Incorporation of T4 bacteriophage in electrospun fibres

Authors


Correspondence

John Kadla, Department of Wood Science, Forest Sciences Centre, University of British Columbia, Vancouver, BC Canada V6T1Z4. E-mail: john.kadla@ubc.ca

Abstract

Aims

Antibacterial food packaging materials, such as bacteriophage-activated electrospun fibrous mats, may address concerns triggered by waves of bacterial food contamination. To address this, we investigated several efficient methods for incorporating T4 bacteriophage into electrospun fibrous mats.

Methods and Results

The incorporation of T4 bacteriophage using simple suspension electrospinning led to more than five orders of magnitude decrease in bacteriophage activity. To better maintain bacteriophage viability, emulsion electrospinning was developed where the T4 bacteriophage was pre-encapsulated in an alginate reservoir via an emulsification process and subsequently electrospun into fibres. This resulted in an increase in bacteriophage viability, but there was still two orders of magnitude drop in activity. Using a coaxial electrospinning process, full bacteriophage activity could be maintained. In this process, a core/shell fibre structure was formed with the T4 bacteriophage being directly incorporated into the fibre core. The core/shell fibre encapsulated bacteriophage exhibited full bacteriophage viability after storing for several weeks at +4°C.

Conclusions

Coaxial electrospinning was shown to be capable of encapsulating bacteriophages with high loading capacity, high viability and long storage time.

Significance and Impact of the Study

These results are significant in the context of controlling and preventing bacterial infections in perishable foods during storage.

Introduction

Bacterial-associated deaths from tainted meats have increased the need for better food packaging and bacteria prevention. In particular, the inner layer of the meat-packaging material that in contact with the food surface has become a source of concern in the food sector as there is a need to provide better food safety (Sorrentino et al. 2007; Chonticha et al. 2011). This layer needs to be engineered in such a way that it extends the shelf life of food and inhibits the growth of pathogens on the surface of meat. Electrospinning has been shown to be an effect process to incorporate bioactive components into fibre mats (Zheng-Ming et al. 2003; Ramakrishna et al. 2005; Andrady 2008). Electrospun nanofibrous materials are flexible, have high specific surface area and superior directional strength and ideal for packaging materials. Moreover, a sustained delivery of bioactive antimicrobial components can be achieved through the selection of suitable polymers (Andrady 2008). Thus, electrospun fibrous materials with microbial resistibility can be highly beneficial for food preservation.

Bacteriophages are viruses that can kill prokaryotes (Ackermann and DuBow 1987). Bacteriophages have been used for species-specific control of bacteria during different phases of food production and storage (Greer 2005). For example, bacteriophages are used to control the growth of pathogens such as Listeria monocytogenes in fruit and dairy products (Greer 2005), Escherichia coli in calves and lambs (Smith and Huggins 1983), Salmonella in broiler chickens (Atterbury et al. 2007) and Campylobacter in red meat (Goode et al. 2003). Typically, bacteriophages are either administered by oral gavage for infected animals or by a simple spraying technique for the treatment of surface infections (Yongsheng et al. 2008). In the surface treatment of meat, the early inoculation in the spraying technique results in deactivation of the bacteriophage; unlike conventional drugs, the pharmacokinetic principles of bacteriophages are quite different (Payne and Jansen 2003). Therefore, a delivery vehicle that can encapsulate bacteriophages in a viable form and release them in a sustained fashion is desirable.

Encapsulation of bacteriophages in the form of microcapsules and electrospun nanofibres have been investigated (Lee and Belcher 2004; Yongsheng et al. 2008). Of the two, nonwoven or three-dimensional network morphologies of electrospun fibres are more applicable for food packaging. In fact, bacteriophage incorporated electrospun nanofibres have been prepared using aqueous suspensions of M13 bacteriophage and polyvinyl pyrrolidone (Lee and Angela 2004). The bacteriophage-encapsulated fibres instantly released the M13 bacteriophage, which exhibited some activity, and were able to infect the bacterial host. However, the viability of the encapsulated M13 was never reported (Lee and Angela 2004). Similarly, the same suspension electrospinning process was used to investigate the encapsulation of T4, T7 and λ bacteriophages in polyvinyl alcohol (PVA) fibres (Salalha et al. 2006). Although exposure of bacteriophages to a PVA aqueous solution showed no effect on their viability, a very low activity (T4: 1%, T7: 2%, λ: 6%) was reported after release from the PVA fibres. The loss of activity was mainly attributed to the rapid dehydration of the bacteriophages and solvent evaporation during fibre formation (Salalha et al. 2006).

