World Journal of Microbiology & Biotechnology (2005) 21:723728 DOI 10.

1007/s11274-004-4798-0

Springer 2005

Micro-encapsulation of Bidobacterium lactis for incorporation into soft foods

Keywords: Beverages, Bidobacterium lactis, microencapsulation, probiotics, rheology

Summary Micro-encapsulation of the probiotic micro-organism Bidobacterium lactis isolated from a bio-yoghurt starter culture, was carried out using a mixture of hydrated gellan and xanthan gums. Rheological studies showed that the gum mix was suitable for encapsulation of B. lactis, for incorporation into soft foods/beverages. The gel behaved as a non-Newtonian material, and the ow curve tted well to the HerschelBulkley model. The average yield stress of the gum was 1.515 Pa, indicating gum stability, and the yield stress range was 1 Pa over a temperature range of 3550 C. Almost constant minimum gum viscosity occurred between 46 and 61 C. Oval/round capsules were synthesized manually using a monoaxial gum ow through a 27.5 G bevelled needle, together with a superposed air stream (air knife technique). The diameter of the capsules, measured using laser diractometry, varied from 20 to 2200lm, with 50% of the capsules having a diameter of 637 lm. Numbers of viable B. lactis in the capsules were estimated using high power ultrasound (20 kHz). By using a concentrated inoculum of B. lactis, microcapsules containing log10 1112 c.f.u. g)1 were synthesized. Apart from the anaerobic culturing of B. lactis, all other work was done in the presence of atmospheric oxygen. The organism exhibited a high degree of oxygen tolerance. A 21-day survival study of immobilized cells in 1 M sodium phosphate buer (pH 7) stored at either 4 or 22 C indicated that B. lactis survived in excess of log10 11 c.f.u. g)1 microcapsule. This technique represents a suitable means of supplying viable probiotics to the food and/or pharmaceutical industries. Mathematical symbols HerschelBulkley model: s0 sy jn y s0 yield stress sy shear stress jy uid consistency index n ow behaviour index

Introduction The consumption of foods containing probiotic organisms is becoming increasingly popular. Probiotic micro-organisms, generally bacteria, are dened as micro-organisms which when consumed in certain numbers, exert health benets beyond nutrition (Lactic Acid Bacteria Industrial Platform, LABIP, cited by Guarner & Schaafsma 1998). One group of organisms gaining credibility as probiotics is the genus Bidobacterium. The signicance and development of probiotic foods, and their use as alternatives to antibiotics, is predicted to increase in the future (Doyle 2003; Kroger 2003). In the European Union, consumption of probiotic foods increased 40% during the rst quarter of 2004 (Ross

2004). However, the provision and supply of foods containing viable Bidobacterium in the numbers required for a probiotic organism to be benecial (RDA log10 8 c.f.u.), has long been recognized to be problematic (Hughes & Hoover 1991; Klaver et al. 1993; Temmerman et al. 2003). Two technologies used to supply Bidobacterium in foods are freeze- and spraydrying. Although improvements in these methods have been reported (Siuta-Cruce & Goulet 2001), the use of spray- and freeze-drying of bidobacterium for incorporation into foods for human consumption has shown that both the Bidobacterium spp. and the carrier medium used in process inuence survival of the organism (Lian et al. 2002). Bidobacterium cells undergoing these treatments lose viability (Klaver et al. 1993; Lian et al. 2002). In addition, microbial injury during

