Development of Probiotic Formulations for Oral Candidiasis Prevention: Gellan Gum as a Carrier To Deliver Lactobacillus paracasei 28.4

Abstract

Probiotics might provide an alternative approach for the control of oral candidiasis. However, studies on the antifungal activity of probiotics in the oral cavity are based on the consumption of yogurt or other dietary products, and it is necessary to use appropriate biomaterials and specific strains to obtain probiotic formulations targeted for local oral administration. In this study, we impregnated gellan gum, a natural biopolymer used as a food additive, with a probiotic and investigated its antifungal activity against Candida albicansLactobacillus paracasei 28.4, a strain recently isolated from the oral cavity of a caries-free individual, was incorporated in several concentrations of gellan gum (0.6% to 1% [wt/vol]). All tested concentrations could incorporate L. paracasei cells while maintaining bacterial viability. Probiotic-gellan gum formulations were stable for 7 days when stored at room temperature or 4°C. Long-term storage of bacterium-impregnated gellan gum was achieved when L. paracasei 28.4 was lyophilized. The probiotic-gellan gum formulations provided a release of L. paracasei cells over 24 h that was sufficient to inhibit the growth of C. albicans, with effects dependent on the cell concentrations incorporated into gellan gum. The probiotic-gellan gum formulations also had inhibitory activity against Candida sp. biofilms by reducing the number of Candida sp. cells (P < 0.0001), decreasing the total biomass (P = 0.0003), and impairing hyphae formation (P = 0.0002), compared to the control group which received no treatment. Interestingly, a probiotic formulation of 1% (wt/vol) gellan gum provided an oral colonization of L. paracasei in mice with approximately 6 log CFU/ml after 10 days. This formulation inhibited C. albicans growth (P < 0.0001), prevented the development of candidiasis lesions (P = 0.0013), and suppressed inflammation (P = 0.0006) compared to the mice not treated in the microscopic analysis of the tongue dorsum. These results indicate that gellan gum is a promising biomaterial and can be used as a carrier system to promote oral colonization for probiotics that prevent oral candidiasis.

Keywords: Candida albicans; Lactobacillus paracasei; gellan gum; hydrogels; oral candidiasis.

Copyright © 2020 American Society for Microbiology.

Figures

FIG 1

Recovery of L. paracasei 28.4 from the probiotic-gellan gum formulations after storage for 7 days. Shown are concentrations of L. paracasei 28.4 cells (CFU per milliliter) recovered from the probiotic formulations in gellan gum (1% to 0.6% [wt/vol]) after storage at room temperature and at 4°C. Student's t test was used to compare the experimental groups (room temperature × 4°C) for each day of the experiment.

FIG 2

Recovery of L. paracasei 28.4 from the probiotic-gellan gum formulations after storage under different conditions for long periods of time. Shown are the concentrations of L. paracasei 28.4 cells (CFU per milliliter) recovered from the probiotic formulation of 1%, after 14 weeks, storage under the following conditions: nonlyophilized at room temperature, nonlyophilized at 4°C, or lyophilized at room temperature.

FIG 3

Evaluation of delivery system of the probiotic-gellan gum formulations. Shown are the concentrations of L. paracasei 28.4 cells (CFU per milliliter) released from the probiotic formulations (1 to 0.6% [wt/vol]) when in the presence of PBS or artificial saliva. The samples were followed and evaluated continuously from 15 to 60 min. Student's t test was used to compare the PBS and artificial saliva groups. OD570, optical density at 570 nm.

FIG 4

Analysis of C. albicansin vitro biofilms. Biofilms were formed in contact with L. paracasei suspension or probiotic-gellan gum formulations at 1 and 0.6%. (A to C) Numbers of C. albicans viable cells determined by CFU per milliliter count. (D to F) Values of optical density obtained in the biofilm biomass analysis. Student's t test was used to compare the control group with the experimental groups.

FIG 5

(A to F) Scanning electron microscopy images of biofilms formed in vitro on resin surfaces, as follows: biofilms formed by only C. albicans (ATCC 18804) (A), biofilms formed by only L. paracasei 28.4 suspension (B), C. albicans biofilms pretreated with L. paracasei 28.4 suspension (C), biofilms formed only by probiotic formulation at 1% (D), C. albicans biofilms pretreated with probiotic formulation at 1% (E), and C. albicans biofilms pretreated with probiotic formulation at 0.6% (F). It is possible to observe the presence of L. paracasei cells (▾), yeasts (*), and hyphae (→) of C. albicans.

FIG 6

Recovery of C. albicans or L. paracasei from the oral cavity of mice. (A) Evaluation of delivery system of the probiotic-gellan gum formulation in the oral cavity of healthy mice. Concentration of L. paracasei 28.4 (CFU per milliliter) recovered after 10 days from oral cavity of mice treated with L. paracasei 28.4 suspension and probiotic formulation of 1%. Student's t test was used to compare the experimental groups. (B) C. albicans CFU/milliliter recovered from the oral cavity of mice infected by C. albicans and pretreated with PBS (control group), gellan gum formulation of 1% not incorporated with L. paracasei (gellan gum group), and L. paracasei 28.4 suspension or probiotic-gellan gum formulation of 1% (ANOVA and Tukey’s test). (C) L. paracasei 28.4 CFU per milliliter recovered from the mice infected by C. albicans and pretreated with L. paracasei 28.4 suspension or probiotic formulation of 1% (Student's t test).

FIG 7

Quantifications of candidiasis lesions formed on the dorsum tongue of mice infected with C. albicans. Mice were pretreated with PBS (control group), 1% gellan gum formulation without L. paracasei (gellan gum group), and L. paracasei 28.4 suspension with 1% probiotic-gellan gum formulation. (A to D) Scores and median obtained in the quantification of macroscopic lesions (A), count of yeasts and hyphae in histological cuts stained by PAS (B), and epithelial lesions (C) and inflammatory infiltrate (D) determined in H&E cuts (Kruskal-Wallis and Dunn’s posttest; different letters correspond to the statistically significant differences between groups).

FIG 8

Analysis of the dorsum tongue of mice infected with C. albicans. The mice were pretreated with PBS (control group, top row), a 1% gellan gum formulation without L. paracasei (gellan gum group, second row), L. paracasei 28.4 suspension in 1% (third row), and a probiotic-gellan gum formulation (bottom row). (A) Macroscopic aspects of the dorsum tongue are the presence of white patches of candidiasis (→) in the groups not treated with L. paracasei. (B) Images of histological cuts stained by periodic acid-Schiff (PAS), where hyphae and yeast can be observed in the epithelium-keratinized layer (→). (C) Images of histological cuts stained by hematoxylin and eosin (H&E), where intraepithelial microabscesses and infiltrate inflammatory in the connective tissue were observed (→).