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Encapsulation of Living Bifidobacteria in Ultrathin PVOH Electrospun Fibers Amparo Lo´pez-Rubio,*,† Ester Sanchez,‡ Yolanda Sanz,‡ and Jose M. Lagaron† Novel Materials and Nanotechnology and Microbial Eco-Physiology and Nutrition Group, IATA, CSIC, Apdo. Correos 73, 46100 Burjassot, Spain Received June 11, 2009; Revised Manuscript Received July 17, 2009
This study shows the application of the electrospinning technique as a viable method for the encapsulation and stabilization of bifidobacterial strains. Poly(vinyl alcohol) (PVOH) was used as the encapsulating material because it is generally recognized as safe (GRAS), has a high oxygen barrier when dry, and is water soluble, hence allowing easy recovery of the bacteria for viability testing. A coaxial setup was used for encapsulation, and the so-obtained electrospun fibers had a mean diameter of ca. 150 nm. Incorporation of B. animalis Bb12 led to a decrease in melting point and crystallinity of the PVOH fibers and to an increase in the polymer glass transition temperature. The viability tests, carried out at three different temperatures (room temperature and 4 and -20 °C) showed that B. animalis Bb12 encapsulated within the electrospun PVOH fibers remained viable for 40 days at room temperature and for 130 days at refrigeration temperature, whereas a significant viability decrease was observed in both cases when bacteria were not encapsulated (p ) 0.015 and p ) 0.002, respectively).
1. Introduction The intestinal microbiota plays an important role in human health as they contribute to inhibiting pathogen colonization, boosting the immune system, and metabolizing nutrients. Bifidobacteria and lactobacilli strains are widely used as probiotic bacteria for human consumption. Probiotics are defined as living microorganisms which, when administered in adequate amounts, confer health benefits to the host, including the prevention and treatment of some pathologies.1 These include reduction of gastrointestinal infections, improvement of lactose metabolism, reduction of serum cholesterol, and improvement of immune system defenses.2 Bifidobacteria are Gram-positive, pleomorphic, and strictly anaerobic bacteria, which inhabit the human gastrointestinal tract. Immediately after birth, the human gastrointestinal tract is rapidly colonized by bacteria, and bifidobacteria are among the dominant groups particularly in breast-fed infants, representing up to 90% of the faecal microbiota. Although the predominance of this genus is reduced with age, it still represents about 5-7% of the adult’s microbiota.3 However, different factors like diet, antibiotics, and stress are reported to negatively influence bifidobacterial populations in human gastrointestinal tract. Among the 32 Bifidobacterium species described, B. animalis, B. breVe, B. longum, B. bifidum, and B. infantis have been extensively studied for their effects on human health, and as a consequence, they have been incorporated as probiotics into diverse food products, supplements, and pharmaceutical formulations.4 The strain Bifidobacterium animalis subsp. lactis Bb12 has been widely used in the food industry and is marketed under a variety of labels particularly in dairy products and infant formulas.5 This strain has shown to exert beneficial effects in the management of lactose malabsorption, rotaviral diarrhea, antibiotic-associated diarrhea, and Clostridium difficile diarrhea in human clinical trials.6 * Corresponding author. Tel.: +34 963900022. Fax: +34 963636301. E-mail address:
[email protected]. † Novel Materials and Nanotechnology. ‡ Microbial Eco-Physiology and Nutrition Group.
