Anaerocult a

Five lactobacilli strains (L. salivarius MG242 (KCTC18554P), L. fermentum MG901 (KFCC11651P), L. plantarum MG989 (KFCC11650P), L. paracasei MG4272 (KCTC13822BP), and L. rhamnosus MG4288 (KCTC13823BP)), isolated from a healthy Korean woman’s vagina, were supplied by Mediogen Co., Ltd. (Jecheon, Korea). All strains were deposited in the Korean Collection for Type Cultures (KCTC, Daejeon, Korea). Each strain was activated by culture in de Man, Rogosa, and Sharpe (MRS) broth (Difco, Detroit, MI, USA) for 18 h at 37 °C [20 (link)]. The dried bacterial powder used for in vivo testing was supplied by Mediogen Co., Ltd. The lactobacilli strain mixture (LM5) was prepared by combining the five powdered strains in equal ratios.
G. vaginalis (KCTC5096) was obtained from the KCTC and sub-cultured in modified brain heart infusion (mBHI) broth (Difco, Detroit, MI, USA) supplemented with yeast extract (1%), maltose (0.1%), glucose (0.1%), and horse serum (10%) and cultured anaerobically using Anaerocult^® A (Merck, Darmstadt, Germany) in a sealed anaerobic jar.

Fecal samples from day 14, corresponding to the last day of the probiotic consumption (the first subsequent evacuation available was considered for a few samples when no evacuation occurred on the 14th day), were processed to verify the ability of the four probiotic strains to survive gastrointestinal transit. For this purpose, one gram of fecal sample was diluted in Maximum Recovery Diluent (Oxoid, Basingstoke, UK), homogenized in a sterile stomacher bag by using a Colworth Stomacher 400 instrument (Seward, West Sussex, UK) and plated on MRS supplemented with 0.05% cysteine and 5 µg mL⁻¹ streptomycin (scMRS). At least five dilutions per fecal sample were plated. After 48 h of incubation at 37 °C in anaerobic conditions with the use of Anaerocult A (Merck, Kenilworth, NJ, USA), all the colonies from each dilution plate were collected separately, and the biomass from the respective dilution plates was used for total DNA isolation as described above. Afterwards, qPCR with probiotic-specific primers was performed on the DNA isolated from the colony biomasses. The highest dilution giving a positive signal in qPCR and the obtained Cq value were used to calculate the “estimated minimum CFU number” (eCFU) for each investigated strain.

Phosphate buffer at 0.05 M and pH 8.0 was used to release the entrapped strain G4 in alginate-gelatin-genipin matrices because phosphate ions chelate calcium, thereby weakening the alginate for effective release of cells. The homogenized sample was diluted to appropriate concentration and spread on TPY agar (Scharlau-Chemie, Barcelona, Spain). The plates were subsequently incubated anaerobically using Anaerocult ®A (Merck, Darmstadt, Germany) for 48 h to 72 h at 37°C. The encapsulated cells were enumerated as log 10 cfu·mL⁻¹. The encapsulation yield (EY), which is a combined measurement of the efficacy of entrapment and survival of viable cells during the encapsulation procedure, was calculated as follows: $\begin{array}{c}\text{EY}=\left(\frac{N}{N_0}\right)\times 100,\end{array}$ where N is the number of viable entrapped cells released from the beads and N₀ is the number of free cells added to the biopolymer mix during the encapsulation procedure.

10 g of Mozzarella di Bufala Campana (MBC) samples were diluted in 90 mL sodium citrate solution (2% w/v) and homogenized in a BagMixer400 (Interscience, France). 60 μL of homogenate was inoculated in 50 mL of MRS medium (Oxoid Ltd., England) and incubated at 37°C for 48 h under anaerobic conditions (Anaerocult A, Merck, Germany), to obtain a bacterial titer of about 1 × 10¹⁰ Cfu/mL, corresponding to OD₆₀₀ = 3. Bacterial counts were obtained by serial dilution in quarter-strength Ringer's solution, followed by plating on MRS agar. Plates were incubated at 37°C for 48 h under anaerobic conditions. Independent colonies displaying different morphologies were isolated from the plates, grown as described above, and stored at −80°C in 15% (v/v) glycerol. Lactobacillus rhamnosus GG (LGG, ATCC53103) was used as probiotic control where indicated.

After the incubation of the test surfaces and controls in microbial suspensions for 2 h and 72 h, respectively, the samples were washed thrice with 2 mL 0.9% saline solution (NaCl) to remove the non-adherent microorganisms and were subsequently transferred into a 5 mL tube with 1 mL NaCl [43 (link)]. The adherent microorganisms/biofilms were then dislodged from the aligner, enamel and bracket surfaces by sonification for 4 min (5 × 10% cycle, power 60%). Afterward, the suspensions of the aligners and controls were serially diluted up to 1:10⁴ in 0.9% NaCl with the aid of a spiral diluter (EDDY JET V.1.23, IG Instrumenten-Gesellschaft AG, Zurich, Switzerland). The final volume of the microbial suspension amounted to 1 mL. Subsequently, 50 μL of the dilutions were plated on Schedler plates (BD254042; Becton Dickinson, Heidelberg, Germany) so that aerobic and facultative anaerobic bacteria were cultivated at 37 °C and 5% CO₂ for 5 days. Accordingly, anaerobic bacteria were also cultivated on Schedler plates at 37 °C for 10 days (anaerobic chamber; Anaerocult A; Merck, Darmstadt, Germany). The analysis was performed three times as multispecies experiments. The number of CFU per cm² was counted by a single investigator. Each measurement was repeated twice. Two individual experiments were performed, and each group was represented in quadruplicate in each experiment.

All samples were cultured on blood, chocolate, cystine lactose electrolyte deficient (CLED), and Brucella laked blood agar plates and were incubated under aerobic and anaerobic conditions. For the aerobic conditions, the blood and CLED plates were incubated at 37°C in a relevant atmosphere, while the chocolate plate was incubated at 37°C in a 5 to 10% CO₂ environment. For the anaerobic conditions, an anaerobic chamber (Anaerocult A; Merck, Darmstadt, Germany) was used to incubate the blood agar and Brucella lacked blood agar plate with kanamycin and vancomycin at 37°C. All of the agar plates were read twice, after 24 h and 48 h for the aerobic plates, and after 48 h and 6 days for the anaerobic plates. After microbial isolation, the colonies were conveyed to MALDI-TOF MS identification.

For the selection of LAB from the bean samples, about 1 g of bean pods were aseptically transferred to de Man, Rogosa and Sharpe (MRS) broth (Sigma-Aldrich, Missouri, USA) with pH adjusted to 5.7 using HCl (Sigma-Aldrich). The flasks were incubated at 37°C for 4 days in a shaking incubator (Barnstead Lab-Line, ON, Canada) at 190 rpm. Dilutions of the liquid were spread in parallels on MRS agar plates (Scharlab, Spain); these were then incubated at anaerobic conditions using Anaerocult^® A (Merck Millipore, Germany) at 37°C for 72 h. Colonies were picked out and re-streaked on MRS agar plates until isolated bacterial strains were obtained. Strain isolation was made for two subsequent times for each sample.