Preparation of sweetness-enhanced yoghurt and sampling
Raw cow’s milk for all experiments was obtained from the experimental farm (Schädtbek, Germany) of the Max Rubner-Institut in Kiel where a herd of almost 100 dairy cows is kept. Sweetness-enhanced milk for yoghurt production was prepared using the bi-enzymatic system of lactose conversion described by Lorenzen et al. (2013). In a concurrent study, the carbohydrate composition of the regular and sweetness-enhanced milk samples used for yoghurt production were analysed and reported (Luzzi et al. 2020). Yoghurt samples were produced with bi-enzymatically modified as well as regular milk as outlined by Luzzi et al. (2020). The two multi-strain starter culture preparations YoFlex® Premium 4.0 and ABT-100 from Chr. Hansen (Nienburg, Germany) were used for yoghurt production. According to the manufacturer, the traditional YoFlex® Premium 4.0 yoghurt culture contained S. thermophilus and Lb. delbrueckii subsp. bulgaricus strains. The probiotic ABT-100, as indicated in the product data sheet, was comprised of S. thermophilus, Lb. acidophilus and a Bifidobacterium species, which was not specified to species level by the manufacturer (Chr. Hansen, Nienburg, Germany).
Three independent repetitions of each yoghurt production experiment were performed. Yoghurt samples were taken at four time points per experiment: immediately after the pasteurised yoghurt milk was inoculated with starter cultures, after fermentation (at the time point of cold storage when the yoghurt pH had reached 4.5) as well as after five and 21 days of storage at 4 °C. Sample sizes were 20 mL for inoculated milk and 20 g for yoghurt samples.
Genomic DNA isolation
In preparation for DNA isolation, inoculated milk samples (20 mL) were centrifuged twice for 30 min at 6000×g at 10 °C in a Heraeus Multifuge (Thermo Fisher Scientific, Waltham, USA; Rotor: 75002005). The final supernatant (ca. 20 mL) from inoculated milk samples was stored at − 20 °C until further processing. Yoghurt samples (20 g) were mixed with 20 mL of 1 M Tris-HCl (pH 7.5; Carl Roth, Karlsruhe, Germany) and centrifuged twice for 20 min at 300×g at 10 °C in a Heraeus Multifuge. The final supernatant (ca. 40–45 mL) from yoghurt samples was also stored at − 20 °C until further analyses.
For further analyses, inoculated milk and yoghurt samples were thawed at room temperature. Bacterial cell lysis was performed using a lysis buffer comprised of 500 mM NaCl, 50 mM Tris–HCl (pH 8.0), 50 mM EDTA and 4% sodium dodecyl sulphate (buffer components from VWR International GmbH, Darmstadt, Germany). One volume of lysis buffer was added to all samples and these were mixed using an Intelli-Mixer RM-2M (ELMI, Calabasas, CA, USA) in the U2-mode at 80 rpm for 10 min. Following incubation in a 70 °C water bath (Julabo GmbH, Seelbach, Germany) for 20 min with shaking at 196 rpm, samples were centrifuged at 4 °C for 30 min at 6000×g in a Heraeus Multifuge. The fat layer that formed on top of the samples was removed with a sterile pipette tip and discarded, whilst the supernatant was transferred to a fresh 50 mL Falcon® tube. This centrifugation step was repeated twice to obtain a clear lysate.
To each lysate tube, 10 M ammonium acetate (Merck, Darmstadt, Germany) amounting to 10% of the total sample volume was added. Samples were briefly mixed and incubated on ice for 20 min, before centrifuging at 4 °C for 30 min at 6000×g. One volume of 2-propanol (Carl Roth, Karlsruhe, Germany; pre-cooled to 4 °C) was added to the supernatant to precipitate the DNA. Samples were mixed and incubated on ice for 45 min, before centrifugation at 4 °C for 30 min at 6000×g and removal of the supernatant. Nucleic acid pellets were resuspended in 1 mL of 70% HPLC gradient grade ethanol (Carl Roth, Karlsruhe, Germany) and centrifuged at 13,000 rpm for 15 min at 4 °C in a Heraeus Fresco21 centrifuge (Thermo Fisher Scientific, Waltham, MA, USA), air-dried and resuspended in 200 µL 10 mM Tris–HCl (pH 8.0).
