Lab-scale production of postbiotic proteins from Bifidobacterium adolescentis with antiviral and epithelial-protective properties

Abstract

Postbiotics produced by probiotic bacteria are gaining attention as multifunctional, food-derived agents with potential applications in human and animal health. This study investigates the production and biological activity of protein-rich postbiotics from Bifidobacterium adolescentis, cultivated under controlled conditions in a 3-liter bioreactor as a laboratory-scale model for functional ingredient development. Culture parameters were improved, and one representative batch was selected for biological evaluation. The postbiotic preparation was tested for cytotoxicity using MA104 (renal) and C2BBe1 (intestinal) epithelial cell lines through viability and cell death assays, confirming its safety across a range of concentrations. To assess its functional activity, we evaluated its ability to reduce rotavirus infection and preserve epithelial integrity. The postbiotic significantly reduced viral infectivity and maintained cytoskeletal architecture in infected intestinal cells, supporting its potential protective role. These findings suggest that B. adolescentis-derived postbiotics may serve as safe and biologically active compounds with potential applications in intestinal health and viral infection management.

Keywords: Bifidobacterium adolescentis; antiviral activity; bioreactor; cytoskeletal integrity; functional ingredients; gut epithelium; postbiotics; rotavirus.

FIGURE 1

Growth kinetics of Bifidobacterium adolescentis under varying microculture conditions. (A) Effect of agitation speed (50, 100, and 150 rpm) on the growth in a 5 mL MRS broth medium under anaerobic conditions, with a 5% inoculum, at 37 °C for 54 h. (B) Effect of inoculum concentration (5% and 10%) on the growth in a 25 mL MRS broth medium under anaerobic conditions, at 37 °C, 150 rpm, over 54 h. (C) Effect of medium concentration (undiluted MRS, 1:2 diluted MRS, and 1:5 diluted MRS) on the growth of in a 25 mL culture under anaerobic conditions, with a 5% inoculum, at 37 °C, 150 rpm, over 54 h. Data points represent the mean from three independent experiments, with error bars indicating standard deviation. Statistical analysis was conducted to assess significant differences between groups, with the appropriate test (parametric or non-parametric) chosen based on data distribution.

FIGURE 2

Growth characterization of Bifidobacterium adolescentis in a lab-scale anaerobic bioreactor. (A) Biomass dry weight (g/L) over time during batch fermentation in a 3 L bioreactor with 1.5 L MRS medium, inoculated at 5% (v/v) and maintained at 37 °C with 200 rpm agitation under anaerobic conditions for 54 h. Data points represent the mean ± standard deviation of three independent measurements. (B) First-order kinetic model fitted to the biomass growth data from panel A. The red dotted line indicates the linear regression trend line, with the corresponding equation and R² value shown.

FIGURE 3

Effect of single and multiple doses of BaP on cell viability percentage (MTT colorimetric assay, Abs 540 nm) and morphology in MA104 and C2BBe1 Cells. (A) Effect of a single dose of 14 different concentrations of BaP over 24 h. (B) Effect of repeated administration of BaP at the MNTC every 48 h over 14 days. Each bar represents the mean cell viability percentage from three independent experiments; error bars indicate the standard deviation. Comparisons between CC and BaP MNTC and H₂O₂ were analyzed using the Mann-Whitney U test at a 95% confidence level. Significance levels are indicated as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. CC, cell control; H₂O₂, positive control. (C) Photographs of MA104 and C2BBe1 cells, either untreated or treated with BaP at the MNTC, were captured at 100× magnification using an Olympus CKX-41 microscope (Olympus Corporation, Tokyo, Japan) equipped with a Moticam camera and MoticImages software. The images display classic epithelial cells morphology in monolayers and highlight an increase in treated cell clusters.

FIGURE 4

Effect of single doses of 14 different protein concentrations from an Abiotic Control (AC, dark blue) and a Production Control (PC, light blue) on cell viability percentage in MA104 cells, as measured by the MTT colorimetric assay (Abs 540 nm). Each bar represents the mean percentage of cell viability from three independent experiments, with error bars indicating standard deviation. Data were analyzed using an independent samples Mann–Whitney U test. Differences between the evaluated concentrations and the cell control (CC) were significant. Significance levels are indicated as follows: **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. The cell control (CC) corresponds to untreated cells in medium without FBS.

FIGURE 5

Evaluation of cell death markers in MA104 and C2BBe1 cells after BaP MNTC exposure. Evaluation of cell death markers following exposure to BaP MNTC in MA104 (blue boxes) and C2BBe1 (red boxes) cells for 8 days every 48 h and medium was partially refreshed at each point. (A) Phosphatidylserine translocation assessed using Annexin V-FITC (488 nm filter). (B) DNA fragmentation evaluated using propidium iodide (PI) fluorochrome (617 nm filter). Boxes represent the mean and interquartile range from three independent experiments with BaP MNTC exposure. Whiskers indicate the maximum and minimum values. Comparisons between the cell control (CC) and BaP MNTC were analyzed using Student’s t-test at a 95% confidence level. Significance levels are indicated as follows: *p ≤ 0.05. CC, cell control; H₂O₂ positive control.

FIGURE 6

In vitro anti-rotavirus activity in MA104 Cells. Rhesus rotavirus (RRV) was co-incubated with two concentrations of (A) BaP, (B) AC, and (C) PC for 1 h at 37°C. MA104 cells were subsequently exposed to the BaP/AC/PC-RRV mixture and incubated for an additional 9 h. Viral infection levels were quantified using immunofluorescence to measure FFU/mL, with infection percentages calculated relative to the positive infection control (RRV). The scatter plot displays mean values with error bars representing standard deviation from three independent samples. Comparisons between the infection positive control (RRV) and BaP treatments were analyzed using Student’s t-test at a 95% confidence level, with significance levels indicated as follows: *p ≤ 0.05, ***p ≤ 0.001. RRV, infection positive control; MOCK, uninfected control. Photographs were captured using an Olympus CKX-41 inverted microscope (Olympus Corporation, Tokyo, Japan) equipped with a Lumin Epi-Fluorescence Module and a Moticam camera with MoticImages software, at 100× magnification. The images display Fluorescent Focus Units (FFU) in green, representing RRV-infected cells.

FIGURE 7

Effect of BaP at MNTC and half-maximal concentration on cytoskeletal architecture in RV-Infected C2BBe1 cells at 6 and 9 hpi. Photomicrographs display cell morphology and distribution in uninfected control cells (MOCK), infected untreated control cells (+RRV), and each BaP treatment condition. Green fluorescence (F-actin) indicates cytoskeletal organization. Red boxes highlight representative areas where morphological changes and differences in F-actin organization are most evident.