The currently accepted probiotic definition requires that the bacteria maintain its viability from production to consumption. In addition, it is desired that the bacterial strains resist the adverse conditions occurring during the passage through the gastrointestinal tract.7 In this context, the International Dairy Federation (IDF) has recommended that the bacteria must be alive, metabolically active, and abundant in the product to guarantee its efficacy. With this purpose, IDF has recommended the presence of, at least, 107 CFU/g in dairy products until the end of their shelf life. However, several factors influence the bacterial survival during technological processes and the gastrointestinal transit, including low and high temperatures, low pH of fermentative and postfermentative processes, and gastric juices and the bile acids and digestive enzymes. Consequently, the industry demands technologies ensuring bifidobacteria stability for both economical and health reasons.8 The development of delivery systems for biologically active compounds in food systems is an important issue in modern food technology. Microencapsulation has previously been reported as a technology to protect sensitive substances against the influences of adverse environments.9 The term “microencapsulation” designates a defined technology of wrapping solids, liquids, or gases in small capsules, which can release their contents under specific circumstances. In recent years, microencapsulation has also been found to be a useful tool for the stabilization of probiotic cells for functional food applications. Microencapsulation can enhance the viability of probiotic bacteria during processing, storage, and subsequent consumption and gastrointestinal transit.10,11 Electrospinning is a process that produces continuous polymer fibers with diameters in the submicrometer range through the action of an external electric field imposed on a polymer solution or melt. Recently, this technique has received substantial attention, especially in the biomedical area, as the high surface area of the electrospun fibers mimic the extracellular matrix12 and can be used in a variety of applications such as scaffolds for tissue engineering13,14 or drug delivery devices.15 In the food science area, this technique has only very recently been applied to encapsulate antioxidants16
10.1021/bm900660b CCC: $40.75 2009 American Chemical Society Published on Web 08/05/2009
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and to generate antimicrobial fibers,17 but it has great potential as a nano- and microencapsulation technique for applications in the areas of active and bioactive packaging18 and to generate ingredients for functional food products. In this sense, coaxial electrospinning or co-electrospinning offers great potential for the encapsulation and controlled release of biologically active agents.19 The configuration in co-electrospinning, which consists of two concentrically arranged dies connected to two reservoirs containing different spinning solutions, presents a number of advantages, such as the possibility of including as core material a favorable fluid medium for the functional component, which does not have to be spinnable as it becomes entrained by the outer polymeric shell.20 A number of bioactive agents, including whole microbial cells, have already been encapsulated in nanofibers using this electrospinning configuration.19,21 The objective of the present work was to study the suitability of the electrospinning method for the encapsulation and protection of the strain B. animalis subsp. lactis Bb12 using poly (vinyl alcohol) (PVOH) as the encapsulating polymer. The morphological and physical characteristics of the fibers generated were studied and compared to pure PVOH fibers and films. PVOH is a hydrophilic, semicrystalline polymer widely used because of its good chemical resistance, good thermal stability, good physical properties, excellent biocompatibility, and low cost.22,23 Moreover, this polymer is generally recognized as safe (GRAS) and has been selected in the present study due to its water solubility property, which permits easy recovery of bifidobacterial cells without affecting their viability.