To overcome the sensitivity of bacteriophages to the electrospinning process and increase bacteriophage viability, we have investigated two different electrospinning processes: emulsion and coaxial electrospinning. Our aim is to ‘protect’ the bacteriophage from the harsh conditions of the electrospinning process by either pre-encapsulating and/or allocating the bacteriophage to the core of a core/shell fibre. The implications of the two different encapsulation processes on the efficacy of encapsulation and viability of T4 bacteriophage throughout fibre production are reported.

Materials and methods

Media preparation and bacterial strain

Tryptic soy agar (TSA) and Tryptic soy broth (TSB) were each prepared by dissolving 30 g of respective powder in 1 l of distilled water. Tryptic soy broth semisolid (TSBSS) was made by dissolving TSB (30 g) and agarose (4 g) in 1 l of distilled water. Storage media buffer (SM) for T4 bacteriophage storage was made by dissolving NaCl (5·8 g), MgSO4·7H2O (2 g), 1 mmol l−1 Tris–hydrochloride (pH 7·5, 50 ml) and 1 ml of 10% (w/v) gelatine in 1 l of distilled water. All the above reagents and media were purchased from Sigma–Aldrich. The prepared media and buffer were autoclaved prior to use. Escherichia coli bacteria (E. coli) ATCC# 11303 [The American Type Culutre Collection (ATCC), Rockville, MD, USA] and wild-type T4 bacteriophage were kindly provided by the laboratory of Professor Dr. Mansel Griffiths, University of Guelph, Canada.

Bacteriophage propagation and plaque assay

The bacterial host for T4 bacteriophage propagation was grown by dispersing 100 μl of E. coli in 5 ml of TSB media 37°C overnight under vigorous shaking. An aliquot of 100 μl of the overnight culture was then mixed with 100 μl of T4 bacteriophage stock solution. This mixture was incubated at 37°C for 20 min and was mixed with 4 ml of partially cooled TSBSS. The mixture was then poured onto a cooled TSA plate and incubated at 37°C overnight. Bacteriophage growth was identified as a clear transparent plate in comparison with the cloudy control plate (100 μl of E. coli). To recover the bacteriophages, 4 ml of SM buffer was added to the plates, which were incubated at 4°C for 1 h. The SM buffer containing bacteriophage and TSBSS was then centrifuged for 20 min at 6500 g at 4°C. The supernatant was then passed through a 0·45-μm sterile filter and the lytic activity of the bacteriophage was determined by plaque assay test.

In the plaque assay test, serial dilutions (10 times dilution) of the above-propagated T4 bacteriophage were first prepared. Aliquots (100 μl) for each dilution were added to an equal volume of E. coli host. Each mixture was then added to 4 ml of partially cooled TSBSS, poured onto a TSA plate and incubated at 37°C overnight. A negative control, bacterial host without bacteriophage was prepared in the same manner. Following incubation overnight, the number of plaques was counted for each dilution and used to calculate the number of plaque forming units (PFU). Bacteriophage activity was determined to be 108 PFU ml−1.

Emulsification process

The emulsification process involved dispersing an aqueous solution of the bioactive-agent/alginate in chloroform to form a water in oil emulsion system (w/o) (Hongxu et al. 2006; Yongsheng et al. 2008). First, freeze-dried T4 bacteriophage (dried from 2 ml of bacteriophage suspension) was suspended in 1 ml of 2% (w/v) sodium alginate. This solution was allowed to sit for 10 min at room temperature and then added dropwise to a sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/chloroform solution (~50 mg ml−1) under mechanical stirring at 3220 g at room temperature. It must be noted that AOT has a high calcium tolerance and does not precipitate upon introduction of calcium chloride. Calcium chloride (0·33 ml, 5% w/v) was then added to complete the encapsulation by in situ alginate crosslinking and three-dimensional gel network formation. This relatively mild gelation process yields stable alginate beads and enables the bacteriophage to retain maximum biological activity. This emulsion was found to be stable up to 30 min.

