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Effects of Sterilization Treatments on Some Properties of Alginate Solutions and Gels William J. Leo,+Aiden J. McLoughlin,? and Dermot M. Malone**$ Department of Industrial Microbiology and Department of Chemical Engineering, University College Dublin, Belfield, Dublin, Ireland
Aqueous sodium alginate solutions were subjected to various heat sterilization treatments. Sodium alginate powder was also treated by both y-irradiation and ethylene oxide sterilization. The effects of these treatments on the viscosities of sodium alginate solutions and both the diameter and strength of the beads formed in 0.1 M CaC1, solutions were determined quantitatively. The viscosity of sodium alginate solutions and the gel strength of the calcium alginate beads decreased with increasing sterilization temperature while the bead diameters were found to increase. All these effects can be attributable to a reduction in the degree of polymerization of the alginate molecules as a result of the heat treatments. Ethylene oxide and y-irradiation treatments caused similar effects. Standard conditions for sterilization are necessary for comparative studies with alginate beads.
Introduction Alginate gels have been widely used to immobilize microbial, plant, and animal cells. This has imposed a need for sterile alginate gels that remain stable during longterm use in biochemical reactors. Within this laboratory, Penicillium chrysogenum entrapped in calcium alginate gel is being used to examine the effect of immobilization on microbial growth. In the course of this work, it emerged that degradation of the alginate molecules was occurring during sterilization. Elevated temperatures are known to cause depolymerization of alginates (1). Changes in the degree of polymerization (DP) of alginate will modify the properties of the gel it forms, e.g., mechanical strength and abrasion resistance, diffusion within the gel, and development of entrapped biomass. In order to make useful comparisons between the results of different workers studying cells immobilized in organic gels, careful consideration must be given to the effect of sterilization on the properties of the immobilization matrix. In this paper, the effects of different types of standard sterilization treatments on sodium alginate solutions are reported. The viscosities of sodium alginate solutions of known composition were measured after exposure to various temperatures for a fixed time. The size and compression strength of the beads formed from these solutions were then determined. Comparative results for ethylene oxide and y-radiation treatments on alginate are also presented. Materials and Methods Alginate Bead Preparation. Sodium alginate (Laboratory Reagent Grade, 65% guluronic acid, 35% mannuronic acid) and calcium chloride dihydrate (Analar Grade) were obtained from BDH Chemicals,Ltd., Poole, England. Sodium alginate solutions were prepared at ambient temperature with water purified by reverse osmosis. The water was stirred at high speed and alginate dusted onto the vortex. Spherical gel beads were prepared by extruding the alginate solution through a tube (i.d. = 0.62 mm) Department of Industrial Microbiology.
* Department of Chemical Engineering.
into a 0.1 M CaC1,-2H,O solution at ambient temperature from a height of 60 mm. Beads were allowed to harden for 16 h in the gently stirred CaC1, solution. Sodium alginate solutions were prepared in 100-mL lots at a standard working concentration of 3% (w/v) unless otherwise stated. Measurement of Viscosity, Gel Particle Diameter, and Strength. The viscosities of sodium alignate solutions were measured by using a cone/plate viscometer (Wells-Brookfield, Stoughton, MA) fitted with cone No. CP-40 accepting 0.5-mL samples. Measurements were made at 20 O C . Shear rates were in the range 2.3-45 s-l as appropriate. Bead diameters were determined by direct microscopic measurement of 35 particles. The critical compression force (CCF) required to rupture the beads was determined by a tensile testing machine, Type T5001 (J. J. Lloyd Instruments, Ltd., Southampton, England). Individual wet gel beads were compressed with a 1000 N load cell at a crosshead speed of 0.33 mm s-'. The CCF was calculated from the mean of nine replicates. Sterilization of Sodium Alginate. The effect of sterilization on sodium alginate solutions was determined by autoclaving 1% and 3% (w/v) solutions at 121 "C for 20 min and measuring their viscosity at 20 "C. The temperature sensitivity of sodium alginate solutions was determined to assess the potential benefits of a less severe sterilization treatment. The 