2. Materials and Methods 2.1. Materials. Poly(vinyl alcohol) (PVOH) with an average molecular weight (Mw) of 146 000-186 000 and degree of hydrolysis of 99% was purchased from Sigma Aldrich Co. (Spain). 2.2. Preparation of Polymer Solution and Cast Film. For electrospinning, a 10% w/w PVOH solution was prepared by dissolving the polymer pellets in distilled water at 80 °C under magnetic stirring until complete dissolution. The same solution was also used to prepare a thin polymer film of PVOH by solvent evaporation after casting in a Petri dish. The solvent evaporation casting process took place over 24 h in an oven at 37 °C followed by delaminating of the cast film and subsequent storage at room temperature. 2.3. Characterization of the Polymer Solution. The viscosity of the polymer solution was measured using a rotational viscosity meter (Visco Basic Plus L from Fungilab S.A. (San Feliu de Llobregat, Spain) using pindle no 1 at 2.5 rpm. The surface tension of the polymer solution was measured using a DynoTester tension meter (Neurtek S.A., Eibar, Spain). Both measurements were done at 25 °C. 2.4. Preparation of Bifidobacterial Suspensions. The strain Bifidobacterium animalis subsp. lactis Bb12 (Chr. Hansen Ltd., Hørsholm, Denmark) was grown in Man, Rogosa, and Sharpe (MRS) broth and agar medium (Scharlau, Barcelona, Spain) supplemented with 0.05% (w/v) cysteine (Sigma, St. Louis, MO) (MRS-C), and incubated at 37 °C under anaerobic conditions (AnaeroGen; Oxoid, Basingstoke, UK). B. animalis Bb12 cells were collected by centrifugation, washed twice in phosphate-buffered saline (PBS, 130 mM sodium chloride, 10 mM sodium phosphate, [pH 7.2]), and resuspended in skimmed milk to approximately 1010 cell/mL. One aliquot of this cell suspension was used to the electrospinning assay, and the rest of non-encapsulated cells were aliquoted and stored at three different temperature conditions (20, 4, and -20 °C) for viability testing. 2.5. Coaxial Electrospinning Equipment Setup. The electrospinning apparatus, equipped with a variable high-voltage 0-30 kV power supply, was assembled in-house.17 The anode was attached to a coaxial stainless steel needle arrangement with each needle connected, through
Lo´pez-Rubio et al. PTFE wires, to a couple of 5 mL plastic syringes. The syringes were placed horizontally in the cradle of a digitally controlled syringe pump able to deliver flow rates in the range of 10-3 to 10 mL h-1 (KD Scientific Inc., Holliston, U.S.A.). B. animalis Bb12 cells suspended in skimmed milk were pumped through the inner needle (L 0.8 mm) and the encapsulating PVOH polymer solution was pumped through the outer needle (L 1.5 mm). The disk-shaped copper ground electrode was held 13 cm in line with the central axes of the coaxial needle arrangement. The experimental setup was housed in a laminar flow safety cabinet. A single needle (L 0.9 mm) configuration was used to obtain neat PVOH fibers for comparison purposes. 2.6. Preparation of PVOH/Bb12 Fiber Mats. For the collection of the PVOH electrospun fibers containing B. animalis Bb12 (PVOH/ Bb12 fibers), the collector was covered with an aluminum sheet. The electrospun PVOH/Bb12 nanofibers were prepared in a similar manner to that reported previously for the encapsulation of viruses and bacteria.24 The electrospinning conditions of voltage and tip to collector distance were fixed at 11 kV and 13 cm, respectively. Using these conditions, a stable jet mode was attained. Once in stable jet mode, the flow rate was gradually increased from 0.06 mL/h to 0.3 mL/h. The as-spun nanofibers were dried overnight at 38 °C before SEM observation. 2.7. Staining of Encapsulated Bifidobacteria. In order to check whether bifidobacterial cells were properly encapsulated within the PVOH fibers, the bacterial cell solution was stained with 5-cCFDA 10 mM (5-carboxyfluorescein diacetate) (Sigma, St. Louis, MO)) for 30 min at 37 °C.25 Cells were then washed and resuspended in skimmed milk for electrospinning. Labeled B. animalis Bb12 cells were encapsulated in dark conditions in order to preserve the 5-cCFDA activity of the cells. The presence and distribution of the PVOH/labeled-B. animalis Bb12 fibers obtained were immediately observed using a digital microscopy system (Nikon Eclipse 90i) fitted with a 12 V, 100 W halogen lamp and equipped with a digital imaging head which integrates an epifluorescence illuminator. A digital camera head (Nikon DS-5Mc) with a 5 megapixel CCD cooled with a Peltier mechanism was attached to the microscope. Nis Elements software (Nikon Instruments Inc., Melville, USA) was used for image capturing and the Adobe Photoshop CS3 extended software was used for image processing and analysis. 2.8. Differential Scanning Calorimetry (DSC). DSC experiments were carried out in a Perkin-Elmer DSC-7 calorimeter. The heating and cooling rate for the runs was 10 °C/min, and the typical sample weight was around 2 mg. Calibration was performed using an indium sample. All tests were carried out at least in duplicate. 2.9. Attenuated Total Reflectance Infrared Spectroscopy (ATRFTIR). ATR-FTIR experiments were recorded in a controlled chamber at 21 °C and 40%RH coupling the ATR accessory GoldenGate of Specac Ltd. (Orpington, UK) to a Bruker (Rheinstetten, Germany) FTIR Tensor 37 equipment. All spectra were collected within the wavenumber range 4000-600 cm-1 by averaging 10 scans at 4 cm-1 resolution. Analysis of the spectral data was performed by using Grams/AI 7.02 (Galactic Industries, Salem, NH, USA) software. 