Streptococcus thermophilus

We first used MetaCRT [33 (link)], which we modified from CRT [34 (link)] (to allow detection of partial repeats at the ends of CRISPR arrays), to predict the CRISPR arrays in complete bacterial and archaeal genomes. The genomes were downloaded in October 2016 from the NCBI ftp website (ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq). We focused on complete reference genomes in this study, as CRISPR–Cas systems may be found in separate contigs when draft genomes are used. However, for a few species we analyzed in detail, we augmented the list of genomes with draft genomes: including 13 draft genomes for Streptococcus thermophilus and 4055 draft genomes for Staphylococcus aureus. In some cases, a long CRISPR may be split into multiple ones because of repeats containing excessive mutations or long spacers. To avoid such cases, CRISPRs that are close to each other (<=200 bps) and share very similar repeat sequences were considered to be in the same locus. We then collected the consensus repeat for each putative CRISPR array. We clustered these consensus repeats at 90% sequence identity using CD-HIT-EST [35 (link)]. In this way, a “cluster” contains more than two CRISPR arrays, and a “singleton” refers to the repeats exclusively found within their corresponding CRISPR array.
We then used hmmscan [36 (link)] to search putative proteins found in the genomes against a collection of Cas families to predict putative Cas proteins (using the gathering cutoff). In total, the collection contains 403 Cas families, among which eight were identified from the human microbiomes (using a combination of context-based and similarity-search approaches) [37 ], and 395 were from a recent study [14 (link)]. Since Koonin and colleagues did not build models for the Cas families they curated [14 (link)], we used hmmbuild to construct hmm models for all of their families. Considering that gene prediction is far from perfect for many genomes, for the genomes/contigs that contain CRISPRs but lack cas genes, we further used the FragGeneScan [38 (link)], a gene predictor we have developed for predicting complete as well as fragmented genes in genomic sequences, to re-predict the genes, and then performed cas gene prediction to rule out the possibility of missing cas genes because the genes were not predicted in the first place.
A cas locus defined in this study should contain at least three cas genes, at least one of which belongs to the universal cas genes for CRISPR adaptation (cas1 and cas2) or the main components of interference module including cas7, cas5, cas8, cas10, csf1, cas9, cpf1 [14 (link)].

We first adapted the S. cerevisiae PCR TAP-tagging vector pEB1340 [34 (link)] for its use in C. albicans by substituting the S. cerevisiae HIS3 marker with the C. albicans URA3, HIS1 and ARG4 genes from the previously published pFA-GFP plasmid series [26 (link)] (Fig. 1A). Subcloning of the C. albicans auxotrophic markers was done by ligation of AscI-PmeI fragments in AscI-PmeI digested pEB1340. We then derived triple HA or MYC epitope-tagging vectors in the same pFA plasmid backbones by cloning oligonucleotides encoding the HA or MYC epitope tags and containing XmaI and AscI sites (Table 3) between the XmaI and AscI sites of the pEB1340 plasmid. Auxotrophic markers URA3, HIS1 and ARG4 from plasmids pFA-XFP [26 (link)] were then subcloned into the HA and Myc constructs between AscI and PmeI.
The beta-galactosidase reporter was constructed by subcloning a PstI-MluI fragment corresponding to the Streptococcus thermophilus lacZ ORF from plasmid placpoly [35 (link)] between the PstI and AscI sites of plasmid pFA-XFP-URA3 [26 (link)].
PCR reactions were performed in 50-μl volumes containing 1 ng of plasmid template with the Expand Long Template polymerase following manufacturer's instructions (Roche, Germany). PCR parameters were 1 cycle at 94°C, 5 min followed by 35 cycles at 94°C, 30 sec; 58°C, 1 min; 68°C, 3 min.

The incorporation of yoghurt with Bouea macrophylla fruit extract was conducted based on Fidelis et al. 2021 [27 (link)]. In order to make yoghurt, 100 mL of fresh cow’s milk was brought to a temperature of 72 °C for thirty minutes before being lowered to 45 °C. Approximately, 0.06 g (0.06% w/w) of starter culture (Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) were added. The mixture was incubated using a yoghurt maker at 40–45 °C for 7–10 h until pH dropped to 4.5–4.6 [27 (link)]. After that, the yoghurt was stabilised by cooling at 4 °C overnight. 2 g of Bouea macrophylla flesh powder extraction was added into 100 mL yoghurt to obtain an approximate final concentration of 2000 mg/100 mL (20 mg/mL).

In total, 112 samples, consisting mainly of raw ovine, bovine, and caprine milk (Supplementary Table S1) were spread on several media for the isolation of putative lactic acid bacteria, as described previously ([31 (link)]). Briefly, samples were streaked or serially diluted and plated on Streptococcus thermophilus selective agar; M17 agar with 10% lactose; de Man, Rogosa, and Sharpe (MRS) agar containing 30 μg·mL⁻¹ vancomycin, MRS adjusted to pH 5.4; Lactobacillus selective agar (LBS); and transgalactosylated oligosaccharide (TOS) agar, supplemented with 50 μg·mL⁻¹ lithium mupirocin, and incubated for 24 to 72 h at 42 °C, 30 °C, and 37 °C, aerobically, and 42 °C, 30 °C, and 37 °C, anaerobically, respectively. All isolates were screened for bacteriocin production by overlaying with sloppy MRS agar (0.75% wt/vol agar), pre-tempered to 50 °C and seeded with 0.25% (vol/vol) of an overnight Lactobacillus delbrueckii ssp. bulgaricus LMG6901 culture. Colonies producing distinct zones of inhibition were triple-streaked for purity and cultured in broth overnight to produce a cell-free supernatant (CFS) for subsequent well diffusion assays. Overnight cultures were centrifuged at 16,000× g for 3 min and the resulting supernatant was filtered through a 0.2 µm filter (Sarstedt, Wexford, Ireland), yielding CFS. For well diffusion assays, 20 mL volumes of sloppy MRS agar seeded with L. bulgaricus LMG6901 were poured into petri dishes and allowed to set. Six-millimeter wells were bored in the agar using glass Pasteur pipettes, into which 50 µL CFS was added. Plates were examined for zones of inhibition following overnight incubation. Supernatants producing zones of inhibition (active supernatant) were treated with 20 mg·mL⁻¹ proteinase K (Merck) for 3 h to digest proteinaceous compounds, and the well diffusion assays were repeated. Loss of activity denoted a proteinaceous compound. Potential bacteriocin producers were subject to MALDI-TOF mass spectrometry, as previously described ([31 (link)]).

Drinking yogurt was prepared in 7 different batches including control. The treatments included 6 yogurt samples each containing 1 g of dried extract from Majdool (MF) and Sukari (SKF) date flesh obtained using 3 different extraction techniques (SFE, SCE and SX). These two varieties were selected due to their high phenolic contents and better antioxidant properties [11 (link)]. A control sample was also prepared to which no extract was added. The date extracts were mixed with 14 g of skim milk powder followed by the addition of distilled water (100 mL). After 5 min of homogenization, samples were placed in a water bath for 30 min to pasteurize at 85 °C followed by cooling to 44 °C. Afterwards, bacterial starter culture (2.5% v/v) consisting of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Hansen, Hoersholm, Denmark) was added in a 1:1 ratio. The samples were then incubated at 44 °C for 4 h until they were fully coagulated (pH 4.6). The curd so formed was first cooled down to 4 °C in a refrigerator and then homogenized to form the liquefied drinkable yogurt. The final yogurt samples were then stored at 4 °C for carrying out various quality studies.