force to cooling solutions of agarose during gelation. These microparticulate fluid gels could be used as bulking agents in food products to create a sensation of satiety due to their breakdown in the GI tract (Norton et al., 2006). While the procedure used by Norton and co-workers (Brown et al., 1990) is a very innovative and inexpensive method to produce microparticles, the process has a few limitations such as a poor control of the polymer concentration in the microparticles and difficult control of material encapsulation. In this study, gel microparticles were prepared by breaking down a quiescently cooled bulk agarose gel dispersed in water and this economical and ingenuous procedure could be of use in many areas in a developing food industry. The actual underlying science behind the manufacture and properties of the microparticles produced by shearing a gel have not been previously studied in great detail and therefore are the main focus of this work. By using a high-speed rotor–stator mixer, microparticles can be produced while controlling both the bulk agarose concentration and the microparticle to water volume ratio. The effect of bulk gel concentration and speed of the rotor on the size, shape, and polydispersity of the resulting microparticles was investigated. Finally, the effect of microparticle phase volume on the flow behaviour of the dispersion was examined.
A. Ellis, J.C. Jacquier / Journal of Food Engineering 90 (2009) 141–145
2.1. Materials Gelagar CT type 1.0 from the Gracilaria rhodophyta species of red algae was obtained from B&V, Gattatico, Italy and was used as is. Distilled water was used to prepare microgel suspensions. Sodium azide (200 ppm) from Sigma, Ireland was added to all bulk gel batches as a preservative. Blue Dextran (2,000,000 average molecular weight, from Sigma, Ireland) was used to determine the microgel phase volume. 2.2. Methods 2.2.1. Bulk gel concentration The bulk gels were prepared by dispersing the powered agarose in distilled water while stirring, before being placed in an autoclave (MP 24 Control, by Rodwell Scientific Instruments) for 15 min at 121 °C to ensure complete dissolution of the polysaccharide. Dry weight measurements were carried out at 105 °C in order to establish final bulk gel concentration. A known amount of bulk gel was placed in pre-weighed vessels and dried to constant mass. Triplicates of each sample were prepared. 2.2.2. Deformation tests Compression test experiments were carried out on an Instron 5544 (High Wycombe, UK) fitted with a 500 N load cell. The cylindrical samples (15 mm diameter, 15 mm length) were compressed at a deformation rate of 50 mm/min. The samples were lubricated with oil to avoid shearing due to friction between the sample and the load plates and at least 15 replicates were tested for each bulk gel concentration. Assuming incompressibility of the agar sample (Normand et al., 2000), the true stress and true or Hencky strain were estimated according to the following equations:
true stress
r¼
FH
pr2 H0
true strain e ¼ ln
H H0
where F is the applied load, H0 is the specimen height and H is the compression displacement. The Young’s Modulus (E) was calculated in the initial linear region of the stress–strain curves. 2.2.3. Generation of microparticles The microparticle suspensions were prepared by placing the bulk gel in distilled water (1:2 gel:water by volume) and blending initially using a standard food blender. A Silverson high speed rotor–stator mixer (model L4RT, Silverson Machines Ltd. Chesham, England) equipped with a fine emulsor screen (800 lm mesh size) was used to further reduce the gel particle size by shearing at speeds of 500–7800 rpm. In order to prevent any melting of the gel during shearing, the temperature of the suspensions were kept below 10 °C by placing the container in iced water. 2.2.4. Particle size analysis Particle size analysis was carried out in distilled water using a Malvern Mastersizer S (Malvern Instruments Ltd., Morchester, UK) fitted with a 300 RF range lens (0.05–900 lm) and a small volume sample dispersion unit. The Fraunhofer optical model was used as particle size was large enough for this model to give a good approximation of particle distribution ISO13320, 1999.
2.2.5. Rheological measurements Steady stress sweep tests were carried out on the microparticle suspensions using a stress controlled rheometer (model SR-2000, Rheometric Scientific, Piscataway, NJ, USA) with couette geometry (bob diameter 29.5 mm and internal diameter of cup 32 mm) at 25 °C. 2.2.6. Particle phase volume Particle phase volume (/) of all samples was established using blue dextran according to Tester and Morrison (1990). As agarose gel does not swell in excess water (Frith et al., 1999) it can therefore be assumed that the concentration of the gel within the microparticle is that of the initial bulk gel. 3. Results and discussion 3.1. Structural properties of bulk agarose gel Compression tests were carried out on agarose gels, ranging in concentration from 1–8% w/w. The evolution of Young’s modulus (E) as a function of increasing bulk gel concentration is shown in Fig. 1 together with the evolution of the stress at failure in the same concentration range. Both these parameters seem to increase linearly with the bulk agar concentration (linear regression coefficients r2 P 0.99 in both cases), which tends to indicate a proportional strengthening of the gel network with the amount of polymer strands in the medium. On the other hand, the effect of concentration on strain at the failure point (Fig. 2) shows a levelling from 4% w/w onwards. The initial increase in strain at failure for the 1–3% w/w gels, seem to indicate shorter network connections at these low concentrations. From 4% w/w onwards, the values of the strain at failure plateau at 0.35, consistent with the results found by Normand et al. (2000). Overall, these compression results would seem to indicate that at low concentration the agar network develops first at short distances, and that the increase in concentration up to 4% w/w increases the occurrence of long distance gel points. Above 4% w/ w, the network is fully established and all additional agar chains seem to only reinforce the existing junction fibres (Guenet, 1992). 3.2. Manufacture of agarose microparticles The mechanism by which the microgel suspension is formed hinges on the suction of the bulk gel pieces and water into the rotor head. Centrifugal forces push the components towards the edge
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Failure Stress σ (kPa )
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Agarose concentration (% w/w) Fig. 1. Evolution of Young’s modulus ( ) and true stress at failure ( ) with agar concentration (% w/w). The dashed lines are linear regressions of the experimental points.