50-mL lots of 3% (w/v) sodium alginate were heated for 15 min at each of the following temperatures: 20, 60, 80, 100, 116, and 121 OC. The viscosity of the sodium alginate solution subjected to each treatment was measured. The diameter and CCF of the calcium alginate gel particles formed from each solution were also determined. Three methods of sterilizing sodium alginate were compared t o assess their effect on the material; i.e., (1)sodium alginate solutions were sterilized by saturated steam in an autoclave at 121 "C for 15 min; (2) a 50-g lot of dry sodium alginate powder was treated with a standard ethylene oxide sterilization cycle involving exposure for a period of 7 h at 57 "C to an ethylene oxide concentration fo 560 mg/L; and (3) a 50-g amount of dry sodium alginate powder was also sterilized by y-radiation from
8756-7938/90/3006-0051$02.50/0 0 1990 American Chemical Society and American Institute of Chemical Engineers
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Table I. Effect of Autoclaving a t 121 O C for 20 min on the Viscosity of Sodium Alginate Solutions sodium alginate concentration, '70 (w/v) 1.0 3.0 viscosity of solution at 20 O C , Pa s before autoclaving 0.054 1.028 after autoclaving 0.012 0.137
a cobalt-60 source (a standard sterilization dose of 23.15 kGy (2.32 Mrad) was applied at a dosage rate of 14.4 kGy/h (1.44 Mrad/h). The viscosities of 3% (w/v) sodium alginate solutions prepared with ethylene oxide treated with y-irradiated sodium alginate were compared to that of a similar solution sterilized by autoclaving (121 "C, 15 min). The diameter and CCF of the resulting gel particles were then measured.
Results and Discussion Effect of Autoclaving on Alginate. Table I shows the effect of autoclaving on the viscosity of sodium alginate solutions of different concentrations. A 78% decrease in the viscosity of 1 % alginate solution was recorded, compared to a reduction of at least 86% for 3% alginate. The viscosity data clearly demonstrate a decrease in the degree of polymerization of the alginate molecules. McDowell (I) and Ohlson et al. (2) found that solutions of all alginates undergo a decrease in viscosity at elevated temperatures due to a degree of depolymerization. This is particularly true of high molecular weight alginates. It is also clear that the degree to which molecular weight is affected by autoclaving is dependent on the concentration of the sodium alginate solution. Effect of Heat Treatments on Alginate. The effect of less severe heat treatments on the alginate are summarized in Figures 1-3. Figure 1 shows the effect of 15min exposure at various temperatures on the viscosity of sodium alginate solutions. The viscosity of alginate solutions remains relatively constant after treatment at 20,60, and 80 "C in the range 0.75-0.84 Pa s. Above 80 "C, a marked decrease in solution viscosity is observed. Figures 2 and 3 show the diameter and CCF of the calcium alginate gel particles formed from these heattreated sodium alginate solutions. The decline in alginate solution viscosity is accompanied by a gradual increase in the diameter of the gelled beads and by a reduction in particle strength. The results highlight the sensitivity of alginate to temperatures above 100 "C. A marked level of depolymerization occurred above this point. Sterilization for shorter time periods or at lower temperatures such as those used by Ketel et al. (3) and Eikmeier et al. ( 4 ) would improve the mechanical strength of the gel particles formed. The greater bead diameter at higher temperatures is probably due to the formation of a more open gel structure that resulted from the cross-linking of shorter polymer chains. The formation of a gel matrix with a greater porosity should lead to increased nutrient and product diffusion rates within the particle. This increase in gel porosity should also lead to more rapid development of entrapped biomass. The growth of P. chrysogenum within gel particles typically results in a nonuniform distribution of biomass and a tendency toward growth at the particle surface. Changes in porosity and mass-transfer resistance within the gel due to sterilization may modify this pattern of growth. However, the lower gel strength and higher microbial growth rates increase the likelihood of
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Figure 1. Effect of temperature (15-min exposure time) on the viscosity of 3% (w/v) sodium alginate solutions. 4.0,
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F i g u r e 2. Effect of temperature (15-min exposure time) on the diameter of calcium alginate gel beads (prepared from 3% (w/v) sodium alginate solution).