724 both of these processes is common, and injured cells require time for repair in a suitable recovery medium (Ray 2001). Bacteria supplied in foods as both freezeand spray-dried cells require time to rehydrate and reconstitute in the human host. The human gastrointestinal tract (GIT) is not an ideal environment for resuscitation of injured bacteria; hence it is likely that Bidobacterium present as spray- or freeze-dried cells in probiotic foods do not all respond optimally under conditions associated with the gastro-intestinal tract. An alternate means of delivering living bidobacteria directly to the GIT is through the use of micro-capsules. Unlike the freeze- and spray-drying processes where the dormant micro-organism is delivered directly into the environment, the capsule matrix surrounding the bacteria protects the viable organisms from the gastric juices of the upper gastro-intestinal tract, and once in the colon, capsules break down to release the living organisms over a period of time (Krasaekoopt et al. 2003). Micro-encapsulation of probiotic bidobacteria is reported in some cases to improve survival of the organism in various foods. Edible gums such as alginate, locust bean gum, j-carrageenan, xanthan and gellan are often used to immobilize bacterial cells (Sun & Griths 2000; Siuta-Crus & Goulet 2001; Kailasapathy 2002; Truelstrup-Hansen et al. 2002; Krasaekoopt et al. 2003). These gums form a soft microcapsule, suitable for inclusion into foods. The aim of this investigation was to provide microcapsules of suitable dimensions synthesized from edible gums, in which a high concentration of living Bidobacterium was present, for incorporation into soft foods and beverages. To achieve this, we undertook a preliminary rheological study of the gum/s of choice before proceeding with micro-encapsulation. We also wished to establish the best method for synthesising micro-capsules with a view to upgrading production to an industrial scale. For technological reasons, it was important to establish oxygen tolerance of the selected Bidobacterium spp. A further objective was to establish the viability of immobilized cells when stored in a buer, as a possible means of supplying the microcapsules to industry for incorporation into foods.

L.D. McMaster et al. Agar (1.5% (w/v) (Biolab) was added for solid media. Bidobacterium lactis was isolated from a yoghurt starter culture as described by Trindade et al. (2003). Rheology of the gum mix Based on work done elsewhere, gellan and xanthan were selected as the gums of choice, and were used as a mixture of 0.75% w/v gellan and 1% w/v xanthan (Sun & Grifths 2000). Immediately prior to the rheological studies, the gums were prepared and re-hydrated as described below. A Paar Physica MCR 300 rotational rheometer with a cone-plate measuring system was used for the gellan:xanthan gum mix to determine a ow curve in controlled shear rate mode, at shear rates of 0.1100 s)1, and a temperature sweep at a constant shear rate of 5 s)1 between 30 and 60 C. In addition, a time dependency test, at a constant shear rate of 5 s)1 at temperatures of 30, 40 and 45 C was done. Micro-encapsulation of B. lactis A variation of the method proposed by Sun & Grifths (2000) was used. Gellan (0.1 g), (Sigma) and xanthan (0.2 g) (Sigma) were added to 20 ml distilled water. The solution was mixed using a magnetic stirrer (Ikamag) and heated to 80 C for 1 h. The gel mix was then autoclaved at 121 C for 15 min. B. lactis was grown in a Bactron 1.5 anaerobic chamber at 37 C overnight in 250 ml TYG broth to an OD600 0.91.1. All subsequent procedures were done under aerobic conditions. Cells were harvested by centrifugation at 8000 g for 10 min in a Beckman J21 centrifuge at 4 C. The pellet of cells was washed three times by resuspension in 20 ml aliquots of sterile distilled water, followed by centrifugation. Finally, the cells were suspended in sterile distilled water, to give a volume of 2.5 ml. One ml of this concentrate was mixed into 20 ml sterile gellan:xanthan gum at 55 C. Microcapsules were generated by a bead entrapment method, using a superposed airow together with mono-axial extrusion of the gum/bacteria mixture (Huebner & Buccholz 1999; Park & Chang 2000). The gum/cell mix was manually extruded through a 27.5 G bevelled needle (Teruma) tted on to a sterile 20 ml syringe (Promex). Gel and sterile air ow rates were 10 ml min)1 and 250 ml s)1 respectively. Microdroplets formed by this method were hardened into spheres by free fall into a sterile 0.1 M CaCl2 solution (100 ml). After 1 h, beads were separated from the solution by aseptic ltration through sterile Whatman lter paper No 1. Microcapsules remaining on the lter paper were washed thoroughly with sterile 0.1 M CaCl2. All procedures were carried out in a laminar ow hood. Estimation of microcapsule size The diameter and size distribution of microcapsules containing B. lactis were estimated by laser

