Isobutyric acid

Chromatographic analysis was carried out using an Agilent 7890a GC system with a DB23 column (Agilent Technologies, Santa Clara, CA, United States) coupled to a flame detector (FID). The temperature at the injection port and at the FID was 250°C. Helium was used as carrier gas at a flow rate of 1.6 mL/min. For the run, 1 μL of the sample was injected with a 1:25 split and a temperature program that consisted of a ramp from 100 to 200°C at a speed of 8°C/min, keeping constant at 200°C for 3 min was used. Calibration curves with standards of glacial acetic acid (1–50 mM), propionic acid (0.5–20 mM), butyric acid (0.5–20 mM), iso-butyric acid (0.5–20 mM), and iso-valeric acid (0.5–20 mM) were prepared (Sigma-Aldrich, San Luis, MO, United States). Organic acids were identified by comparison to standard retention times and quantified with the corresponding peak area using the calibration curve. Differences were statistically tested using One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (p < 0.05) conducted by the GraphPad Prism^® software.

Acetic acid was purchased from Thermo Fisher Scientific (Fair Lawn, NJ). Propionic acid, isobutyric acid, butyric acid, 2-methylbutyric acid, isovaleric acid, valeric acid, 2-methylpentanoic acid, 3-methylpentanoic acid, isocaproic acid, caproic acid, 2-methylhexanoic acid, 4-methylhexanoic acid, heptanoic acid, hexanoic acid-6,6,6-d₃ internal standard, N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), and methoxyamine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO).

POP of 98% purity was purchased from Shaanxi Ciyuan Biotechnology Co. (Xi’an, China). SIM from Mo Sha Dong (Hangzhou, China) was used as the reference hypolipidemic drug. High-purity SCFA standards—including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid—were purchased from Sigma Aldrich Chemical, Co. (St. Louis, MO, USA). All other chemicals used were of analytical grade.

Probiotic Lactobacillus reuteri DPC16 culture was kindly provided by Bioactives Research New Zealand. Yogurt starter culture Lyofast Y 450B (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) was obtained from SACCO System, Italy. Local purchases were made for invert syrup (IS) and manuka honey with different antibacterial gradings: Drapac Manuka Honey Blend (MB), Comvita Manuka Honey UMF^TM 18⁺ (UMF18), and Drapac DrKiwi AMF^TM 15⁺ (AMF15) and AMF 20⁺ (AMF20). Yogurts for the sensory and physicochemical analysis were prepared with fresh full-cream Anchor Blue milk bought from a supermarket. Microbiological enumeration and NMR analyses were conducted with yogurts made from nil-fat UHT milk. Difco^TM MRS (de Mann, Rogosa and Sharpe) broth and agar were utilised as media for bacterial growth and plate counts respectively. Analytical standards for lactic acid, acetic acid, butyric acid, propionic acid, isobutyric acid and succinic acid were obtained from Sigma Aldrich (Christchurch, New Zealand).

The tested choline ILs were synthesized by mixing choline bicarbonate (Sigma-Aldrich, St. Louis, USA, 80% in H₂O) with carboxylic acids (acetic acid, lactic acid, malonic acid, isovaleric acid, isobutyric acid, 2-methylbutyric acid; all from Sigma-Aldrich, used as received) in aqueous solutions [34 (link)]. The chemical identity of the obtained ILs (for structures, names and abbreviations, see Table 1) was confirmed by ¹H-NMR spectroscopy (Bruker Avance II 400 MHz, Bruker Corporation, Billerica, MA, USA) using D₂O as a solvent. The water content was determined by Karl Fischer’s coulometric titration (Metrohm 756 KF Coulometer, Metrohm AG, Herisau, Switzerland). All ILs were dried under high vacuum and stored in a glovebox under an argon atmosphere (water content below 0.5 ppm) prior to use.