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F i g u r e 3. Effect of temperature (15-min exposure time) on the mechanical strength of calcium alginate gel beads (prepared from 3% (w/v) sodium alginate solution).
an outgrowth of biomass from the particle. This results in the growth of free cells in the suspending medium and product stream and negates many of the benefits that should accrue from the immobilization process. Effect of Alternative Sterilization Treatments. Since steam sterilization caused a significant deterioration in gel strength, alternative methods of sterilizing the alginate were examined. Table I1 summarizes the comparative results for autoclaving, ethylene oxide treat-
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Table 11. Effect of Sterilization Treatments (A-D)' on the Viscosity of 3% (w/v) Sodium Alginate Solution and on the Properties of the Gel Particles Formed property viscosity at 20 'C, Pa s gel particle diameter, mm critical compression force (CCF), N
C D 0.33 0.035 2.79 (*0.05) 2.90 (k0.05) 11.9 (f0.7) 3.5 (i0.7) a Column A corresponds to the control, B to sterilization by autoclaving, C to ethylene oxide treatment, and D to y-irradiation. A
0.83 2.65 (*0.05) 13.5 (i0.7)
ment, and y-radiation on both sodium alginate and calcium alginate beads formed from these solutions. All of the alginate samples yielded firm gel particles of consistent shape except for those undergoing y-irradiation. The beads formed in this case tended to be weak with an extended "tail". Solution viscosities were all significantly different. The viscosity of irradiated sodium alginate solution was markedly lower than unsterilized sodium alginate solution. The CCF and particle diameter displayed a similar trend. None of the sterilization procedures used preserved the DP of the untreated alginate. y-Irradiation caused serious depolymerization of the polysaccharide as indicated by the viscosity decrease. Antoni (5) states that irradiation of polysaccharides can split the oxygen bridges within the molecule. Similarly, a decrease in the mean molecular weight of dextran molecules was also observed after exposure to 5-50 kGy (0.5-5.0 Mrad), indicating degradation (6). The difference between the standard autoclaving and ethylene oxide treatments was marginal but suggested that slightly less polymer damage resulted from the former treatment. The mechanism by which ethylene oxide reduced solution viscosity is not clear. Ethylene oxide acts as an alkylating agent, adding -CH,CH,OH groups in place of labile hydrogen atoms in reactive groups, e.g., -COOH, -SH, -NH,, -OH. Combination with acid residues gives rise to ethylene glycol esters. Alginate esterified with propylene glycol has been examined under alkaline conditions (1). Hydrolysis of the ester occurs with some degradation of alginate by @-elimination. Since both methods proved to be similar with regard to the integrity of the alginate polymer, doubts over the safety of ethylene oxide sterilization would favor autoclaving for many applications in the food, pharmaceutical, and related industries.
B 0.36 2.61 (*0.05) 11.1 (i0.7)
Conclusions Heat treatments cause a significant decrease in the degree of polymerization of alginate and possibly of other organic gelling agents. Sterilization by standard cycles of autoclaving, ethylene oxide treatment, and y-irradiation all caused a reduction in gel strength. Autoclaving at lower temperatures reduced the loss of gel stability. Calcium alginate gels are being widely used for cell immobilization and are subjected to a variety of sterilization regimes. Sterilization modifies the gel structure with consequences for mass transfer, cell growth, and product formation. Comparisons between the data derived from immobilized cell systems should therefore take into account the influence of sterilization on the gel matrix. Ideally, an effort should be made to standardize the sterilization treatments applied.
Literature Cited (1) McDowell, R. H. Properties of Alginates; Alginate Industries: London, 1977. (2) Ohlson, S.; Larsson, P. 0.;Mosbach, K. Eur. J . Appl. Microbiol. Biotechnol. 1978, 7, 103. (3) Ketel, D. H.; Hulst, A. C.; Gruppen, H.; Breteler, H.; Tramper, J. Enzyme Microb. Technol. 1987, 9, 303. (4) Eikmeier, H.; Westmeier, F.; Rehm, H. J. Appl. Microbiol. Biotechnol. 1984, 19, 53. (5) Antoni, F. Manual on Radiation Sterilization of Medical and Biological Materials; IAEA Report Series No. 149; International Atomic Energy Commission: Vienna, 1973. (6) Jacobs, G. P. Radiat. Phys. Chem. 1985,26(2), 133.
Accepted October 26, 1989. Registry No. Sodium alginate, 9005-38-3; calcium alginate, 9005-35-0; ethylene oxide, 75-21-8.