2.10. Scanning Electron Microscopy (SEM). SEM was conducted on a Hitachi microscope (Hitachi S-4100) at an accelerating voltage of 10 kV and a working distance of 20 mm. The electrospun fibers were sputtered with a gold-palladium mixture under vacuum before their morphology was examined using SEM. Fiber diameters of the electrospun fibers were measured by means of the Adobe Photoshop CS3 extended software from the SEM micrographs in their original magnification. 2.11. Viability of Encapsulated Bifidobacteria. As-spun ultrathin fibers were detached from the sheet. Aliquots of the PVOH/Bb12 fibers were weighed and stored at three different temperatures (room temperature and 4 and -20 °C) for viability testing, and a small sample was taken for scanning electron microscopy (SEM). Aliquots of non-
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encapsulated Bb12 cells (milk/Bb12) were also stored at room temperature and 4 and -20 °C, and growth ability was also tested. The resistance of B. animalis Bb12 to electrospinning and storage conditions (room temperature and 4 and -20 °C) was evaluated at different times by plate counting. For this purpose, one fresh PVOH/ Bb12 aliquot was diluted in PBS at room temperature for 1 h, in order to dissolve the polymeric fibrilar capsules, and serial 10-fold dilutions were made and plated on MRS-C agar. After incubation at optimal conditions for 48 h, the ability of electrospun B. animalis Bb12 cells to growth was determined in comparison with nonelectrospun cells. This test was made in duplicate. 2.12. Statistical Analysis. The mean of two individual determinations was used to calculate cell counts. A single factor ANOVA and student t test (R ) 0.05) was used to analyze the cell counts.
3. Results and Discussion This study presents, for the first time, the encapsulation of bifidobacterial strains by means of the electrospinning technique. The removal of water by rapid evaporation upon electrospinning is thought to cause a drastic change in the bacterial osmotic environment, and therefore, a strain generally considered resistant to adverse environmental conditions, B. animalis Bb12, was chosen for this first study. The PVOH encapsulating matrix was selected because it is a biocompatible and water-soluble material which is not expected to affect the bioactivity of the bacteria and, moreover, allows for an easy recovery of the bacterial cells to carry out viability studies. 3.1. Electrospraying of Bacterial Suspensions. The viability and morphology of the bifidobacteria after applying the electrical voltage was tested before conducting the encapsulation in PVOH of B. animalis Bb12 using the electrospinning technique. In this preliminary experiment, a concentration of B. animalis Bb12 of 1010 cfu/mL was resuspended in skimmed milk. Due to the nonspinnable character of the bacterial suspension in skimmed milk, instability in the jetting process is expected and a phenomenon known as electrospraying takes place. For electrospraying, B. animalis Bb12 cells were syringed through a needle kept at a positive potential with respect to the ground electrode; this process charged the skimmed milk containing the cells, whereupon the medium was immediately dispersed on entering the external electric field. A voltage of 11 kV was applied and a volume of 0.45 mL of the bifidobacterial solution was electrosprayed. A glass Petri dish covered with aluminum with the same diameter as the ground electrode (about 15 cm) was used to collect the bacteria. Some cells were also collected in an SEM sample holder for the morphological study. Electrosprayed B. animalis Bb12 cells were detached from the sheet and dissolved in PBS for viability control, and the SEM sample holder was kept inside a sterile Petri dish with the cover until SEM observation to avoid cross-contamination. No differences were found in the viability of B. animalis Bb12 before and after the electrospraying process, and SEM observation showed that the morphology of the bifidobacterial cells after the application of a high voltage remained the same. Figure 1 shows the bacteria morphology before (Figure 1a) and after the electrospraying process (Figure 1b). As observed from the SEM micrographs, the morphology of the bacterial cells is not affected by the process, but the dimensions of the cells were slightly affected by the electrospraying process (P < 0.05 by unpaired Student t test). B. animalis Bb12 has a rod shape, and the mean size was calculated by measuring the length and width of at least 50 different cells in both the control and the electrosprayed bifidobacteria. Control cells have a mean size of 2.22 ( 0.41 µm long and 0.60 (
Figure 1. Bifidobacterium animalis Bb12 before (a) and after electrospraying (b) from a skimmed milk solution.