Emulsion electrospinning

The above-prepared emulsion system was added to a polyethylene oxide (PEO; Mv = 300 000)/chloroform solution (40 g l−1), to obtain a final polymer concentration of 2·0% (w/v). The prepared polymer emulsion system was then applied to electrospinning process, and it was carried out at 25°C using a vertical spinning apparatus and a 0·9-mm flat needle. The spinning voltage was set at 1 kV cm−1, and the distance between the spinneret and collector plate was 20 cm. The flow rate was set at 0·2 ml min−1 using a 1-ml syringe with a 5 mm internal diameter. The fibres were collected on a sterilized aluminium plate.

Coaxial electrospinning

Coaxial electrospinning was performed using a PEO solution (Mv = 300 000, 2·5% (w/v) in chloroform) to form the shell and T4 bacteriophage/buffer suspension (108 PFU ml−1) for the core. The electrostatic field used was 1 kV cm−1, and the distance between the spinneret and the collector plate was 20 cm. Two syringe pumps controlled the flow rates of both the core and the shell solutions: 0·01 ml min−1 for the shell and 0·001 ml min−1 for the core. The fibres were collected on a sterilized aluminium plate. The collected electrospun fibre was freeze-dried overnight to avoid fibre dissolution due to the presence of buffer in the core.

In vitro release

The bioactivity of the T4 bacteriophage after alginate pre-encapsulation was determined prior to electrospinning by removing the chloroform using rotary evaporation (25°C), followed by lypholization of the remaining water. The freeze-dried alginate/bacteriophage capsules (~0·5 g) were then suspended in 2 ml SM buffer (pH 7·5) under vigorous shaking at room temperature for 2 h. The corresponding emulsion and coaxial electrospun fibre mats (~0·5 g) were handled in the same manner (~0·5 g suspended in 2 ml SM buffer at pH = 7·5 and shaken at room temperature). In all of the release experiments, a 50 μl aliquot of the released T4 bacteriophage was removed from medium at predetermined time intervals and replaced with 50 μl of fresh SM buffer. The T4 bacteriophage activity was determined by the plaque assay and expressed as PFU ml−1.

Microscopy analysis

The morphology of the electrospun fibres were examined by scanning electron microscopy (SEM). Fibres were mounted on an aluminium stub, coated with a 5-nm gold layer and imaged using a Hitachi S-2600N SEM (Hitachi High Technologies America, Inc., Dallas, TX, USA) operating at 25 kV. For transmission electron microscopy (TEM) analysis, the specimens were prepared by direct deposition of the electrospun fibre onto a carbon film–coated copper grid. Images were collected using a Hitachi H-800 (Hitachi High Technologies America, Inc.) TEM at 200 kV without any sample staining. All fibre and alginate capsule diameters were obtained from a minimum of 100 measurements taken from multiple samples using Image J software (National Institute of Health, Bethesda, MD, USA) and reported as average diameters ± standard deviation.

Results

Suspension electrospinning of T4 bacteriophage

The electrospinning of T4 bacteriophage (2 ml) suspension from 10% (w/v) PEO solution resulted in uniform nanofibres with an average diameter of 500 ± 100 nm (Fig. 1a). TEM analysis of the resulting fibres revealed that the embedded T4 bacteriophages are nonuniformly distributed throughout the fibre matrix; not along the fibre nor from core to surface (Fig. 1b). The encapsulated T4 bacteriophage nanofibres were immediately dissolved when submerged in SM buffer, instantly releasing the T4 bacteriophage. However, the plaque assays showed a large drop in T4 bacteriophage activity from 108 to 103 PFU ml−1 (Table 1). To further improve the bacteriophage activity, we investigated the pre-encapsulation of the T4 bacteriophage and their incorporation into the core of the fibre prior to electrospinning.

Table 1. Lytic activity of T4 phage after each process of encapsulationa
T4 phage activityOriginal stockAfter freeze dryingAfter suspension E-spinningReleased from Ca-alginate capsule, after emulsificationReleased from Ca-alginate capsule, after E-sprayingReleased from fibre, after emulsion E-spinningReleased from fibre, after coaxial E-spinning
  1. E-spinning, electrospinning; E-spraying, electrospraying.