Materials and methods Bacterial strains and culture conditions Bacterial cultures were propagated anaerobically at 37 C in a Bactron 1.5 anaerobic chamber (Shel Lab) at 37 C in an environment consisting of hydrogen (4%), carbon dioxide (10%) and nitrogen (86%). Cells were grown in tryptone yeast glucose (TYG) media which contained the following, in g l)1: tryptone 10 (Biolab); yeast extract, 5 (Biolab); glucose, 5 (Saarchem); Tween 80, 1.0 (Sigma); NaCl, 4.5; KCl, 0.25; MgCl26H2O, 0.15; KH2PO4, 0.4; K2HPO4, 0.2; NH4Cl, 0.4 (Saarchem); cystein HCl, 0.5 (Sigma); rezasurin, 0.001% (Sigma).

Micro-encapsulation of B. lactis diractometry using a Malvern Mastersizer S, version 2.19. The analysis model used was Polydisperse, and the presentation 30HD standard wet for use with the Mie theory. Enumeration of B. lactis To estimate numbers of B. lactis in microcapsules, 0.1 0.2 g of capsules were weighed into 10 ml of 1 M sodium phosphate buer (NaH2PO4 46.3 g l)1, NaH2PO4 53.7 g l)1), pH 6.8. At predetermined time intervals, the mix was subjected to sonication to release B. lactis cells from the gel matrix. A Vibracell Ultrasonic Processor VCX750 (20 kHz) (Materials and Sonics) was used with a standard probe, with a tip diameter of 13 mm generating a sound wave amplitude of 105 lm for 15 s. All samples were treated in 20 ml sterile cylindrical Polytop glass cylinders immersed in an ice water bath. After completion of the treatment, to estimate the number of released B. lactis, standard serial dilution and plate counts were done. Cells were diluted in 2% buered peptone water (Difco). Representative 0.1 ml volumes from dilutions were spread in triplicate/dilution onto TYG agar plates. These were incubated anaerobically at 37 C for 48 h, and colonies were counted. Values obtained were expressed as either log10 c.f.u. g)1 or ml)1 or as log surviving fraction. The log surviving fraction was calculated as Nt/N0, where N0 and Nt are the average number (N) of c.f.u. ml )1 (free cells) or g)1 (microcapsules) at the start of the experiment (0), and at time (t) respectively. Survival of immobilized B. lactis in 1 M sodium phosphate buer (pH 6.8) Sodium phosphate buffer (1 M) pH 6.8 was prepared as described. Known weights (0.2 g) of entrapped B. lactis were added to 10 ml of sterile buer. The solution was stored aerobically at either 4 or 22 C for 21 days. Viable numbers of entrapped B. lactis were determined using the Vibracell Ultrasonic Processor VCX 750 followed by serial plate dilution. Rheology of the gellanxanthan gum mix

725

We elected to use gellan (0.75%) and xanthan (1%) for immobilizing materials as a combination of these gums has been reported to have better technical properties for micro-encapsulation than do alginate, j-carrageenan and locust bean gums (Sun & Grifths 2000). Although alginate is frequently used to microencapsulate probiotics, it has undesirable attributes, such as susceptibility to degradation by acids (Sun & Grifths 2000; Truelstrup-Hansen et al. 2002; Krasaekoopt et al. 2003). Hence alginate micro-capsules are likely to erode in the hydrochloric acid of the stomach and not reach the colon intact. Rheological studies used to determine the shear stress data of the gellan:xanthan gum indicated that the gel behaved as a non-Newtonian material, and the ow curve tted well to the HerschelBulkley model s0 sy)k)n, which is used to describe the ow behavy iour of a yield pseudoplastic material (Figure 1). For microencapsulation, Newtonian liquids such as water or honey are unsuitable as encapsulating materials, as liquid droplets, not solid capsules, are formed during extrusion. In order for successful encapsulation of micro-organisms to occur, the selected material should exhibit non-Newtonian properties i.e. be a relatively viscous material with solid properties. The data reported here indicated that the gum mix of gellan and xanthan satised this requirement. The apparent yield stress calculated for the gellan xanthan mix indicated that the gums would only ow or deform plastically when external forces were greater than the internal structural forces inherent in the gum. The average yield stress of the gum mix was 1.515 Pa, a value similar to that of raw meat batter (Steffe 1996). This value indicated that the gum mix was stable and that at lower stresses, the gel would behave as a solid. The yieldstress range was approximately 1 Pa over a temperature range of 3550 C (Figure 2). Any material selected for micro-encapsulation should be able to traverse the upper gastro-intestinal tract (GIT) intact to protect the bacteria, but on arrival in the colon, the yield stress of the gum should be such that the immobilized bacteria are released into the human host.

