Oxgall

The strains were washed twice in PBS, resuspended in a
simulated pancreatic juice (mMRS broth with different
concentrations of bile salts (Oxgall, Merck, USA) - 0.3%,
0.5%, 1%, 1.5%, 2%, and 3% v/v) and incubated at 37 °C
for 9 h. A sample in MRS (pH 6.5) without bile salts was
used as a control probe. The resistance to bile salts was
determined by plate count method as described previously
and expressed as log 10 values of colony-forming units per
ml (CFU ^–1).

Flaxseed oil cake (FOC) was purchased from ACS Sp. z o.o. (Bydgoszcz, Poland). The proximate composition of FOC (based on supplier information) was: solids—80.50%, including: proteins—41.97%; carbohydrates—27.99%; fiber—6.29%; fat—6.11%; ash—4.50%. Lacticaseibacillus rhamnosus GG (ATCC53103), was procured from ATCC (Manassas, VA, USA). Buffered peptone water, microbiological agar, MRS agar and broth were purchased from Oxoid (Basingstoke, UK). Potassium chloride, sodium chloride, potassium thiocyanate, disodium hydrogen phosphate, monosodium dihydrogen orthophosphate, calcium chloride, sodium hydrogen carbonate, hydrochloric acid, ammonium chloride, sodium peroxide, urea, α-amylase, uric acid, mucin from porcine stomach (type II), glucose, glucuronic acid, glucosamine hydrochloride, bovine serum albumin (BSA), pepsin, pancreatin, oxgall, Triton X-100, cholesterol, ethanol, ninhydrin, glacial acetic acid, cadmium chloride and hexadecane were purchased from Merck Chemical (Saint Louis, MI, USA). All reagents were of analytical grade.

Bile tolerance ability of bacterial strains was determined by using MRS broth supplemented with (0.3 or 0.5% w/v) bile salts (Oxgall, Merck) and MRS without bile salts served as control. Overnight grown bacterial cells (A₆₀₀ = 1.5) were harvested, washed with distilled H₂O, resuspended in MRS broth and incubated at 37 ± 2°C. One mL sample was withdrawn after 3 and 5 h, subsequently plated onto MRS agar. Bacterial viability was assessed as mentioned in the previous experiment.

The isolates were analyzed for their ability to thrive separately in mMRS having 0.3% oxgall (Merck) and mMRS with pH adjusted to 3.9 using hydrochloric acid by CFU/ml counts after three-hour exposure. The ability of the strains to utilize various sugars was assessed by growing them in minimal mMRS medium containing 2% (w/v) sugars viz., lactose, maltose, arabinose, melezitose, melibiose, raffinose, salicin, sorbitol, trehalose, sucrose, mannose, and fructose and comparing OD₆₀₀ with those obtained with 2% (w/v) glucose. Nitrate reduction was examined on nitrate broth using sulphanilic acid and α-naphthylamine with zinc dust. H₂S production, arginine dihydrolase activity, and urease activity were analyzed on TSI agar, arginine dihydrolase broth, and urea agar, respectively. Catalase activity was assessed using 15% H₂O₂ on a glass slide. The pH reduction was examined after 3 days of the growth in 10% skim milk media at 37 °C.

Approximately 100 mg of the beads was mixed by gentle agitation and then incubated in 20 ml of a low pH of PBS solution (pH 2.0) (Haghshenas et al., 2015; Picot & Lacroix, 2004) for 2 hr at 37°C to assess the survival rate of the encapsulated and free probiotic bacteria. Phosphate buffer (pH 7.4) was used to disintegrate the treated beads. The CFU of the bacterial cells was counted on MRS agar using the pour plate technique.
The viability of the encapsulated E. durans IW3 at high bile salt solution was evaluated according to the method described by Ma et al. (2008). The encapsulated probiotic products were transferred into 1 g/100 ml of bile salt solution (0.5% w/v oxgall; Merck, Germany) and then incubated at 37°C with gentle shaking at pH 7.4 for 2 hr. The number of viable cells was counted following the procedure described in the section of “Free and encapsulated bacterial enumeration.” Finally, the survival rate (%) of the bacteria was measured as follows: $\text{Survival rate}(\%)=(\text{log CFU/g capsules after treatment}/\text{log CFU/g capsules before treatment})\times 100.$

To determine the tolerance of LAB strains to artificial gastric juice and bile salts, which are similar to the conditions of the human upper gastrointestinal tract, an in vitro methodology was applied. Briefly, the fresh cultures were centrifuged at 6,000 × g for 2 min and washed two times with sterile saline for future use. The bacterial pellets were suspended in artificial gastric juice (MRS broth, pH 2.5, supplemented with 1% (w/v) pepsin) and incubated at 37°C for 4 h. After that, the pellets were harvested and washed two times in sterile saline and resuspended in MRS supplemented with 0.4% (w/v) bile salts (Oxgall, Sigma-Aldrich, Germany). After being incubated at 37°C for 12 h, the bacteria were serially diluted and plated on the MRS agar medium. The bacterial colony counts were calculated to determine the tolerance to artificial gastric juice and bile salts.