Mrs broth

LAB strains were streaked twice on De Man, Rogosa, and Sharpe (MRS) agar (Oxoid, Oslo, Norway), incubated anaerobically in anaerobic incubation containers with GasPak EZ Anaerobe container system sachets w/indicator (BD, Oslo, Norway) at 25 °C for 2–5 days, transferred into MRS broth (Oxoid, Oslo, Norway), and cultivated at 15 °C in 15 mL tubes with MRS broth to assess their ability to proliferate at 15 °C. Cell growth was monitored until the cultures reach the stationary phase (approx. 120 h of cultivation) by measuring the optical density at 600 nm (OD₆₀₀; Shimadzu UV 1800; Shimadzu, Duisburg, Germany) after proper dilution. For preparation of LAB inoculums for the coculture assay, a colony was transferred from the MRS agar plate into MRS broth for overnight incubation at 15 °C to adapt to the lower temperature in the inhibitory assay. The cultures were standardized to a concentration of 10⁸ CFU/mL by proper dilution in MRS broth to obtain an optical density of 0.22 at 600 nm (Shimadzu UV 1800; Shimadzu, Duisburg, Germany).

L. sakei DSM 15831 was obtained from DSM, Germany. The bacteria were cultured in de Man Rogosa Sharpe (MRS) broth (Oxoid Ltd, Cambridge, UK) medium at 30°C in a microaerophilic chamber for 48 h. Microbial cells were harvested at 7000×g for 10 min and subsequently added to either to MRS broth or phosphate buffer saline (PBS) to achieve a target population of ca. 10⁷ cfu/mL for subsequent ultrasound treatment and analysed further. MRS broth and PBS (Oxoid Ltd, Cambridge, UK) were prepared as per the manufacturer’s instruction.

The D. hansenii strains were grown on MEMA at 25 °C for 48–72 h. Suspensions were prepared by adding a loopful of cells to peptonized water (0.7% NaCl and 0.1% peptone in 1000 mL of water). The density of the yeast cultures was determined spectrophotometrically at an optical density at 600 nm (OD₆₀₀) of 0.1, after which serial dilutions were prepared to obtain the concentrations used for the experiments. The L. buchneri strains were maintained at −80 °C in vials with MRS broth + 30% (v/v) glycerol (Oxoid, Italia). The strains were grown in MRS broth (Oxoid, Italia) at 37 °C for 24 h. Then, the suspensions were standardized to an optical density at 600 nm (OD₆₀₀) of 0.1, and their microbial counts were evaluated. Specifically, serial dilutions were performed in peptonized water, and 0.1 mL of each dilution was spread onto MRS plates (Oxoid, Italy). The plates were incubated at 37 °C for 48 h, and the resulting colonies were counted. Each suspension contained approximately 7 log CFU (colony-forming unity) mL⁻¹. A mixed suspension of both microorganisms was generated by mixing 5 mL of 7 log D. hansenii mL⁻¹ to 5 mL of 7 log L. buchneri mL⁻¹ to obtain a final suspension at 7 log CFU mL⁻¹.

To prepare Lplant-B80 freeze-dried pellets, 50 mL of de Man, Rogosa, and Sharp (MRS) broth (Oxoid) was inoculated with 24-hour colonies of Lplant-B80 and incubated at 37°C for 24 hours which was inoculated onto 1-L MRS broth. Bacteria were harvested after 24 h by centrifugation (4400 ×g, 15 minutes, 4°C) and washed 1x with phosphate buffered saline. For freeze-drying, 6% dextran and 0.9% of NaCl solution (Gentran 70®) was used to resuspend bacteria (1 : 1 w/v). The suspensions (aliquoted as 2.5 mL in 4-mL glass test tubes) were frozen at −80°C for 24 h prior to freeze-drying at −50°C. The resulting freeze-dried pellets were stored sealed in the tubes with a rubber cap at −80°C and used within 3 weeks of preparation. The concentration of live Lplant-B80 was verified in random pellets. Culture of leftover milk from feeding bottle confirmed viability at ingestion. A set of sealed tubes containing the pellets were left at room temperature (23°C; 70% humidity) and monitored for bacterial viability at 6, 12, and 48 months.

The skimmed milk powder used for manufacturing the Yakult-like fermented skimmed milk was Skimmillac, purchased from Millac Foods Pvt Ltd. (Karachi, Sindh, Pakistan). Other ingredients, including glucose, sugar, and natural flavor (citrus), were purchased from the local supermarket in Faisalabad, Punjab, Pakistan. All chemicals were purchased from Merck (Merck KGaA, Darmstadt, Germany) and Sigma-Aldrich (Sigma-Aldrich, Tokyo, Japan). The probiotic strains Lacticaseibacillus casei (Lc) and Lacticaseibacillus rhamnosus (Lr) were isolated from lactase. Lc and Lr were cultivated on de Man, Rogosa and Sharpe (MRS) broth (Oxoid, Ltd., Basingstoke, Hampshire, UK) and incubated for 15 to 18 h at 37 °C anaerobically, and the activated culture was again inoculated into MRS broth at 37 °C for 18 h. The microbial cultures were kept in broth medium in a cold store (4 °C) with tight caps until they were needed, in order to keep the cultures fresh for later use.

Lactobacillus rhamnosus strains grown in MRS broth (Oxoid, Milan, Italy) at 37°C were taken in the mid-exponential phase and centrifuged at 7,500 rcf for 15 min at 4°C (Centrifuge Eppendorf, 5804R). The pellet was washed 2 times with 1X phosphate buffer (1X PBS) and 1% resuspended in Erlenmeyer flasks containing 500 mL of sterilized modified MRS (final pH 6.2) prepared following the standard formula (MRS broth, Oxoid, Milan, Italy) with glucose and citrate omitted, and added of 10 g/L (final concentration) of each filter-sterilized prebiotic (Filter Unit Red 0.22-μm pore size; Schleider&Schuell, Dassel, Germany). Filter-sterilized glucose (Sigma-Aldrich, Italy) at the same concentration was used for comparative purposes. The growth was assessed by plate counts on MRS agar (Oxoid) at regular time intervals. Two replicates were made for each experiment. The growth kinetic parameters were estimated with the D-model of (Baranyi and Roberts, 1994 (link)) using the excel add-in DMFit v.3 (Baranyi and Le Marc, 1996 ). In detail, maximum specific grow rate (μmax), lag phase, initial load values (y_0) and final load values (y_end) were evaluated. Moreover, the load increase was evaluated as reported by the Equation 1.
Three independent experiments were performed and the results were reported as average.

Both cocci and rod-shaped strains were phenotypically characterized according to the following criteria: appearance by Gram staining; ability to grow after incubation in MRS broth (Oxoid, Basingstoke, UK) at 10 °C and 45 °C for cocci and 15 °C and 45 °C for rods; production of CO₂ from glucose using inverted Durham tube in MRS broth and incubation at 30 °C. For cocci, distinguished extra tests were specifically used: tolerance at 6.5% NaCl, ability to grow in MRS substrate with vancomycin, and esculin hydrolysis [20 ,21 ]. The ability to ferment carbohydrates was studied by observing the gas production and the color changes of the tubes that contained the sugar substratum for fermentation (37 °C for 24 h) (Sigma-Aldrich, St. Louis, MO, USA).