  2. a

    Based on results from at least three different batches.

PFU ml−1108108103107103106108
Figure 1.

(a) Scanning electron microscopy micrograph of polyethylene oxide (PEO) fibers after suspension electrospinning, (b) transmission electron microscopy (TEM) micrograph of a T4 bacteriophage, (c) TEM micrograph of plain PEO electrospun fibers, and (d) TEM micrograph of T4 bacteriophage incorporated in PEO electrospun fibers.

Pre-encapsulation using an emulsification process

In the emulsification process, the T4 bacteriophage is concentrated within an alginate capsule to protect them from the harsh electrospinning conditions. SEM images demonstrate that the alginate beads were of uniform size with smooth rounded external surfaces (Fig. 2a). Higher magnification did not reveal the presence of T4 bacteriophage or any components there from on the surface of or between alginate capsules. The average size of the alginate capsules after the emulsification process was found to be 800 ± 150 nm (Bhowmik et al. 2006). Such capsule sizes could readily be incorporated into electrospun fibres. To confirm the pre-encapsulation of T4 bacteriophages in alginate capsules, a lytic activity test was performed prior to emulsion electrospinning.

Figure 2.

Scanning electron microscopy images of (a) freeze-dried T4 bacteriophage/calcium-alginate capsules isolated after emulsification, (b) electrosprayed T4 bacteriophage/calcium-alginate emulsion, and (c) T4 bacteriophage/calcium-alginate/polyethylene oxide emulsion electrospun fibers.

As our approach is to protect the bacteriophage through pre-encapsulation prior to electrospinning, it is important to understand the impact of the emulsification process on the stability and activity of T4 bacteriophage. It was found that the T4 bacteriophage activity was not affected by exposure to surfactant, mechanical stirring or freeze-drying. The T4 bacteriophage activity after the emulsification process was tested by dispersing the T4 bacteriophage-alginate capsules in SM buffer for 2 h at 25°C under vigorous shaking. The plaque assay exhibited a drop in the T4 bacteriophage activity by one order of magnitude from 108 to 107 PFU ml−1 after the w/o emulsification process.

The physical stability of the crosslinked alginate capsules was evaluated by electrospraying the emulsion system. SEM analysis clearly shows that the alginate capsules cannot survive the electrospraying process (Fig. 2b); T4 bacteriophage exhibited a large drop in activity from 108 to 103 PFU ml−1 (Table 1). Typically, the alginate capsules are coated and protected with another polymer shell layer. Coating the alginate capsules likely enhances the mechanical strength and avoids the leaking of encapsulated material. This is of particular importance in the case of processes such as electrospinning (Fig. 2c).

Emulsion electrospinning of T4 bacteriophage

In emulsion electrospinning, the calcium–alginate capsules serve as a reservoir for T4 bacteriophage. Therefore, their fluidity and activity are predicted to remain viable after immobilization in the electrospun fibre. PEO is an ideal polymer for emulsion electrospinning. PEO is biocompatible and can be dissolved in both organic and aqueous solvents, making it a suitable carrier for encapsulating and releasing the T4 bacteriophage. SEM images of electrospun fibres produced from PEO/bacteriophage/alginate emulsions are illustrated in Fig. 3. Continuous fibres could be produced from the PEO solutions (Fig. 3a). The fibres had an average fibre diameter of 1·00 ± 0·5 μm with a bead-on-string morphology; indicative of bacteriophage–alginate capsule incorporation (Fig. 3b). Similarly, TEM images of the emulsion electrospun fibres showed the presence of darker areas within the fibre (Fig. 3d,e), which were not observed in the fibres produced without bacteriophage/alginate capsules (Fig. 3c).

Figure 3.

SEM images of electrospun fibers from (a) polyethylene oxide (PEO) solution and (b) PEO/bacteriophage/alginate emulsion system, as well as transmission electron microscopy images of electrospun fibers from (c) PEO solution and (d) and (e) bacteriophage loaded beaded PEO fibers from the PEO/bacteriophage/alginate emulsion system.