Results and discussion Bacterial strain isolated Using an AB yoghurt starter culture, Bidobacterium lactis was isolated and used for all subsequent studies (Trindade et al. 2003). B. lactis was chosen as the probiotic organism for encapsulation, as it is commonly found in yoghurt starter cultures. The organism possesses attributes important in selecting a probiotic for commercial use. These include oxygen tolerance reported for up to 24 h exposure, fermentation of a wide variety of carbohydrates (Trindade et al. 2003) and survival at pH 23 (Truelstrup-Hansen et al. 2002).
Shear stress (Pa)
30 25 20 15 10 5 0 0 20 40 60 80 100 120

Shear rate (s-1)

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7 6

L.D. McMaster et al. such as 13 C. The latter values would require critical temperature control to maintain a constant shear viscosity immediately prior to the extrusion process during which microdroplets are formed. Maintenance of a constant minimum shear viscosity would enhance control over microcapsule size and shape, as well as permit optimization/standardization of mixing procedures necessary for even dispersal of B. lactis in the viscous gum held at these temperatures, prior to extrusion.
0 50 100 150 200 250 300 350

Viscosity (Pa)

5 4 3 2 1 0

Micro-encapsulation Using the methods described above, micro-encapsulation of B. lactis in the gellanxanthan matrix was successful. Estimation of viable c.f.u. in the capsules over periods of time indicated that oxygen present in the superposed airow during microencapsulation was not lethal to B. lactis (data not shown). Visual examination of microcapsules using a light microscope indicated that capsules were generally oval/ round in shape, with a dense core comprised of B. lactis cells (Figure 4). When the capsules were broken open and stained by the Gram method, bacterial cells isolated from the core stained gram positive and demonstrated a typical bidus morphology. The shape of the capsules was variable. Using the manual air knife technique described, we were unable to generate a constant capsule shape. Although the irregularity of microcapsule shape often reported for probiotics immobilized in edible gums is related to process, this problem requires further elucidation, as microcapsule shape could inuence both viability and numbers of entrapped bacteria. Shape will also determine ow properties of the capsules, important for industrial process. The use of a constant temperature and gum

Time (s)
Figure 2. Time sweep of shear viscosity at 5 s)1 at dierent temperatures for the gum. Temperature range: 30 C (s); 40 C ()); 45 C (*).

The immobilizing material should not be comprised of materials with a high yield stress, as probiotics encapsulated in such a material would not be released. The value obtained in this study for the gellanxanthan gum mix indicated that stresses associated with GIT movements/peristalsis, could release the bacteria in the colon. The average yield stress of the gum indicated that gellan:xanthan would behave as a solid under low mechanical stress conditions, but would easily be broken by shear stresses such as those associated with chewing. Therefore B. lactis enclosed in a gellan:xanthan microcapsule would have to be supplied in soft foods not requiring mastication, to prevent breaking of the capsule in the mouth. The yield stress value is also important when considering the means of addition of the capsules to foods such that the capsules remain intact and are not disrupted during process. The shear viscosity of the gum mix maintained an almost constant minimum value at temperatures between 46 and 61 C (Figures 2 and 3). These temperatures are within the range for survival of Bidobacterium. It is recommended that encapsulation using gellan:xanthan as the support matrix should be done between 46 and 61 C. For industrial application of micro-encapsulation it is of importance that the shear viscosity is constant over a broad 15 C range, as opposed to a limited range
4.5 4 3.5