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38
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Fig. 2. Effect of agar concentration (% w/w) on true strain at failure.
of the rotor blades and the inside stator wall. Shear forces then push the gel pieces and water at high velocity through the fine emulsor screen. The resulting microparticles are angular and jagged in shape as shown in Figs. 3a and b with the low concentration agarose microparticles (Fig. 3a) seemingly more serrated and oblong than the more concentrated ones (Fig. 3b). In order to understand the effect of rotor speed on the shearing process, the size distribution of the generated microgel particles was examined at fixed bulk gel concentration (5% w/w) on the entire speed range of the Silverson mixer (500–7800 rpm). In Fig. 4 the size distribution of the 5% w/w agarose bulk gel is shown after
1000
Sizes (μm) Fig. 4. Particle size distribution for each rotor speed (rpm) on the 5% agarose bulk gel. 500 rpm , 1000 rpm , 3000 rpm , 5000 rpm , 6000 rpm , 7800 rpm .
it had been ‘sheared’ over time at a range of speeds. For each given speed, shearing of the gel was ceased when repeated shearing showed no change in particle size. Therefore it should be noted that particle size was dependent on the speed at which the mixture was being broken down rather than the length of time it was under this stress for. At speeds of up to 3000 rpm there is a broad distribution of sizes, with a greater volume of large particles (above 100 lm) and a small second shoulder representing particles of smaller sizes than the rest of the mixture. Within this low speed range, the shearing process seems to reduce the size of the larger particles while the small second shoulder seems unaffected. As speed increases up to 6000 rpm the particle size becomes smaller with the second shoulder becoming part of a narrowing size distribution. At 7800 rpm, speed has caused the size distribution to narrow significantly with the particle size being more uniform than was seen at the lower speeds. Polydispersity of the samples, expressed as the ratio of the De Brouckere to the Sauter Diameters (D[4, 3]/D[3, 2]), reflects this narrowing of the size distribution as it decreases from 2.153 at 500 rpm to 1.322 at 7800 rpm. This high speed of 7800 rpm, the highest speed that could be obtained from the Silverson, will therefore be used for all bulk gel concentrations. In order to determine an effective shearing time, samples of the microparticle dispersions were taken at varying time intervals over the shearing period (until no further change in particle size was recorded after repeated shearing). Fig. 5 shows the decrease in size (D[3, 2]) of the microparticles with time. The microparticle size seems to decrease in size following a pseudo-first order kinetic 200 180
D[3,2] (μm)
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Time (min) Fig. 3. Light microscopy of microparticles.
Fig. 5. Effect of shearing time on Sauter diameter (D[3, 2] (lm). 1 (% w/w) – d, 2 (% w/w) – s, 4 (% w/w) – ., 8 (%w/w) – 4.