Further support for the encapsulation of the bacteriophage within the fibres came from measuring bacteriophage activity. Specifically, the collected fibres were immersed in SM buffer and the bacteriophage activity was measured by the plaque assay test. The solution was left for 2 h as per the previous samples to ensure complete bacteriophage release from the calcium–alginate capsules and electrospun fibres. The plaque assay reported a lytic activity of 106 PFU ml−1, a two order of magnitude drop from the original stock (Table 1).

Coaxial electrospinning of T4 bacteriophage

Core/shell electrospun fibres were produced using a PEO/chloroform solution for the shell and a T4 bacteriophage/buffer suspension for the core. The idea is to fully incorporate T4 bacteriophages into the core of the fibre and thereby avoid the rapid dehydration during electrospinning. To ensure the complete encapsulation of T4 bacteriophages in the core of the fibre, the feeding ratio of the core was set to 10 times slower than the polymer shell solution. Although the T4 bacteriophage suspension is not electrospinnable by itself and it has a tendency to spray, a uniform electrospun fibre was obtained in the coaxial electrospinning. SEM images did not show the presence of T4 bacteriophage spray or any components on the fibre surfaces or within the fibre (Fig. 4a). A TEM image (Fig. 4c,d) confirmed the core/shell fibre structure with the T4 bacteriophage incorporated in the core. The average diameter of the fibres was determined to be about 2·0 ± 0·2 μm. Due to the relatively low contrast between the polymer matrix and the unstained virus particles, the relatively narrow tail could not be seen, although the capsid was clearly observed. The lytic activity of the encapsulated T4 bacteriophage was found to be 108 PFU ml−1 (from three batches); the initial bacteriophage titre before encapsulation was 108 PFU ml−1 (Table 1).

Figure 4.

(a) Scanning electron microscopy micrograph of core/shell electrospun polyethylene oxide (PEO) fibers, (b) transmission electron microscopy (TEM) micrograph of a plain PEO fiber, (c) TEM micrograph of a core/shell electrospun PEO fiber, and (d) PEO fibers with encapsulated T4 bacteriophages.

Stability of core/shell encapsulated T4 bacteriophage

The encapsulation of bacteriophage in a dry form, while preserving their activity is important to prolong storage in many applications including food packaging. Therefore, the stability of powdered and encapsulated T4 bacteriophage was investigated during storage at 20, 4 and −20°C. Freeze-drying had little or no effect on the viability of T4 bacteriophage (Table 1). The stability of freeze-dried T4 bacteriophage during 4 weeks of storage at 20, 4 and −20°C is reported in Fig. 5. The survival of freeze-dried bacteriophage powders decreased rapidly from 108 to 102 PFU ml−1 after 1 day of storage at 20°C. While those stored at 4°C had their activities decreased relatively slower from 108 to 105 PFU ml−1 during 1 month of storage, the most significant losses occurring in the first week. Storage at the lowest temperature tested (−20°C), revealed no decrease in viability over the 4 weeks of storage.

Figure 5.

Activity-time plots for (a) freeze-dried T4 bacteriophage powders and (b) encapsulated T4 bacteriophage in freeze-dried core/shell electrospun fibers at three temperatures (20, 4, and −20°C). Error bars indicate standard deviation; = 3.

Interestingly, the coaxial electrospun PEO/T4 bacteriophage fibres showed significantly different storage behaviour. As shown in Fig. 5b, a significantly slower decrease in viability was observed for the samples stored at room temperature, and those stored at 4 and −20°C were essentially stable for up to several weeks.

Release profile and fibre morphology

The release profiles of viable bacteriophage from the emulsion and coaxial electrospun PEO fibres along with that of the calcium–alginate capsules are shown in Fig. 6. Although the amount of loaded T4 bacteriophage was different for the emulsion and coaxial electrospinning systems, in all the cases, a burst release was first observed within the first 15 min. After the initial burst release, a period of slow, but continuous, phase was observed for all formulations. The emulsion electrospun PEO fibre exhibited a slightly slower release rate of T4 bacteriophage in comparison with the alginate capsules and core/shell electrospun fibre (Fig. 6). For the core/shell formulation, nearly the full amount (99%) of encapsulated T4 bacteriophage was released after 60 min; however, there was a 25% reduction in the release of T4 bacteriophages from the emulsion electrospun formulation. This decrease is attributed to the loss of bacteriophages during the emulsion electrospinning process.