Viscosity (Pa)

3 2.5 2 1.5 1 0.5 0 26 31 36 41 46 51 56 61

Temperature (C)
Figure 3. Shear viscosity of the aqueous gum mix at 5 s)1, as a function of temperature. Figure 4. Phase contrast view of a single capsule ( 500), with a densely packed core of B. lactis surrounded by the capsule support material.

Micro-encapsulation of B. lactis ow rate during extrusion, not possible in our encapsulation technique, should improve uniformity of shape. Despite having a constant rate of air ow, a wide variation in the size distribution of the capsules was noted. This ranged from a maximum diameter of 2200 lm to a minimum of 20 lm, with 50% of the capsules having a diameter of <637 lm, measured using laser diractometry (Table 1). Variables, including temperature, gum- and air ow rates, needle gauge etc, will inuence capsule diameter. Although we commenced with a constant temperature of 55 C (i.e. minimum gum viscosity), we were unable to maintain a constant temperature immediately prior to or during extrusion. Hence gum ow rate, as inuenced by viscosity, varied. For industrial production of capsules, a uniform bead size is important. Data obtained from the rheology of the gum mix, will assist in attaining this. Sun & Griths (2000), using B. infantis, reported an average gellan:xanthan bead diameter of 3 mm produced by a simple dropping of the mixture through a syringe with a 21 G syringe needle. Krasaekoopt et al. (2003), reported an average diameter of 25 mm for capsules synthesized by extrusion. By using a smaller gauge needle with a bevelled tip, and a superposed airow, we were able to reduce the average size of gellan:xanthan beads by 80%. Measurement of particle size using laser diractometry identied capsules with a diameter as small as 20 lm. Adikhari et al. (2003), using co-acervation/emulsion to micro-encapsulate B. longum B6 in j-carrageenan, reported capsule diameters of 22350 lm, measured using laser diractometry. It is generally recognized that capsules generated by co-acervation are smaller than those manufactured by extrusion. However, there are disadvantages associated with co-acervation. These
Table 1. A single analysis of microcapsule diameter and size distribution as determined for 0.2 g capsules (wet weight) using a Malvern Mastersizer S. Diam Volume Diam (lm) (%) (lm) 4.88 5.69 6.63 7.72 9.00 10.48 12.21 14.22 16.57 19.31 22.49 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.02 0.03 26.20 30.53 35.56 41.43 48.27 56.23 65.51 76.32 88.91 103.58 120.67 Volume Diam (%) (lm) 0.05 0.08 0.12 0.18 0.25 0.35 0.47 0.62 0.78 0.97 1.18 140.58 163.77 190.80 222.28 258.95 301.68 351.46 409.45 477.01 555.71 647.41 Volume Diam (%) (lm) 1.42 1.71 2.06 2.49 3.04 3.71 4.52 5.42 6.33 7.16 7.85 754.23 878.67 1023.66 1192.56 1389.33 1618.57 1885.64 2196.77 2559.23 2981.51 3473.45 Volume (%) 8.37 8.81 8.38 7.47 6.17 4.70 3.22 1.75 0.27 0.00 0.00