0.08 µm wide, while after electrospraying, the mean values obtained were 2.04 ( 0.35 µm long and 0.64 ( 0.08 µm wide. Probably, the material that was surrounding the bifidobacterial cells after electrospraying (Figure 1b) was made of milk components (proteins, carbohydrates, and minerals) left after water evaporation. 3.2. Encapsulation of B. animalis Bb12 in Ultrathin PVOH Fibers. For the encapsulation of B. animalis Bb12 using the electrospinning technique, a coaxial setup was chosen. In this specific case, as the polymer used for encapsulation (i.e., PVOH) is water-soluble, direct mixing of the polymer and the bacterial cells was possible, and a single needle configuration of the process was also carried out for comparison purposes. However, the majority of the experiments in the present work, including the viability testing, were done using the coaxial setup, because it intuitively seemed to be the most efficient method to entrap the cells and cells medium. Moreover, in case other encapsulating biopolymers are used which are not water-soluble, this seems the most adequate methodology to apply. The coaxial setup will also be the preferred processing method for encapsulation of living organisms, as the biological solution goes through an independent circuit and only comes into contact with the encapsulating matrix upon contact with the needle tip. At this specific point, i.e., near the needle tip, it has been observed that the electrical properties of the outer polymer solution become important when the jet emerges and that the electric force acts only on the free surface.26 The inner fluid, which in this specific case contains the solution of bifidobacteria, is entrained into the emerging jet only by the viscous forces generated by the outer polymer.26 As water is the solvent of both the PVOH and the bacterial solution, a fast solidification of PVOH is expected, while the inner solution can remain wet for a certain time.27 Solution concentration determines the limiting boundaries for the formation of electrospun fibers due to variations in viscosity
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and surface tension.28 In general, low-concentration solutions form droplets due to high surface tension, while too high concentration prohibits fiber formation due to higher viscosity. The concentration of the polymer solution influences the spinning of fibers and controls the fiber structure and morphology.29 On the other hand, the surface tension decreases with increasing polymer concentration and molecular weight of the polymer in the solution.30 A low surface tension is desirable in electrospinning, as it reduces the critical voltage needed for the ejection of the jet from the Taylor’s cone as shown below:31
V2c )
4H2 2L 3 ln - 0.117πγR 2 R 2 L
(
)
Where H is the separation distance between the needle and the collector, L is the length of the needle (or capillary), R is the radius of the needle, and γ is the surface tension of the solution. Given the Mw of the PVOH used, the concentration was adjusted so that the minimum voltage was needed for the ejection of the jet, while keeping the viscosity low enough so as not to block the flow through the external needle. The viscosity and surface tension of the 10% PVOH solution used for encapsulation of the bifidobacteria, measured at 25 °C, were 2280 cP and 53.5 mN/m, respectively. The presence and distribution of labeled B. animalis Bb12 cells within and along the PVOH nanofibers was observed by fluorescence microscopy. After staining the bacterial solution with 5-cCFDA, electrospinning was carried out in dark conditions to avoid inactivation of the dye. Some electrospun PVOH/ labeled B. animalis Bb12 fibers were collected on a microscope slide and taken directly for observation under a microscope with a fluorescence source. Figure 2 shows the microphotographs of some fibers taken under polarized light (Figure 2a) and illuminated with the fluorescence source (Figure 2b). In Figure 2, it is observed that labeled B. animalis Bb12 cells were successfully encapsulated along the fibers. B. animalis Bb12 cells were localized along the fibers; however, some more agglomerated cells were also observed in certain areas of the fibers. In all cases, the cells were completely encapsulated inside the polymeric matrix and were aligned longitudinally along the nanofiber axis. The distance between the thick agglomerated cells in the