Regular Article
Design of a potentially prebiotic and responsive encapsulation material for probiotic bacteria based on chitosan and sulfated β-glucan

https://doi.org/10.1016/j.jcis.2016.10.019Get rights and content

Abstract

Hypothesis

Chitosan and sulfated oat β-glucan are materials suitable to create a prebiotic coating for targeted delivery to gastrointestinal system, using the layer by layer technology.

Experiment

Quartz crystal microbalance with dissipation (QCM-D), spectroscopic ellipsometry (SE) and atomic force microscopy (AFM) were used to assess the multilayer formation capacity and characterize the resulting coatings in terms of morphology and material properties such as structure and rigidity. The coating of colloidal materials was proven, specifically on L. acidophilus bacteria as measured by changes in the bacterial suspension zeta potential. Viability of coated cells was shown using plate counting method. The coatings on solid surfaces were examined after exposure to mimics of gastrointestinal fluids and a commercially available β-glucanase.

Findings

Successful build-up of multilayers was confirmed with QCM-D and SE. Zeta potential values proved the coating of cells. There was 2 log CFU/mL decrease after coating cells with four alternating layers of chitosan and sulfated β-glucan when compared to viability of uncoated cells. The coatings were partially degraded after exposure to simulated intestinal fluid and restructured as a result of β-glucanase treatment, mimicking enzymes present in the microflora of the human gut, but seemed to resist acidic gastric conditions. Therefore, coatings of chitosan and sulfated β-glucan can potentially be exploited as carriers for probiotics and delicate nutraceuticals.

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.

References (53)

  • F. Devlieghere et al.

    Chitosan: antimicrobial activity, interactions with food components and applicability as a coating on fruit and vegetables

    Food Microbiol.

    (2004)
  • H.K. No et al.

    Antibacterial activity of chitosans and chitosan oligomers with different molecular weights

    Int. J. Food Microbiol.

    (2002)
  • R. Kivelä et al.

    Oxidative and radical mediated cleavage of β-glucan in thermal treatments

    Carbohydr. Polym.

    (2011)
  • P. Murphy et al.

    Effects of cereal β-glucans and enzyme inclusion on the porcine gastrointestinal tract microbiota

    Anaerobe

    (2012)
  • R.G. Boot et al.

    Identification of a novel acidic mammalian chitinase distinct from chitotriosidase

    J. Biol. Chem.

    (2001)
  • a. Planas

    Bacterial 1,3–1,4-beta-glucanases: structure, function and protein engineering.

    Biochim. Biophys. Acta

    (2000)
  • A. Shah et al.

    β-Glucan as an encapsulating agent: effect on probiotic survival in simulated gastrointestinal tract

    Int. J. Biol. Macromol.

    (2016)
  • a. Lazaridou et al.

    Molecular aspects of cereal beta-glucan functionality: physical properties, technological applications and physiological effects

    J. Cereal Sci.

    (2007)
  • R.V. Klitzing

    Internal structure of polyelectrolyte multilayer assemblies

    Phys. Chem. Chem. Phys.

    (2006)
  • M. Schönhoff

    Layered polyelectrolyte complexes: physics of formation and molecular properties

    J. Phys.: Condens. Matter

    (2003)
  • G. Decher et al.

    Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials, 2nd ed.

    (2012)
  • X. Qiu et al.

    Studies on the drug release properties of polysaccharide multilayers encapsulated ibuprofen microparticles

    Langmuir

    (2001)
  • X. Zhao et al.

    PH-sensitive fluorescent hepatocyte-targeting multilayer polyelectrolyte hollow microspheres as a smart drug delivery system

    Mol. Pharm.

    (2014)
  • Z. Tang et al.

    Biomedical applications of layer-by-layer assembly: from biomimetics to tissue engineering

    Adv. Mater.

    (2006)
  • K.M. Vårum et al.

    Structure-property relationship in chitosans

  • L. Illum et al.

    Chitosan as a delivery system for the transmucosal administration of drugs

  • Cited by (30)

    • The encapsulation of probiotics by polysaccharides

      2022, Polysaccharide Nanoparticles: Preparation and Biomedical Applications
    • Bioderived materials that disarm the gut mucosal immune system: Potential lessons from commensal microbiota

      2021, Acta Biomaterialia
      Citation Excerpt :

      More recent approaches utilize biomaterial encapsulation strategies to couple and protect probiotic and prebiotic components and enhance localized delivery to the gut. For instance, probiotic L. acidophilus has been encapsulated in layers of chitosan and β-glucan for gastric protection and prebiotic effect, respectively [282]. In addition, alginate has been used to co-encapsulate Lactobacillus plantarum with the prebiotic oligosaccharide arabinoxylan to form GI-stable microspheres, and the same probiotic has also been encapsulated with docosahexaenoic fatty acid using alginate-pectin-gelatin biocomposites.

    • Application of Pleurotus ostreatus β-glucans for oil–in–water emulsions encapsulation in powder

      2020, Food Hydrocolloids
      Citation Excerpt :

      For the microencapsulation of sunflower oil by spray drying, a mixture of MD and acacia gum (AG) is frequently used (Gharsallaoui, Roudaut, Chambin, Voilley, & Saurel, 2007; Munoz-Ibanez et al., 2016; Tolun, Artik, & Altintas, 2020). Since the field of encapsulation is expanding, research is required to study the possibility of using alternative and naturally occurring compounds, with health effects and available at low cost as support materials or emulsifiers for a wide variety of active compounds (Falco, Sotres, Rascón, Risbo, & Cárdenas, 2017). In this context, the basidiomycete P. ostreatus can make a valuable contribution as it can be cultivated on different by-products with a limited capital investment and technical skills.

    View all citing articles on Scopus
    View full text