727 include the undesirable trapping of the emulsifying oil in the microcapsule, which can alter food avour and reduce bacterial viability. In addition, co-acervation is costly, and bacterial numbers in these capsules is low and random (Krasaekoopt et al. 2003). Diameter of microcapsules is inuenced by many variables, and obtaining a uniform size of capsules within a batch when operating under non-standard conditions is dicult. To manufacture capsules of both equal and optimum diameter will be complex, but necessary for successful incorporation into foods to minimize problems associated with cell viability and food texture. Little has been reported with regard to optimum capsule size for addition to foods. It is known that particles with a diameter below 3 lm are undetected by the tongue (Singer & Dunn 1990). Therefore, above this value, capsules can impart a gritty texture to foods not normally associated with this sensation. Adikhari et al. (2003) reported that when microcapsules were added to yoghurt at 10% (w/v), there was an unfavourable consumer response due to the resultant grainy texture. The 10% content of encapsulated B. longum was necessary to provide the RDA of the organism. Ideally, a uniform capsule diameter is such that sensory qualities of the food are not altered, whilst simultaneously delivering therapeutic doses of probiotic. The diameter must not adversely aect viability of the organism. TruelstrupHansen et al. (2002) reported that alginate capsules should have a diameter of at least 100 lm to prevent a reduction in Bidobacterium viability in simulated acidic gastric juices. With milk as the surrounding medium, this was not noted. Hence determination of optimum uniform capsule size is complex. The optimum/ideal size of gellan:xanthan microcapsules is unknown, but we have demonstrated that it is possible to generate capsules of dierent diameters, and to based on results obtained, we are currently developing a novel temperature-controlled procedure, without superposed airow, to produce microcapsules of 100200 lm diameter. We intend to extend rheology studies to these capsules to gain insight into storage conditions which inuence gum integrity, and therefore shear strength of the capsules. Enumeration of B. lactis in the capsules The use of the Vibracell VCX 750 to release viable cells from the immobilizing matrix was successful. The method is reproducible, rapid and there was no detectable reduction in c.f.u. often reported for methods requiring lengthy extraction procedures for entrapped cells (Sun & Grifths 2000; Truelstrup-Hansen et al. 2002; Adikhari et al. 2003). Using concentrated washed B. lactis cells in the stationary phase of growth, with the microencapsulation technique described, we consistently achieved a high loading of log10 1012 c.f.u. B. lactis g)1 microcapsules (results not shown). Hence to consume the RDA of B. lactis required 0.1 g microcapsules as a single serving. This amount would exert minimum eect on the texture of a soft food or beverage.

D (v, 0.1) 186.1; D (v, 0.5) 637.00; D (v, 0.9) 1387.15. D [3, 2] 367.71; D[4, 3] 658.76. D (v, 0.1) 10% of the microcapsules were below 186.1 lm in diameter. D (v, 0.5) 50% of the microcapsules were below 637.00 lm in diameter. D (v, 0.9) 90% of the microcapsules were below 1387.15 lm in diameter. D [3, 2] surface area mean diameter of the sphere having the same surface area as the real particle.

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10
1

L.D. McMaster et al. with Malvern diffractometry analyses. We are grateful to Ms L. Francis, Dept of Civil Engineering, Cape Technikon, for rheological analyses. Financial assistance from the National Research Foundation and Cape Technikon Seed Research Fund is gratefully acknowledged.