Figure 6.

T4 phage release profiles as measured by plaque assay from emulsified alginate capsules, emulsion electrospun polyethylene oxide (PEO) fibers and coaxial electrospun PEO fibers. Error bars indicate standard deviation; = 3. Phage counts of zero were recorded at 100 PFU ml−1 (or 2 log of PFU ml−1), which is the limit of the detection in the assaying test. (image) Emulsification; (image) emulsion electrospinning and (image) coaxial electrospinning.

The change in morphology of the electrospun PEO fibre upon exposure to buffer is shown in Fig. 7. During the first 5 min, the PEO electrospun fibre began to swell, followed by complete disintegration within 10 min, confirming the rapid release of the T4 bacteriophages in a short period of time.

Figure 7.

Scanning electron microscopy (SEM) images of polyethylene oxide electrospun fibers exposed to buffer at increasing time intervals. (Samples were freeze-dried prior to SEM analysis).

Discussion

Bacteriophages are sensitive biomolecules that are usually damaged upon exposure to harsh organic solvents, acidic or basic condition, dehydration, and/or high temperature (Joerger 2003). Our experiments showed that T4 bacteriophages could easily survive the high shear vortexing and freeze-drying processes but not the air-drying process associated with electrospinning. The large drop or complete loss of T4 bacteriophage activity in the suspension electrospinning is likely due to the rapid dehydration of T4 bacteriophage during the spinning process (Lee and Angela 2004; Salalha et al. 2006). The rapid water evaporation likely affected the nonuniform distribution of T4 bacteriophages throughout the nano-diameter electrospun fibre matrix. The embedded bacteriophages closer to the surface of the fibre were less shielded and, therefore, the rapid dehydration during the spinning process resulted in low activity after release.

Incorporating T4 bacteriophages in alginate capsules dramatically improved the activity of the bacteriophages during the emulsification and emulsion electrospinning processes. After the w/o emulsification process, the lytic activity of the T4 bacteriophage showed only a slight drop in activity from 108 to 107 PFU ml−1. This drop in bacteriophage activity could be due to a deficiency in bacteriophage loading in the alginate capsules and/or leaching of bacteriophage from the capsules prior to crosslinking. Although previous studies have looked at the encapsulation of bacteriophages in suitable aqueous media (Moser et al. 1998; Singh 2007; Yongsheng et al. 2008), our emulsification process involves the encapsulation in the presence of an organic solvent environment, chloroform. It is well known that the chloroform–water interface can strongly denature proteins, and therefore, it can easily destroy the bacteriophage structure (Van de Weert et al. 2000). Thus, the presence of alginate as a pre-encapsulating material is needed to protect the T4 bacteriophage structure from such a harsh solvent system. Calcium–alginate crosslinking represented an example of a simple and inexpensive technique, which is very suitable for maintaining T4 bacteriophage viability due to its relatively mild and nontoxic effects on the viral particles (Crcarevska et al. 2008; Hammad 1998).

However, the calcium–alginate material has a relatively low mechanical strength and the entrapped T4 bacteriophage could leak out when high stress is applied to the alginate beads. The SEM analysis clearly showed that the alginate capsules could not survive the emulsion electrospraying process due to the low physical stability of the crosslinked alginate capsules. That could explain the large drop in T4 bacteriophage activity, from 108 to 103 PFU ml−1 as result of emulsion electrospraying. Typically, the alginate capsules are coated and protected with another polymer shell layer (Zhongyi et al. 2007; Yongsheng et al. 2008). Coating the alginate capsules likely enhances the mechanical strength and avoids the leaking of encapsulated material. This is of particular importance in the case of processes such as electrospinning.

