Lysozyme

The DNA of yeast isolates from Miang, which had the ability to produce ethanol, and have tannin tolerance, was identified by sequence analysis of the D1/D2 domain of the LSU rRNA gene. Each isolate was grown in YPD broth at 30 °C for 24 h, then the cells were harvested by centrifugation (Velocity 14R, Dynamica Scientific Ltd., Livingston, UK) at 14,000× g for 1 min at 4 °C and resuspended in 480 μL of 50 mM ethylenediaminetetraacetic acid (EDTA). A volume (40 μL) of a 50 mg/mL solution of lysozyme (Bio Basic Inc., Markham, ON, Canada) was added to the cell suspension. The genomic DNA was extracted using a Wizard Genomic DNA purification kit (Promega, Madison, WI, USA) following the manufacturer’s protocol for genomic DNA from yeast. Then, extracted DNA was amplified using universal primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) [27 (link)]. The polymerase chain reaction was undertaken in an MG-96 MyGene^TM Thermal Cycler (LongGene Scientific Inc., Changzhou, China). The PCR products were purified and sequenced by a sequencing service provider (1st BASE Pte Ltd., Singapore). The pairwise sequence alignment of the D1/D2 sequences was compared to other genes in the GenBank databases, and the phylogenetic tree was established based on the neighbor-joining method by MEGA version 4.0 software.

The pellet of cells of mutant and two wild-type probiotics strains was re-suspended in 10.0 mL of sterile saline solution with two different concentration of lysozyme (100 and 200 mg L⁻¹, Bio Basic Canada INC) and incubated for 3 days at 37 °C according to Pinto et al. [22 (link)]. The survival rate was calculated according to the following equation: $$\mathrm{S}\mathrm{u}\mathrm{r}\mathrm{v}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\%=\mathrm{Final}({\mathrm{Log}}^{10}\mathrm{cfu}/\mathrm{mL})/\mathrm{Initial}({\mathrm{Log}}^{10}\mathrm{cfu}/\mathrm{mL})\times 100.$$

Human serum albumin (HSA) was purchased from Akron Biotech, USA. Lysozyme was obtained from Bio Basic Inc., Canada. Rutin, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 1,1-Diphenyl-2-picrylhydrazyl (DPPH), gallic acid, ascorbic acid, fructose, sodium phosphate monobasic, sodium phosphate dibasic, ferrozine, iron (II) chloride, ethylenediaminetetraacetic acid (EDTA), folin-Ciocalteu phenol reagent, aluminium chloride, sodium acetate, glycerol, glacial acetic acid, methanol, ethanol, streptozocin (STZ), and sodium azide were acquired from Sigma Aldrich, USA. Tetramethylethylenediamine (TEMED), sodium dodecyl sulfate (SDS), bromophenol blue, ammonium persulfate, acrylamide solution, Coomassie brilliant blue, glycine, and β-mercaptoethanol were acquired from Bio-Rad Laboratories, USA. Tris (hydroxymethyl)-aminomethane was purchased from Scharlau, Spain, while molecular markers were obtained from Thermo Scientific, USA.