Lactobacillus rhamnosus gg

Two probiotic dietary supplements commercially available in Poland were purchased:

LacidoEnter capsules (Institut Rosell, Montreal, Canada; batch numbers HG09241 and HI17731; expiry date 01/2017 and 03/2017, respectively) containing lyophilized Saccharomyces boulardii CNCM I-1079 in a declared amount of 5 × 10⁹ CFU per dose,

Dicoflor 60 capsules (Dicofarm, Rome, Italy; batch numbers SM513 and SM514; expiry date of both batches 03/2017) containing Lactobacillus rhamnosus GG (ATCC 53103) in a declared amount of 6 × 10⁹ CFU per dose.

In order to obtain indistinguishable formulations, the content of the original capsules were transferred to the new gelatin capsules (ACG Associated Capsules, Maharashtra, India) and filled with a mixture of maltodextrins (Pepees, Łomża, Poland) quantum satis. Placebo products were obtained by filling the same capsules with the mixture of maltodextrins only. The formulations were prepared using a manual capsule filling device (Capsunorm; Eprus, Bielsko-Biała, Poland).
At the completion of the study, a sample of each product was subjected to internal quality control to determine the viability of probiotic organisms, and the obtained results were compared to the recommended minimum daily doses of the probiotics [21 (link),27 (link),28 (link)]. Details on the methods and results of this quality control are described in Supplementary Material S2.

To perform longitudinal host–probiotic co-cultures in a Leaky Gut Chip, we used two live biotherapeutic products, Lactobacillus rhamnosus GG (ATCC 53103) and over-the-counter VSL#3 formulation (Sigma-Tau Pharmaceuticals, Inc.). For an LGG pre-culture, we grew LGG cells in a 15-mL sterilized test tube containing 3 mL autoclaved Lactobacilli MRS Broth (MRS; Difco). For a VSL#3 pre-culture, we inoculated VSL#3 power in the mixture (3 mL) of autoclaved MRS broth and Reinforced Clostridial Medium (RCM; Difco) at 1:1 (v/v) in a 15-mL sterilized tube. Next, we incubated the inoculated test tubes in a GasPak EZ container (BD Diagnostics) containing two anaerobic gas-generating GasPak EZ sachets (BD Diagnostics) without shaking for 12–18 h. After a seed culture, we centrifuged the culture broth at 10,000×g for 1 min, aspirated the supernatant, resuspended the cell pellet with an antibiotic-free, l-cysteine-containing culture medium and adjusted the final cell density at 1 × 10⁷ CFU/mL. Using a 200P micropipette, we inoculated bacterial cells into the upper microchannel of a germ-free Leaky Gut Chip that was pre-conditioned with an antibiotic-free, AOI-created culture microenvironment, then incubated the setup without flow at 37 °C for 1 h. After incubation, we resumed the flow (50 μL/h) and physical deformations until the experiments were finished.

All chemicals and monosaccharide standards were at least of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). The dextran M_w narrow standards were supplied by Pharmacosmos A/S (Holbaek, Denmark). In addition, 50%_w/v sodium hydroxide extra pure solution for anion exchange chromatography eluent preparation was sourced from Acros Organics (Geel, Belgium).
Bacteroides galacturonicus (DSM 3978) and Bacteroides xylanisolvens (DSM 18836) were sourced from the DSMZ (Braunschweig, Germany). Lactobacillus rhamnosus GG (ATCC 53103), Bifidobacterium longum subsp. infantis (ATCC 15697), and Lactobacillus plantarum subsp. plantarum (ATCC 14917) were sourced from the ATCC (Manassas, VA, USA). Bacteroides thetaiotaomicron and Bifidobacterium faecale were isolated from human stool samples, as described in [30 (link)].

Lactobacillus rhamnosus and Caco-2/HT29-MTX tri-culture methods have previously been described (Richter et al., 2018 (link)). Briefly, Lactobacillus rhamnosus GG (L. rhamnosus) was purchased from American Type Culture Collection (ATCC 53103). Lactobacillus rhamnosus stock was serially diluted in 0.9% saline solution, plated on MRS agar (Difco^TM) and allowed to grow in a 5% CO₂ incubator at 37°C for 24 h. The OD of each dilution was quantified at a wavelength of 600 nm. To correlate absorbance with concentration (CFU ml⁻¹), CFUs of L. rhamnosus at each dilution were counted and plotted to make a growth curve. Prior to each experiment, bacterial concentrations were determined using OD and added to the apical chamber of the Transwells in high- or low-glucose DMEM at a concentration of 10³ CFU ml⁻¹.

Escherichia coli (ATCC 11775) and Lactobacillus rhamnosus GG (ATCC 53103), both originally isolated from humans, were purchased from American Type Culture Collection. A tri-culture model was constructed by introducing E. coli or L. rhamnosus [described by (14 (link))] into the Caco-2/HT29-MTX co-culture model. Overnight cultures of E. coli were grown in nutrient broth (Difco^TM) while L. rhamnosus were grown in MRS broth (Difco^TM). Prior to inoculation, bacterial concentrations of the overnight cultures were estimated using OD₆₀₀, using an established calibration curve. Bacteria were introduced into the wells or apical chamber of the Transwells, in high or low glucose DMEM, at a concentration of 10³ or 10⁴ CFU/mL. For combination studies with bacteria and food additives, the bacterial inoculum concentrations were prepared in the respective food grade or chemical grade samples. Establishment of the calibration curve was obtained by correlating the absorbance with viable counts (CFU/mL). Each bacterial culture was serially diluted, and to each dilution, measurements of OD₆₀₀ and viable counts were performed. Viable counts were performed by drop plating each dilution of E. coli onto nutrient agar (Difco^TM) and L. rhamnosus onto MRS Agar (Difco^TM), which were then incubated for 24 to 48 h, at 37°C with 5% CO₂.