After emulsion electrospinning, the T4 bacteriophage activity significantly improved as compared to the activity measured from the suspension electrospun fibres and emulsion electrospraying of the calcium–alginate capsules. The biological activity of the T4 bacteriophages was relatively maintained in high feasibility when the alginate capsules were protected by and electrospun polymer shell layer. The bead-in-string fibre morphology clearly showed that the alginate capsules are incorporated and shielded by the PEO polymer fibre. The decrease in bacteriophage activity relative to the original bacteriophage activity could be attributed to the low mechanical strength of the alginate capsules, wherein some were likely disrupted during the electrospinning process. The mechanical stresses during electrospinning could be high enough to rupture alginate capsules in regions close to the fibre surface or to imperfections in the core/sheath fibre structure. The emulsion electrospinning of the same alginate system without addition of calcium chloride exhibited no bacteriophage activity (data not shown). This demonstrates the important role of calcium–alginate crosslinking and its physical strength to protect the bacteriophages in the alginate capsules during emulsion electrospinning.

The coaxial electrospinning was found to be the most reliable process to encapsulate T4 bacteriophages and maintain full bioactivity. Coaxial electrospinning produced a continuous core/shell morphology bead-free fibre, and the T4 bacteriophage was uniformly incorporated in the core of the fibre. Generally, the characteristic time associated with mutual diffusion at the liquid–liquid boundary is much slower than the millisecond timescales associated with bending instability during the electrospinning process (Ramakrishna et al. 2005; Andrady 2008). In our system, the use of immiscible solvents in the core and shell (water and chloroform, respectively) contributed towards the stability of the jet and the formation of a core/shell electrospun fibre morphology. Thus, the T4 bacteriophages were uniformly incorporated in the core of the fibre and fully protected by a 2-μm PEO polymer shell layer. In this process, solvent evaporation from the core is very slow, and therefore, the drastic change in the osmotic environment of the water-based core is prevented.

A key parameter in the utilization of encapsulated bacteriophage in the treatment of infected meat is the bacteriophage release profile. The in vitro release test was employed to study the release profile of the encapsulated T4 bacteriophages from each formulation. In all systems, a burst release was first observed followed by a plateau after about 15 min. The burst release is attributed to the rapid dissolution of the PEO polymer in the aqueous buffer. The emulsion electrospun PEO fibres exhibited a slightly slower T4 bacteriophage release rate in comparison with the alginate capsules and the core/shell electrospun fibres. In this case, the T4 bacteriophages are liberated initially from the PEO electrospun fibres, followed by a complete release from the alginate capsules. Therefore, a slower release was expected from the emulsion electrospun fibres. In the case of the coaxial electrospun fibres, the PEO fibre dissolution is accelerated by the presence of aqueous SM buffer in the core of fibre, and therefore, a faster release was observed upon exposure to the release media.

In practice, that is, in the meat packaging, a slower release rate of the T4 bacteriophages is expected as the electrospun fibre mats will be exposed to much lower moisture conditions. However, the very high solubility of the PEO-based fibres in water will help to ensure bacteriophages are released and in fact may help prevent the undesired early inoculation that could arise from the rapid and complete burst release observed under the conditions used in this report. The lower moisture conditions along with the lower temperatures encountered in food storage, that is, refrigeration, will also slow the rate of bacteriolysis. But it is important to note that it only takes one bacteriophage to infect one bacteria and the lytic replication process will rapidly produce ~200 bacteriophage that can go on to infect other bacteria present. This fact may help to alleviate the slow release of bacteriophage under low moisture conditions. We are currently investigating this.

Although our system can maintain bacteriophage viability for prolonged periods, this does raise a potential concern over the survival and exposure of bacteriophage to the consumer. We do know that while encapsulated in the electrospun fibres the viability is enhanced and bacteriophage can survive indefinitely under refrigeration conditions. However, that is not the case for the progeny bacteriophage, which like the freeze-dried bacteriophage will have a significantly reduced survival rate (except at −20°C). As such they would behave very similar to bacteriophage applied using a spraying technique, which is starting to receive some acceptance (FDA, 2006). Regardless, the use of any such materials will have to overcome a potentially bigger obstacle, the negative perception of viruses by the public.

Acknowledgements

The authors thank the NSERC Sentinel Bioactive Paper Network and its supporting member companies for supporting this research, and Dr. Mansel Griffiths from the University of Guelph for providing T4 bacteriophage and E. coli.

Ancillary