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model, where most of the size decrease happens in the first four minutes while no significant change occurs after 16 min (p < 0.05) of shearing for all bulk gel concentrations. There is a significant difference between the 1% w/w and 2% w/w gels while the 4% w/w and 8% w/w have final D[3, 2] (lm) values that are overlapping. Above 2% w/w, all bulk gels seem to decrease in size at the same rate (k = 0.35 ± 0.05 min1) while the 1% w/w gel has a higher k value of 0.52 min1 and no significant change in size was detected for this low concentration after 8 min (p < 0.05). The evolution of final particle size with bulk agarose concentration is shown in Fig. 6 and reveals that the particle sizes obtained for the l% and 2% w/w microgel suspensions are significantly lower than for the more concentrated gels (3–8% w/w) which are statistically identical (p < 0.005) at 103 ± 2 lm, irrespective of concentration. It is noteworthy to mention here that these final sizes are considerably smaller than the mesh size of the Silverson grid (800 lm) so that the physical extrusion of the gel through the screen is not the main force behind the generation of small particles. However, the plateau in yield strain with bulk gel concentration seen in Fig. 2 is very similar to the observed plateau in particle size seen in Fig. 6. It seems therefore that the ability of the gels to withstand large deformation strain before breaking down into smaller particles may explain the final D[3, 2] values obtained. As mentioned previously, the bulk gels of higher concentrations could withstand larger strain before fracture than the 1% and 2% w/w gels. It could therefore be rationalised that, as the gel is being forced through the emulsor screen at high speed, it is subjected to tremendous shearing strain before breaking down in smaller particles. As the higher concentration gels can withstand higher strain before failure, there final size will be larger than the lower concentration gels. 3.3. Rheological properties of the microparticles The influence of the microparticle volume fraction on the dispersion viscosity was also investigated. Samples were prepared over a range of phase volumes (/) for each bulk gel concentration. In Fig. 7 the effect of shear stress on a 6% w/w bulk gel concentration is shown. At dispersion volume fractions up to 30%, the flow behaviour of the microparticles in solution is essentially Newtonian with low viscosity (0.010 Pa s). The flow behaviour of the microgel dispersions changes dramatically as the volume fraction increases towards the maximum packing fraction /m. The flow curve at 37% shows a very high viscosity at rest (50 Pa s at 0.2 Pa stress) indicative of a solid-like structure that is easily disrupted with increasing stress. As the applied stress increases above an apparent yield stress, the viscosity of the dispersion plummets nearly four orders of magnitude to reach 0.017 Pa s at stresses above 30 Pa. This final low viscosity is similar to the more dilute
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1 0.1 0.01 0.001 0.1
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σ [Pa] Fig. 7. Flow curves for 6% w/w microparticle suspensions with different microparticle volume fractions (/) where d 28.5%, s 37%, . 54%.
and liquid-like dispersions. This apparent breakdown in the dispersion viscosity is replicated at higher volume fractions up to 54%, where the initial viscosity at rest reaches 3000 Pa s. At this very high dispersion volume fraction, probably above the maximum packing fraction /m, the apparent yield stress is quite high (above 20 Pa) and the sample is solid like, resembling a gel. Although shear thinning follows at higher shear stress values, making the suspension quite fluid, this dispersion never reaches the low viscosity values obtained at lower dispersion volume fractions, probably because of our instrument limitations. The relationships between the minimum viscosity of the dispersions at different bulk gel concentrations and their particle phase volume are shown in Fig. 8 as well as their fit to the Krieger– Dougherty model (Krieger and Dougherty, 1959)
g ¼ gf 1
/ /m
½g/m
where gf is the suspending fluid viscosity, / is the true volume fraction of the solid particles, /m is the maximum packing fraction and [g] the intrinsic viscosity. The dispersions made from highly concentrated bulk gels (4–8% w/w) seem to follow the same evolution of viscosity with phase volume, with a calculated maximum packing fraction /m of 46.4%, while the dispersions made from less concentrated bulk gels (3% down to 1% w/w agarose) show distinct patterns with decreasing values of maximum packing fraction (40.8%, 34.8% and 30.7% for the 3%, 2% and 1% w/w gels, respec-
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Fig. 8. The evolution of the solution viscosity at high shear as a function of the microparticle phase volume for different bulk gel concentrations ( 1%, 2%, 3%, 4–8%).
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tively). Overall these maximum packing fraction values are quite low (theoretically, /m should be close to 60% for monodisperse spherical dispersions), all the more so if the polydispersity of the present dispersions is taken into account. The angularity of the broken gels might explain these low /m values, resulting in interlocking particle aggregates preventing flow at intermediate shear values. This behaviour would be increased with the angularity of the dispersion particles, increasing from low to highly concentrated gels as seen in Fig. 3 and discussed previously. But, this behaviour is quite complex, and more investigation is needed to fully explain this phenomenon. 4. Conclusion The overall diameter of the microparticles following shearing was approximately 100 lm for the more concentrated (4–8% w/ w) bulk gel concentrations, with smaller sizes found for the 1% and 2% w/w bulk gels. The small size of these particles means that they could be incorporated into a food product as a bulking agent and texture modifiers for a wide range of beverages. The results from the mechanical testing of the bulk gel indicate that their soft texture and high elastic nature will not impact negatively on the mouth feel of the product. Initial testing of the rheological properties of the microgels has shown that even at high solid content (<30% v/v), the viscosity is low and should have little effect on the overall flowability of the final food product. At higher volume fractions (>35%), the dispersion becomes solid-like at rest, with the characteristics of a fluid gel. Overall, a simple and lowcost procedure has been developed to produce an array of agarose microparticles that have a range of functionalities and could be of use in many areas throughout the food industry, for example as fat replacers or as micro-encapsulation vehicles for functional ingredient protection and delivery. Acknowledgements Authors would like to acknowledge funding of this FIRM project by the Department of Agriculture and Food under the National Development Plan 2000–2006.
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