Simultaneous Encapsulation of Probiotic Bacteria (Lactococcus lactis, and Lactiplantibacillus plantarum) in Calcium Alginate Hydrogels

Abstract

Encapsulation in alginate hydrogel microspheres is an effective method for protecting and improving the survival of lactic acid bacteria in different environments. This research aims to expand the knowledge about the structure/property relationship of calcium alginate microspheres loaded with a mixture of autochthonous probiotic bacteria (Lactococcus lactis and Lactiplantibacillus plantarum). A novel hydrogel formulation (FORMLAB) was prepared by ionic gelation and the molecular interactions between the FORMLAB constituents, surface morphology, structure, swelling degree, and release profile were characterized. The simultaneous encapsulation of two bacterial cultures in the same compartment does not diminish their viability. The binding of calcium ions to bacterial cells creates favorable conditions for the propagation of the encapsulated bacteria. The molecular interactions between the FORMLAB constituents are complex, involving mainly hydrogen bonds and electrostatic interactions. With a very high degree of swelling followed by low crosslinking, the surface of the microspheres covered with bacterial cells and diffusion through the hydrogel matrix allow for the delivery of probiotics at the right time. The findings suggest that bacterial cells are efficiently delivered from calcium alginate microspheres, offering promising applications in the development of functional foods, especially in cheese production.

Keywords: Lactiplantibacillus plantarum; Lactococcus lactis; calcium alginate hydrogel; simultaneous encapsulation.

Figure 1

Microphotographs of L. plantarum and Lc. lactis cell mixture obtained with LM (a) without and (b) with staining. SEM images of (c) L. plantarum cells and (d) Lc. lactis cells. SEM microphotograph of L. plantarum and Lc. lactis cell mixture: (e) surface morphology and (f) surface elemental analysis using dispersive X-ray spectroscopy (expressed in the atomic weight percent). AFM of L. plantarum and Lc. lactis cell mixture: (g) detailed 2D topographic height image with dimensions of 15 × 15 µm², and (h) 3D-AFM height image with dimensions of 5 × 5 µm², respectively. Bars are indicated on each image.

Figure 1

Microphotographs of L. plantarum and Lc. lactis cell mixture obtained with LM (a) without and (b) with staining. SEM images of (c) L. plantarum cells and (d) Lc. lactis cells. SEM microphotograph of L. plantarum and Lc. lactis cell mixture: (e) surface morphology and (f) surface elemental analysis using dispersive X-ray spectroscopy (expressed in the atomic weight percent). AFM of L. plantarum and Lc. lactis cell mixture: (g) detailed 2D topographic height image with dimensions of 15 × 15 µm², and (h) 3D-AFM height image with dimensions of 5 × 5 µm², respectively. Bars are indicated on each image.

Figure 2

Variation in (a) average zeta potential (ζ) and (b) average hydrodynamic diameter (d) of particles with calcium chloride concentration (c(CaCl₂)) of suspended mixtures of lactic acid bacteria cells. Standard deviations are denoted. Inserted microphotographs illustrate the growth of aggregates of bacteria with an increase in calcium ion concentration.

Figure 3

FTIR spectra of freeze-dried LAB (black line), ALG/Ca (red line), and FORMLAB (blue line) microspheres.

Figure 4

LM microphotographs of (a) ALG/Ca and (b) FORMLAB microspheres. SEM microphotograph of (c) ALG/Ca (Lc. lactis marked with the red line and L. plantarum with the white line) and (d,e) FORMLAB surface, and (f) surface elemental analysis using dispersive X-ray spectroscopy (expressed in the atomic weight percent). Bars are indicated on each image.

Figure 5

Variation in LAB strain abundance (log CFU g⁻¹) in the FORMLAB microspheres with time (t).

Figure 6

Fraction of LAB (f) release from FORMLAB microspheres with time (t). The data are shown as a mean value with standard deviation.

Figure 7

pH change with time (t) in a model system. The data are shown as mean values with standard deviation.