fibers was around 20-30 µm. The morphology of the electrospun fibers containing B. animalis Bb12 was also examined by SEM. Although the polymer concentration was high enough to attain a stable jetting process, the coaxial setup led to beaded fibers (see Figure 3). The diameters of the fibers obtained were in the submicrometer range. Thus, the fibers’ diameters span between ∼70 nm (nanofibers) and ∼300 nm (ultrathin fibers), with the average fiber diameter being ∼150 nm. A control sample was also prepared without bifidobacteria using the coaxial setup, i.e., pumping PVOH through the outer needle and milk through the inner needle, and beaded fibers were also obtained (see Figure 4). In fact, the morphology of the so-obtained fibers was much less homogeneous with fiber diameters ranging from less than 50 nm to more than 1 µm. The beads were also more heterogeneously distributed, and a huge variation in size was also observed. Probably, as PVOH is water-soluble and milk is a water suspension, this causes instability during the electrospinning process and both materials are mixed together at the tip of the needles. Ribbon-like
Figure 2. Optical micrographs of PVOH electrospun fibers containing encapsulated Bifidobacterium animalis Bb12 under polarized light (a) and using a fluorescence source (b). 5-cCFDA was used to reveal/ show bacteria within the fibers.
Figure 3. SEM micrographs of electrospun fibers containing Bifidobacterium animalis Bb12 obtained through coaxial electrospinning.
structures are seen in Figure 4, which could be ascribed to the collapse of the fibers. This phenomenon has been previously explained on the basis of considerable amounts of solvent remaining inside the fibers.32 The presence of bifidobacteria at
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Figure 4. SEM micrograph of PVOH fibers coaxially electrospun with milk.
high concentrations in the milk pumped through the inner needle may act as a stabilizer promoting a more stable electrospinning process and, thus, more homogeneous fibers. For comparison purposes, electrospinning was also carried out using the single needle configuration, i.e., making a suspension of the bifidobacteria in the polymer solution. From Figure 5a, it can be seen that nonbeaded homogenously distributed fibers are obtained by this means, having an average diameter of about 200 nm (ultrathin fibers). Distribution of bacteria along the fibers was investigated, as previously, by staining the bifidobacterial solution with 5-cCFDA prior to the electrospinning process. The average distance between B. animalis Bb12 cells along the fibers was similar to that observed in the coaxially electrospun fibers, i.e., 20-30 µm. Figure 5b,c shows, as an example, the optical micrographs of some of the ultrathin fibers obtained by direct mixing of the polymer with the bacterial solution observed under polarized light and using a fluorescence source, respectively. 3.3. Thermal Properties of the Ultrathin Fibers. Table 1 gathers the melting point, melting enthalpies, and glass transition temperatures of the electrospun fibers and of a PVOH film calculated from the first heating run. When comparing pure PVOH fibers with a PVOH film, it can be seen that the glass transition temperature (Tg) of the latter is lower than the Tg of the electrospun fibers. Moreover, both the maximum of melting and the melting enthalpy are considerably higher for the electrospun fibers, indicating more perfect crystallites and/or molecular orientation and a higher crystallinity, respectively. However, when the fibers are produced through coaxial electrospinning incorporating either milk or a suspension of B. animalis Bb12 in milk, a considerable decrease in melting temperature and enthalpy is observed, indicating that incorporation of milk components and B. animalis Bb12 into the fibers distorts the polymer crystallinity. These effects seem to be more pronounced in the control PVOH fibers with milk, but the standard deviations of the DSC parameters from the electrospun fibers with B. animalis Bb12 are greater, pointing out to the uneven distribution of the bacterial cells within the materials. Regarding the Tg, while an increase is seen for the control fibers with milk, the glass transition temperature remains similar when incorporating the bifidobacteria. These results seem to indicate that milk particles (like proteins and sugars) could enhance the rigidity of the chains in the amorphous phase of the fibers. Attenuated total reflectance infrared spectroscopy (ATRFTIR) was used to study the molecular structure of the electrospun fibers. The PVOH film was also analyzed, displaying a very similar spectrum to that of pure PVOH fibers (result not shown). In Figure 6, the spectra of pure PVOH fibers, control
