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Yoghurt and cheese starter cultures were obtained from Chr. Hansen (Nienburg, Ger- many; see Table 3 and Table 4) and were analysed as outlined in the following chapter.

3.2.1.1 Strain isolation from mixed cultures

Individual strains of SLAB were isolated from the mixed-strain cultures for subsequent classification and characterisation, as well as for use as reference strains for microbial ecology studies of product samples (see chapter 3.2.4). The isolation of pure cultures was achieved by dilution streaking three times using the culture medium and incubation conditions outlined in Table 3 and Table 4.

Strain isolates were verified based on colony morphology and microscopy, and then used to inoculate broth media, which was incubated for 24 h. After incubation, 800 µL of broth culture were mixed with 200 µL of 85 % glycerol to create stocks cultures, which were stored at - 70 °C. For subsequent usage of these stock cultures, a sterile loop was used to aseptically scrape off a small portion of the frozen culture for inoculation of fresh broth medium.

3.2.1.2 Identification of starter cultures by 16S rDNA sequencing

For further strain isolate classification, bacterial DNA was extracted from overnight broth cultures of all isolates obtained from the commercial yoghurt and cheese cultures (see chapter 3.2.1.1). For this the ‘PeqGOLD Bacterial DNA Mini Kit’ was used with 2 mL of log-phase bacteria in broth culture and according to the manufacturer’s guidelines for gram-positive bacteria (VWR International GmbH, Darmstadt, Germany). Briefly, the ‘PeqGOLD Bacterial DNA Mini Kit’ is based on a DNA-binding column technology, whereby bacterial cell walls are first treated with lysozyme (10 mg/mL), proteinase K (20 mg/mL) and RNase A (20 mg/mL), before the DNA of lysed cells is reversibly bound to a column matrix where it undergoes a series of wash steps to remove salt and protein contamination. The DNA is then eluted from the column using the elution buffer provided. The elution step for DNA isolated from all yoghurt and cheese samples was performed twice using 50 µL of elution buffer per elution.

To confirm DNA extraction from bacteria was successful, polymerase chain reaction (PCR) amplification from DNA of all strains isolated from yoghurt and cheese cultures was performed using the ‘High efficiency Taq Polymerase with Buffer E’ kit (Genaxxon Bioscience GmbH, Ulm, Germany) together with the universal 16S bacterial DNA primers

27f and 1540r (Table A 6). These primers amplify a fragment of approx. 1500 bp, which indicates successful extraction and amplification of bacterial DNA from the samples. The PCR reaction consisted of a 10x amplification buffer constituted with 2.5 mM MgCl2, 10 mM of each nucleotide triphosphate (dNTP), 10 pmol of each primer, 10 µL of non- adjusted DNA sample (for a 50 µL reaction), 1.5 U of Taq-Polymerase, and made to a final volume of 50 µL with sterile, type 1 ultrapure H2O. A Touchdown PCR reaction was performed on a PeqSTAR thermocycler with an initial denaturation step at 94 °C for 3 min; followed by 10 cycles of denaturation at 94 °C for 30 s, annealing at 62 °C (- 1 °C/cycle) for 30 s, and extension at 72 °C for 90 s; followed by 22 cycles of denatur- ation at 94 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 90 s; completed by a final extension step at 72 °C for 5 min.

Following the initial classification steps of colony morphology and PCR confirmation of the bacterial DNA isolates, Sanger sequencing of 16S rRNA gene PCR amplicons was used to confirm the identity of isolated strains. For this, 5 µL of each PCR product were run on a 1.5 % agarose gel containing a 1:20 dilution of GelRed™ Nucleic Acid Gel Stain for 1.5 h at 100 V in 1x TAE buffer. The gel was visualised under UV light on a BioDo- cAnalyze gel documentation system. Samples showing successful amplification of the expected 1500 bp fragment length were cleaned using the ‘NucleoSpin® _{Gel and PCR} Clean-up Kit’ and samples were sent to Eurofins Genomics (Ebersberg, Deutschland) for Sanger sequencing using the company’s LightRun Tube sequencing with the univer- sal 16S (27f and 1540r) primers outlined in Table A 6.

3.2.1.3 Carbohydrate utilisation tests

Miniaturised sugar fermentation tests were used to assess carbohydrate fermentation by the yoghurt culture isolates obtained in chapter 3.2.1.1. These tests aimed to determine the ability of different strains of LAB in the yoghurt cultures to metabolise a variety of mono- and disaccharides. They were performed using the basal medium for fermentation studies (Table 1), based on the method proposed by Jayne-Williams in 1975.182 _The colour indicator chlorophenol red contained in this medium helped determine whether the bacteria being tested could successfully ferment the provided sugar, producing acidic compounds. Upon successful fermentation of the added sugar of interest, the basal me- dium turned from red to yellow. Glucose was used as a positive control whereas sterile, type 1 ultrapure H2O served as a negative control.

For the sugar fermentation tests, 1 mL of overnight broth culture of the strain to be tested was centrifuged for 10 min at 4,000 rpm in a Heraeus Fresco21 centrifuge and the su- pernatant was subsequently removed. The pellet was resuspended in 1 mL basal

medium and this suspension was then further diluted 1:50 in basal medium. A 96-well plate was prepared with 25 µL per well of each carbohydrate solution to be tested (see chapter 3.1.3), working under sterile conditions in a class 2 safety cabinet and using ‘PCR clean and sterile filter tips’. Thereafter, each well was inoculated with 100 µL of the previously prepared 1:50 bacterial dilution in basal medium. The 96-well plates were incubated for 48 h at the relevant incubation conditions for the individual strains (see Table 3), with initial evaluation after 24 h.