Bromcresol Green

Yeast and bacterial strains were isolated from liquid and pellicle samples of black tea kombucha from the company Biomère (Paris, France). WallersteinLab (WL) agar medium from Thermo Fisher Scientific (Waltham, MA, USA) was used to isolate yeasts [19 (link)]. Bromocresol Green allowed a macroscopic discrimination of colonies based on their aspect and color for the set of yeasts isolated in the present study [20 (link),21 ]. De Man Rogosa and Sharpe (MRS) (pH 6.2) from Condalab (Madrid, Spain), LAC (pH 5.1), and Mannitol agar media [22 (link)] were used for the isolation of bacteria that may have different nutritional requirements in both aerobic and anaerobic conditions of incubation [23 (link)]. All reagents used in agar media were purchased from Merck (Darmstadt, Germany), if not otherwise specified.
Five yeast colonies per colony morphotype were separately picked-up for inoculation in Yeast Peptone Dextrose (YPD) liquid medium (48 h at 28 °C). Twenty-five bacterial colonies per agar medium were separately picked-up for inoculation in LAC medium (48 h at 28 °C in the aerobic condition of incubation), since all species identified on Mannitol agar medium were also identified on LAC agar medium. After incubation, culture media were eliminated by centrifugation (14.500× g, 3 min at 15 °C). A half volume of appropriate liquid medium with a half volume of 40% (v/v) glycerol was then added to the samples to make a stock kept at −20 °C.
DNA extraction from each isolated strain was performed using an Instagen Matrix kit (Bio-Rad, Hercules, CA USA). For yeasts, the 26S rDNA region ribosomal non transcribed spacer 2 (NTS 2) was amplified using the following primers: NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GTCCGTGTTTCAAGACGG-3′) [24 (link)]. Two microliters of extracted DNA were added to 25 µL PCR mix (1.8 mM MgCl₂, 0.25 mM dNTPs, 1.25 µM of each primer, and 0.025 U Taq polymerase) (Promega Corp., Madison, WI, USA). A Biorad (Hercules, CA, USA) thermocycler was used as described previously [25 (link)].
For bacteria, 16S ribosomal DNA was amplified using the following primers: E517F (5′-GCCAGCAGCCGCGGTAA-3′) and E106R (5′-CTCACGRCACGAGCTGACG-3′). Amplification was performed in the same conditions as for the yeasts, except for the annealing temperature, which was changed to 58 °C [26 (link)]. Both strands Sanger sequencing was performed on amplified DNA by Genewiz^® (Leipzig, Allemagne) using NL1 and NL4 primers for yeasts and E515F and E106R primers for bacteria. The sequences obtained were then analyzed using the software Geneious R7 (version 7.1.5) and the Basic Local Alignment Search Tool (BLAST) available on NCBI’s website (https://blast.ncbi.nlm.nih.gov/Blast.cgi), thus returning genus and species names.

