Tri ammonium citrate

Bifidobacterium bifidum YIT 10347 (BF-1) was used as a wild-type (WT) strain. The complete genome sequence (2,149,912 bp) of BF-1 (GenBank: AP024712) was found to be similar to that of B. bifidum PRL2010 (Turroni et al., 2010 (link)). BF-1 and its mutants were anaerobically cultured at 37°C in de Man–Rogosa–Sharpe (MRS) medium (Difco Laboratories, Detroit, MI, USA) supplemented with 0.05% L-cysteine HCl or in modified ILS (m-ILS) medium containing 10 g trypticase peptone (Difco Laboratories), 5 g yeast extract (Difco Laboratories), 3 g tryptose (Difco Laboratories), 10 g lactose, 3 g KH₂PO₄, 3 g K₂HPO₄, 2 g tri-ammonium citrate, 1 mL pyruvate, 0.3 g L-cysteine HCl, 1 mL Tween 80 (Sigma-Aldrich, St. Louis, MO, USA), 0.575 g MgSO₄·7H₂0, 0.12 g MnSO₄·4H₂0, and 0.034 g FeSO₄·7H₂O in 1.0 L distilled water (pH 6.8).
To count viable bacteria (CFUs), serial dilutions of the samples were plated on agar plates containing MRS medium supplemented with 0.05% L-cysteine HCl and cultured at 37°C. Preparation of the dilutions and cultures was carried out anaerobically.
Escherichia coli JM109 (Takara Bio, Shiga, Japan) were used as competent cells for plasmid construction; they were aerobically cultured at 37°C in Luria–Bertani medium (Difco Laboratories).

Poly-ethylene glycol (PEG) 3350 was purchased from Sigma Life Sciences, tri-ammonium citrate was purchased from Sigma Life Sciences, Ampicillin was purchased from GoldBio, dehydrated Luria-Bertani (LB) Broth was purchased from Fisher Scientific, DL-dithiothroitol (DTT) and isopropyl-β-d-thiogalactopyranoside (IPTG) were purchased from GoldBio. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from Fisher BioReagents. Imidazole was purchased from Acros Organics; tris(hydroxymethyl)aminomethane (Tris) was purchased from Fisher Scientific. Sodium chloride (NaCl) was purchased from Fisher Chemical, and bovine serum albumin (BSA) was purchased from Sigma Life Science.

For enumeration of Lactobacillus species, when their presence was indicated on the product specification, 0.1 mL of the product cell suspension was spread on de Man Rogosa Sharpe (MRS) agar (BD Difco, Detroit, MI, USA) and let dry. Only the highest three dilutions were used per sample and plated by duplicate. The plates were incubated for 48 h at 37 °C in anaerobic chamber (Whitley DG250 Anaerobic Workstation, Don Whitley Scientific Limited, Bingley, West Yorkshire, UK). For products indicating Lactobacillus acidophilus as part of its formulation, the sample suspensions were plated on Modified Rogosa agar pH 5.5 and incubated under the same conditions following the same approaches. The formulation for one liter of Modified Rogosa agar consisted of 10 g Bacto™ tryptone, 5 g yeast extract (BD Difco), 20 g D-glucose, 6 g KH₂PO₄, 1 g Tween 80, 2 g triammonium citrate, 0.575 g MgSO₄·7H₂O, FeSO₄·7H₂O (Sigma-Aldrich, St. Louis, MO, USA), 0.110 g MnSO₄·H₂O (Duksan Chemicals) and 15 g agar (LPS Solution, Daejeon, South Korea). The pH was adjusted to 5.5 using glacial acetic acid (Daejung Chemicals, Siheung, South Korea) prior autoclaving at 121 °C for 10 min to avoid hydrolyzation of agar due to low pH. Based on the colony counts the CFU/g was were determined for each product.