Regular ArticleDesign of a potentially prebiotic and responsive encapsulation material for probiotic bacteria based on chitosan and sulfated β-glucan
Graphical abstract
Introduction
Even though it was invented more than two decades ago, the layer by layer self-assembly technique is still today a very exciting method due to the tailorable properties of the end coating such as thickness, structure and surface properties [1]. This rather simple technique is based on the alternate deposition of polyanions and polycations [1] and exploits the gain in entropy due to charge compensation upon complex formation in between oppositely charged polyions that leads to release of numerous small counterions [2]. The physico-chemical properties of such assemblies can easily be modulated by changing some experimental parameters, i.e. polyelectrolyte concentration, degree of charge, salt concentration, pH, temperature and number of assembled layers [3]. Moreover, the layer by layer technology is applicable not only for planar surfaces but also for colloidal particles such as biological cells [4]. These advantages of the layer by layer coatings give possibilities for a broad range of applications in different areas including drug delivery [5], [6], [7], sensors [8], [9], and biomaterial coatings [10], [11]. In the case of coatings for encapsulation technologies, the permeability of the coating is an important property to control. In this respect, layer by layer is specifically a right approach since it allows the permeation of small molecules while halts larger molecules. Moreover, the semi-permeable nature of layer by layer based coatings can be regulated by the experimental parameters upon assembly [3].
Recently, the need to replace traditional materials by sustainable materials from renewable resources has become pressing due to stricter international regulations. In encapsulation technology, one of the most popular choices for natural cationic polyelectrolyte is chitosan. Chitosan is a random linear copolymer of (1–4)-N-acetyl-d-glucosamine and (1–4)-d-glucosamine units, produced by the deacetylation of the naturally occurring chitin under high alkaline conditions [12]. Its polycationic property is due to amine residues that are protonated below pH ∼6.5 [13]. Moreover, chitosan is biocompatible and non-toxic [14]. It is also known to be a biodegradable since it can be metabolized by enzymes such as lysozyme, present in human body fluids [15]. β-glucan is another bio-based natural polysaccharide that so far has not been much exploited in nanotechnology and material science. β-glucan extracted from barley and oat is composed of linear (1–3) - and (1–4)-β-d-glucose units [16]. β-glucan has prebiotic properties [17], [18], [19] as well as blood serum cholesterol and glycemic index lowering properties [20]. Moreover, β-glucan can be chemically modified giving beneficial biological activities e.g. antiangiogenic, antitumoral properties [21] and anticoagulant activity [20].
In the present work, we focus on the fabrication of coatings based on alternating multilayers of chitosan and sulfated β-glucan as a future delivery system that ensures a safe passage through the harsh conditions in the stomach while allows the release of coated ingredients to the human colon. An anionic variant of oat β-glucan was prepared and it was combined with chitosan for the formation of the layer by layer coating. To our knowledge, this is the first time sulfated β-glucan has been used in layer by layer coatings. β-glucan can be fermented by human gut microbiota and chitosan is degradable in the human body. In addition, sulfated β-glucan was employed as an exterior layer to disable gastric mucosa attachment. In the meanwhile, chitosan was used to protected against coating disintegration at acidic gastric conditions. Chitosan was chosen despite its antimicrobial activities [22] since it is the only known bio-based, food grade polymer with cationic nature so far, which makes the use of formulation possible for food applications such as probiotics coating. Besides, lactic acid bacteria is less prone to the antimicrobial effect of chitosan [23], [24]. Our hypothesis is that the sulfated β-glucan and chitosan can produce responsive material suitable for targeted release in the intestine for bioactive molecules and probiotics. The coatings were investigated using in situ quartz crystal microbalance with dissipation (QCM-D), atomic force microscopy (AFM) and spectroscopic ellipsometry (SE) for their ability to form assembled multilayers, as well as for their characterization in terms of structure and stability under gastrointestinal conditions. Besides, coating bacterial cells was studied using zeta potential measurements and confocal laser scanning microscope (CLSM). Viable counts of coated and uncoated cells were demonstrated using plate counting method.
Section snippets
Materials and chemicals
Oat β-Glucan (high viscosity) was acquired from Megazyme, Wicklow, Ireland. Chitosan (extracted and/or purified from Pandalus borealis shell, low molecular weight, deacetylation ⩾75%), dry formamide, (⩾99%, water content of <0.03%), pepsin from porcine gastric mucosa powder (⩾250 units/mg), ethanol (96% v/v) pancreatin from porcine pancreas (⩾3 × USP), acetic acid (glacial, ⩾99.85%) and FITC (Fluorescein 5(6)-isothiocyanate) were purchased from Sigma-Aldrich, Steinheim, Germany. Chlorosulfonic
Multilayers formation: characterization by QCM-D
In situ QCM-D experiments were performed in order to explore the ability of layer by layer formation based on the biopolymers CH and sβG at 25 °C. CH is positively charged below pH 6.5 [12] and oat β-glucan was modified to introduce negative charges by sulfation. Multilayer formation was thus attempted at pH 5.6. The principle of QCM-D implies a frequency shift, ΔF, upon any mass change [29]. The continuous decrease of the 7th overtone shift (from now on ΔF7 which refers to the overtone shift
Conclusions
We demonstrated a reproducible build-up of chitosan and sulfated β-glucan on the planar surface and on the bacteria via layer by layer. QCM-D, SE, AFM, zeta sizer and plate counting for enumeration of viable cells were used in order to characterize these multilayer-based coatings. The influence of rinsing time (1 versus 5 min) on the structure of multilayer coatings was also examined and 5 min of rinsing were found to give denser coatings. Alternating zeta potential values after addition of each
Acknowledgements
The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/under REA grant agreement n° 606713. Marité Cárdenas thanks the Swedish Research Council for funding. This work has been performed under the umbrella of COST actions CM1101 and MP1106.
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