A total of 986 L. lactis strains originating from the Chr. Hansen culture collection were screened for ability to enhance texture in fermented milk. The strains were inoculated in 96 low-well microtiter plates in 200 μl M17 broth (Terzaghi and Sandine 1975 (link)) containing 1% glucose and 1% lactose as C-source and incubated overnight at 30°C. A volume of 10 μl was transferred to 990 μl B-milk containing pH colour indicator ±0.2% yeast extract in 96 deep-well plates. B-milk was prepared by reconstituting low fat skim milk powder to a level of dry matter of 9.5% and pasteurised at 99°C for 30 min, followed by cooling to 30°C. The pH colour indicator milk was prepared in the following way: 50 mg bromocresol purple salt and 50 mg bromocresol green salt (both from Sigma Aldrich, St. Louis, Missouri, United States) were dissolved in a final volume of 40 ml dH₂O, pH was adjusted to 7.0 with NaOH and the final volume was adjusted to 50 ml with dH₂O. The pH indicator was sterile filtered (0.2 μm) and 5 ml was added to 95 ml B-milk. The inoculated pH colour indicator milk samples were incubated for 18–20 h on top of flat-bed scanners (HP ScanJet G4010) with temperature-controlled hoods set at 30°C (Fig. 1). pH-dependent changes in colour were recorded every 6 min, using pH Multiscan software (v.5.1, HNH Consult Aps, 9530 Støvring, Denmark). After 18-h static incubation at 30°C, most samples in the plate had a pH of 4.3–4.5, where the fresh milk was converted to fermented milk gel. The plates were kept at 4°C overnight, and TADM (Total Aspiration Dispense Monitoring) pressure curves were obtained by aspirating the samples using Hamilton liquid robot.
A Hamilton MicroLab Star liquid handling device (Hamilton, Bonaduz, Switzerland) was used to collect pressure versus time data using TADM software of the Hamilton (Camenisch 2001 ). Aspiration pressure curves were used to identify samples with elevated texture. A volume of 500 μl was aspirated (350 μl/s) using wide-bore tips (Hamilton Robotics). Pressure versus time data (TADM) were expressed as a single number by either recording pressure at a particular time point (e.g. 1 s) or as TADM curve area.
Three different tools for carbohydrate active enzyme annotation in dbCAN2 (http://cys.bios.niu.edu/dbCAN2) were combined to classify glucosyltransferases: HMMER search against the dbCAN HMM (hidden Markov model) database, DIAMOND search against the CAZy pre-annotated CAZyme sequence database and Hotpep search against the conserved CAZyme short peptide database (Zhang, Yohe and Huang 2018 (link)).
Lactococcus lactis subsp. lactis strain Lll3 (CHCC11848) was deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 21 August 2014 under the accession no. DSM 29291.

Analysis of alkaline-labile sites in genomic DNA was performed as reported earlier^{1 (link)}. Briefly, overnight cultures were diluted for exponential growth in 50 ml medium and harvested at the optical density of OD₆₀₀ = 0.6. Genomic DNA was isolated with the Qiagen Gentra Puregene Yeast/Bact Kit (Qiagen, 158567). The gDNA concentration was determined by an intercalating fluorescent dye supplied via the Qubit dsDNA broad-range kit (Invitrogen, Q32854). In a final volume of 40 µl, 10 µg of extracted gDNA were treated, shielded from light, for 2 h at 55 °C with 0.3 M KOH or 0.3 M KCl. KOH treated DNA samples were mixed with 8 µl 6× alkaline loading buffer (300 mM KOH, 6 mM EDTA, 18% (w/v) Ficoll Type 400, 0.15% (w/v) bromocresol green, 0.25% (w/v) xylene cyanol) before loading on a 1% alkaline agarose gel; the alkaline gel was casted using the 1× dilution of the 10× alkaline running buffer (0.5 mM NaOH, 10 mM EDTA). The alkaline gel was run in 1× alkaline running buffer. The KCl treated gDNA samples were loaded on a 1% TBE agarose gel with standard loading dye. Both gels were run at room temperature for 5 min at 65 V before reducing the voltage to 26 V for 18 h. The alkaline gel was neutralized in two washes of 250 ml (700 mM Tris-HCl pH 8.0, 1.5 M NaCl) for 45 min each with gentle shaking. Both gels were stained in a 1:10,000 SyBr Gold solution (ThermoFisher, S11494) (diluted in 1×TBE for the neutral gel; diluted in water for the alkaline gel) for 2 h with gentle shaking before image acquisition.

Cerebrospinal fluid (CSF) and plasma samples were collected in the morning after overnight fasting. After centrifugation, the samples were divided into 200 μL aliquots in polypropylene tubes and stored at −80°C until measurements were taken. CSF and plasma Aβ40, Aβ42, P-tau181, and total tau were measured on the HD-X analyzer (Quanterix) using Simoa kits (Quanterix, 103714, 101195). CSF albumin levels were measured using commercial assay kits (Thermo Fisher Scientific). Serum albumin levels were determined using a bromocresol green dye-binding assay (ADVIA 1800; Siemens, Berlin, Germany). The Qalb value was calculated using the following formula: (CSF albumin/serum albumin) × 1,000.