Log surviving fraction g-1

10

References
Adikhari, K., Mustapha, A. & Grun, I.U. 2003 Survival and metabolic activity of microencapsulated Bidobacterium in stirred yoghurt. Journal of Food Science 68, 275280. Doyle, M.P. 2003 Food research trends 2003 and beyond. Food Technology 56, 4445. Guarner, F., & Schaafsma, G.J. 1998 Probiotics. International Journal of Food Microbiology 39, 237238. Huebner, H. & Buccholz, R. 1999 Microencapsulation. In The Encyclopedia of Bioprocessing Technology; Fermentation, Biocatalysis and Bioseparation, vol 4. eds. Flickinger, M.C. & Drew, S. pp. 17851798. New York: John Wiley and Sons. Inc. ISBN 0-47113822-3. Hughes, D.B. & Hoover, D. G. 1991 Bidobacteria: their potential for use in American dairy products. Food Technology 45, 7483. Kailasapathy, K. 2002 Microencapsulation of probiotic bacteria: technology and potential applications. Current Issues of Interest in Microbiology 3, 3948. Klaver, A.M., Kingsma, F. & Weerkamp, A.H. 1993 Growth and survival of bidobacteria in milk. Netherlands Milk Dairy Journal 47, 151164. Krasaekoopt, W., Bhandari, B. & Deeth, H. 2003 Evaluation of techniques of probiotics for yoghurt. International Dairy Journal 13, 313. Kroger, M. 2003 Food research trends 2003 and beyond. Food Technology 56, 3637. Lian, W.-C., Hung-Chi, H. & Chou, C.-C. 2002 Survival of bidobacteria after spray-drying. International Journal of Food Microbiology 74, 7986. Park, J.K. & Chang, H.N. 2000 Microencapsulation of microbial cells. Biotechnology Advances 18, 300319. Ray, B. 2001 Fundamental Food Microbiology, 2nd ed. Boca Raton: CRC Press. ISBN 0849300452. Ross, P. 2004 Overcoming the technological hurdles in the development of probiotic foods. Society for Applied Microbiology Dairy and Food Microbiology. Summer Conference 1215 July. Singer, N. S.& Dunn, J.M. 1990 Protein microparticulation process: the principle and the process. Journal of the American College of Nutrition 9, 388397. Siuta-Cruce, P. & Goulet, J. 2001 Improving probiotic survival rates. Food Technology 55, 3644. Steffe, J.F. 1996 Rheological Methods in Food Process, 2nd edn. Freeman Press. ISBN 0-9632036-1-4. Sun, W. & Grifths, M.W. 2000 Survival of bidobacteria in yoghurt and simulated gastric juice following immobilization in gellanxanthan beads. International Journal of Food Microbiology 61, 17 25. Temmerman, R., Pot, B., Huys, G. & Swings, J. 2003 Indentication and antibiotic susceptibility of bacterial isolates from probiotic products. International Journal of Food Microbiology 81, 110. Trindade, M.I., Abratt, V.R. & Reid, S.J. 2003 Induction of sucroseutilisation genes from Bidobacterium lactis by sucrose and ranose. Applied and Environmental Microbiology 69, 2432. Truelstrup-Hansen, L., Allan-Wotjas, P.M., Jin, Y.-L. & Paulson, A.T. 2002 Survival of Ca-alginate microencapsulated Bidobacterium spp. in milk and simulated gastrointestinal conditions. Food Microbiology 19, 3545.

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14

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Time (days)
Figure 5. Surviving fraction of immobilized B. lactis cells stored aerobically in 1 M sodium phosphate buer (pH 7.0) at 4 C, , or 22 C, n, over 21 days (data representative of two experiments). Average standard deviation log10 c.f.u. 0.15.

Survival of B. lactis in 1 M sodium phosphate buer (pH 6.8) Storage of immobilized B. lactis for 21 days in 1 M sodium phosphate buer under aerobic conditions indicated that loss of viability of the cells stored at 4 or 22 C was less than 0.5 log cycle at both temperatures (Figure 5). Survival was improved by storage at 4 C. At the commencement of the storage time, the number of B. lactis in the capsules was log10 11.6 c.f.u. g)1. Therefore after 21 days storage, the level of viable B. lactis in 0.1 g microcapsules remained in excess of the RDA. Storage of the microcapsules at 4 C in a sodium phosphate buer (pH 6.8) represents a possible means of supply of the capsules to the food industry. In conclusion, we report successful incorporation of high numbers of viable B. lactis in gellan xanthan microcapsules. Rheology of the gum mix provided valuable data for application to standardization of procedures for both small- and large-scale industrial production of microcapsules. Shear stress data showed that the type of foods/beverages suitable as delivery vehicles cannot undergo mastication prior to consumption. Such foods would include beverages as well as soft foods. B. lactis was not inactivated in the presence of oxygen, even after 21 days. As maintenance of anaerobic conditions, required for growth of the organism, is unlikely during both microencapsulation and subsequent industrial processes, this attribute is technologically essential.

Acknowledgements Thanks are due to Ms H. Divey, Dept Chemical Engineering, University of Cape Town for assistance