Figure 5. Hybrid PVOH/Bb-12 fibers electrospun using the single needle configuration: (a) SEM micrograph; (b) and (c) optical micrographs under polarized light and using a fluorescence source, respectively. Table 1. DSC Maximum of Melting (Tm), Melting Enthalpy (∆H), and Glass Transition Temperature (Tg) of PVOH Film and the Various Fibers
PVOH PVOH PVOH PVOH
film fibers fibers + milk fibers + milk + Bb12
Tm (°C)
∆H (J/g)
Tg (°C)
188.5 (1.5) 194.5 (0.1) 182.1 (0.6) 183.2 (1.8)
19.3 (0.5) 35.6 (0.8) 13.15 (0.2) 17.9 (5.2)
50.1 (0.1) 53.1 (0.8) 57.0 (2.3) 52.6 (1.9)
fibers with milk, and those with B. animalis Bb12 are displayed. The main differences among the spectra of the different fibers are seen in the amide region (1800-1500 cm-1), where the amide I and II bands of proteins and peptides from the milk are present (see Figure 6b), and in the polysaccharide region (1200-900 cm-1), where two prominent bands are observed at ∼1048 and ∼995 cm-1 (see arrows in Figure 6c), probably arising from vibrations in the peptidoglycan of the bacterial cell wall.33 The results further support the presence of the cells within the fiber structure.
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Figure 6. ATR-FTIR spectra of (a) PVOH fibers, (b) PVOH fibers coaxially electrospun with milk, and (c) PVOH fibers coaxially electrospun with milk and Bifidobacterium animalis Bb12. Data have been offset for clarity.
3.4. Viability Studies of Encapsulated B. animalis Bb12. Although PVOH may not seem a priori an ideal matrix for the protection of bifidobacterial cell, because of its hydrophilic character, it has, on the other hand, a very high oxygen barrier while dry. Thus, experiments were carried out to check whether the encapsulated probiotics remained viable inside the fibers during storage at different temperature conditions. The viability of the encapsulated B. animalis Bb12 was studied at three different temperature conditions: room temperature (∼20 °C) and 4 and -20 °C. B. animalis Bb12 cells, with an initial cell density of 1010 ufc/mL, were resuspended in 5 mL of skimmed milk and were electrospun using a coaxial setup. The obtained fibers were collected in an aluminum sheet, cut into samples, and divided into Eppendorf tubes containing around 2 mg of fiber-containing bifidobacteria. The survival of the encapsulated B. animalis Bb12 cells was evaluated at different times during storage and compared with the viability in skimmed milk, which is known to act as a protecting system for these cultures.4 There was no decrease in the viability of B. animalis Bb12 cells after the electrospinning assay. This is already a promising result for the preparation of bifidobacterial food adjuncts which are dry, stable, and occupy a small volume. Other techniques used for this purpose, such as spray-drying, lead to a decrease in the viable counts of probiotic bacteria.34 Encapsulation by other means has also proven to be less efficient than the electrospinning method. For instance, homogenization techniques for reducing the size of calcium alginate beads during the microencapsulation cause a significant decrease in viability of different strains of probiotic bacteria.35 Microencapsulation through gelation techniques also leads to a certain viability loss. Encapsulation yields ranging from 10% to 93% have been found in a number of studies dealing with probiotic cells.35-38 Therefore, in terms of encapsulation yields, electrospinning presents substantial advantages when compared with other encapsulation techniques used. As shown in Figure 7a, at room temperature (∼20 °C), the viability of B. animalis Bb12 was decreased 1 log unit after 15 days in both encapsulated and non-encapsulated forms. After 40 days of storage, a significant viability decrease was observed in both encapsulated bifidobacteria, PVOH/Bb12 (6 log units reduction, p ) 0.002), and non-encapsulated bifidobacteria, Milk/Bb12 (>8 log units reduction, p < 0.001); however, the viability of encapsulated bacteria (4 log units) was significantly higher than that of non-encapsulated bacteria (
