Mrs broth

Lactobacillus acidophilus (LA_5) (PTCC1608) and Bifidobacterium bifidum (BB_12) (PTCC1644), stored at the Bacterial and Fungal Collection Center of the Iranian Scientific and Industrial Research Organization, were used in the study.
The strains L. acidophilus and B. bifidum were reactivated and cultured, respectively, in the medium MRS broth (Sigma‐Aldrich, Germany) at 37°C and MRS modified (MRS broth with 0.05 g/100 g of L‐cysteine hydrochloride) at 37°C under anaerobic conditions (using Gazpack System) until exponential phase was reached. After incubation, the grown cells were harvested by centrifugation (4500 × g, 15 min, 4°C) and washed twice with sterilized peptone water solution (0.1 g 100 mL⁻¹).
Each probiotic culture was subsequently added to milk for yogurt preparation at a concentration of about 1 × 10⁸ cfu mL⁻¹ (Mehdizadeh et al., 2019 (link)).

Cultures of Lactobacillus plantarum strains PCS 20, PCS 26 (Bionutritech, France) and Lactobacillus rhamnosus LGG (ATCC, UK) were maintained at − 80°C in 20% (v/v) glycerol (Merck, Darmstadt, Germany) with MRS broth (Merck). Propagation, prior testing on cell lines, was later done in MRS broth (Merck, Darmstadt, Germany) for 24 h at 37°C and under anaerobic conditions by the use of Anaerogen (Oxoid Ltd, Hampshire, UK).

LAB strains were further subjected to bile tolerance. Briefly, bovine bile salt (Sigma, St. Louis, MO, USA) was added to two MRS broth (Merck) volumes to obtain the final concentrations of 0.3% (w/v) and 2% (w/v) separately. The MRS broth (Merck) with the added salt was then distributed into test tubes in 9 mL volumes each and autoclaved at 121 °C for 15 min after which it was cooled at 37 °C. The cooled sterilized tubes were then inoculated with 100 µL of overnight bacterial culture of known cell concentration (10⁶ cfu/mL). The MRS broth culture (Merck) without bile salt was used as a control. The test tubes were incubated at 37 °C in a water bath (GFLD83, Berlin, Germany). The bacterial count was periodically determined at initial stage, after 5 h and finally after 24 h by withdrawing 1 mL from each test tube, to conduct a 10-fold serial dilution then spread onto MRS agar plates. The plates were incubated anaerobically at 37 °C for 48 h. The results were recorded as log counts cfu/mL. All experiments were conducted in triplicate.

Bacteria underwent heat adaptation according to Jewell and Kashket [16 (link)]. Test tubes containing aliquots of 20 ml of 30 hours fresh bacterial culture (37°C and 5% CO₂) in MRS broth (Merck GmbH, Germany) were treated at 60°C for 15 minutes. The survived and heat adapted strains were collected after further incubation of viable strains on MRS agar medium and after 48 hours incubation (temperature, 37°C and 5% CO₂). The experiments were repeated at higher temperatures of 65°C and 75°C and the adapted strains were stored at -80°C for subsequent use in the spray drying. Strains subcultured on MRS broth were enriched with 0.05% L-cysteine (Merck GmbH, Germany), at 37°C for 30 hours [15 (link)]. Following incubation under 5% CO₂ cells were harvested by centrifugation at 2000 rpm for 15 min, and were further re-suspended in sterile PBS-glycerol (20% v/v) solution and finally stored in 1mlcryotubes at -80°C.

Stock cultures of the tested microorganisms (Bifidobacterium bifidum and Lactobacillus casei) were maintained in cryovials at −40 °C with glycerol (20% v/v) used as a cryoprotective agent. For the revival of each microorganism, one cryovial was transferred into 10 mL MRS broth (Merck, 1.10661, Darmstadt, Germany) and incubated at 37 °C for 24 h; for the revival of Bifidobacterium bifidum, 3% v/v of L-cysteine HCl sol. was added to MRS broth to achieve anaerobic environment. For growth and use in the kinetic experiments, 100 μL of the above inocula were transferred individually into 10 mL MRS broth (with 3% v/v of L-cysteine HCl sol. for Bifidobacterium bifidum) and incubated at 37 °C for 18−20 h. The final suspensions were transferred into 90 mL MRS broth with modified pH value to 4.80 (HCl sol. 1 M) or 6.50 (Na₂HPO₄/NaH₂PO₄ buffer sol. 0.1 M), representing the model system of low and high pH value respectively, and served as the inocula for viability loss experiments (microbial cells in stationary phase and initial plate counted at approximately 10⁹ CFU/mL). The selection of MRS growth medium instead of a milk-based medium was based on the fact that the viability of the tested microorganisms will not be affected by the possible presence of any protective agent (e.g., lipids) of the bacterial cells.

Encapsulation materials were food grade, and the other materials for survival and physicochemical analysis were analytical grades. Whey protein isolate 90 (WPI) (JFO store, Jakarta, Indonesia) and gum arabic (GA) (ALMA Chemical, Demak, Indonesia) were purchased using local e-commerce in Indonesia. Microbiological growth media used were de Man-Rogosa-Sharpe (MRS) broth (MERCK, Darmstadt, Germany) and MRS agar (MERCK, Darmstadt, Germany). For GIT simulation, the MRS broth was supplemented with glucose (MERCK, Darmstadt, Germany), KH₂PO₄ (MERCK, Darmstadt, Germany), CaCl₂ (MERCK, Darmstadt, Germany), and KCl (MERCK, Darmstadt, Germany). The rehydration media for spray-dried probiotic and an additional supplement to gastrointestinal simulation media was NaCl (Himedia, Mumbai, India). The pH of gastrointestinal simulation media was maintained by adding HCl (MERCK, Darmstadt, Germany) and NaOH (ROFA, Bandung, Indonesia). P. acidilactici culture was obtained from Universitas Gadjah Mada (UGM), Food and Nutrition Culture Collection. The bacterial identity was confirmed through gram-staining and 16S rRNA sequencing.

The previous study by Marlida et al. [13 ] found a sum of 10 LAB of dadih origin which exhibited a potent capacity to produce GABA-based on thin-layer chromatography (TLC) and spectrophotometer. The 10 LAB were cultivated in 10 ml of MRS Broth (Merck) having a glutamic acid concentration of 50 mM, a pH of 5 and incubated at 30°C for 72 h. GABA content of the culture in the MRS Broth (1 ml) was measured and used for high-performance liquid chromatography (HPLC) analysis.