To each sample, 4 µL of DNAse-free RNase (10 mg/mL; VWR International GmbH, Darmstadt, Germany) was added and samples were incubated at 37 °C for 15 min. Thereafter, 30 µL of proteinase K solution (20 mg/mL; AppliChem GmbH, Darmstadt, Germany) and 200 µL of ‘Buffer AL’ from the QIAamp DNA Stool Mini Kit (QIAGEN GmbH, Hilden, Germany) were added to wash the sample and the mixture was incubated at 70 °C for 10 min. Two volumes of 99% HPLC gradient grade ethanol were added and samples were mixed thoroughly, prior to being transferred to the QIAamp Mini spin columns from the QIAamp DNA Stool Mini Kit (QIAGEN GmbH, Hilden, Germany) and centrifuged at 10,000 rpm for 1 min at room temperature. Subsequent washing steps with Buffers ‘AW1’ and ‘AW2’ were done with 500 µL of each wash buffer. Samples were eluted in 2 × 50 µL of pre-warmed ‘Buffer AE’ with centrifugation at 6000 rpm for 1 min to maximise the eluted DNA. The DNA concentration was measured using a Qubit® 3.0 Fluorometer in conjunction with the Qubit™ dsDNA BR Assay Kit following the manufacturer’s protocol (Thermo Fisher Scientific, Darmstadt, Germany).
Library preparation and sequencing
The bacterial community composition was determined using tag-encoded, MiSeq™ System-based 16S rRNA gene high throughput sequencing. The genomic DNA was diluted to 1.67 ng/µL prior to library preparation. Specific primers for the 16S rRNA gene V3 and V4 regions with Illumina adapter overhangs were used for amplification of the bacterial community in all samples (16S fw-meta 5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3′ and 16S rev-meta 5′-GCT TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3′) (Klindworth et al. 2013). DNA amplification was carried out following the two-stage PCR protocol for 16S Metagenomic Sequencing Library Preparation provided by Illumina (Illumina Inc., San Diego, CA, USA). The size of the 16S rRNA gene PCR products was confirmed by means of automated electrophoresis using the Experion™ Automated Electrophoresis System in conjunction with the Experion™ DNA 12K Assay Kit according to the manufacturer’s instructions (Bio-Rad Laboratories, Inc., CA, USA). 16S rRNA gene sequencing was performed on a MiSeq™ System using the MiSeq Reagent Kit v3 following the manufacturer’s instructions (Illumina Inc., San Diego, CA, USA). Samples with a sequencing coverage of < 10K reads were re-sequenced and the reads from both runs were combined for subsequent analyses.
The raw dataset was analysed using the de novo analysis pipeline of the IMNGS platform for ecology and diversity studies of the Technical University of Munich (Lagkouvardos et al. 2016). The raw fastq files were pre-processed using the Remultiplexor Perl script available through the IMNGS platform and then uploaded to the IMNGS pipeline, which implements the UPARSE algorithm from the USEARCH 8 (32-bit) package (Edgar 2010, 2013). The pipeline produced operational taxonomic unit (OTU) tables for each sample, which were uploaded to the SINA Arb Silva Aligner to compare these with both the Silva and rdp reference databases (Pruesse et al. 2012; Cole et al. 2014). The consolidated and verified OTU tables were used for statistical analysis using RStudio Version 1.1.423 by implementing the set of ‘Rhea scripts’ published by Lagkouvardos et al. (2017). These R scripts were used for 16S rRNA gene bioinformatic analysis of yoghurt samples by processing the OTU tables. Taxonomic binning to visualise the taxonomic composition of samples was performed in Microsoft Excel using the OTU tables generated by the Rhea pipeline. For statistical analysis, the Rhea pipeline applied a permutational multivariate analysis of variance (PERMANOVA) using distance matrices (vegan::adonis) to calculate the significance (p-values) of differences in microbial composition between groups of OTUs (Anderson 2017; Lagkouvardos et al. 2017). Three biological replicates were analysed for all sampling time points with the exception of the enhanced ABT-100 yoghurt sample after five days of storage at 4 °C, where only two biological replicates were analysed as no DNA could be recovered due to a methodological problem during DNA isolation in the third replicate.