Screening L. lactis Strains for Texture Enhancement

A total of 986 L. lactis strains originating from the Chr. Hansen culture collection were screened for ability to enhance texture in fermented milk. The strains were inoculated in 96 low-well microtiter plates in 200 μl M17 broth (Terzaghi and Sandine 1975 (link)) containing 1% glucose and 1% lactose as C-source and incubated overnight at 30°C. A volume of 10 μl was transferred to 990 μl B-milk containing pH colour indicator ±0.2% yeast extract in 96 deep-well plates. B-milk was prepared by reconstituting low fat skim milk powder to a level of dry matter of 9.5% and pasteurised at 99°C for 30 min, followed by cooling to 30°C. The pH colour indicator milk was prepared in the following way: 50 mg bromocresol purple salt and 50 mg bromocresol green salt (both from Sigma Aldrich, St. Louis, Missouri, United States) were dissolved in a final volume of 40 ml dH₂O, pH was adjusted to 7.0 with NaOH and the final volume was adjusted to 50 ml with dH₂O. The pH indicator was sterile filtered (0.2 μm) and 5 ml was added to 95 ml B-milk. The inoculated pH colour indicator milk samples were incubated for 18–20 h on top of flat-bed scanners (HP ScanJet G4010) with temperature-controlled hoods set at 30°C (Fig. 1). pH-dependent changes in colour were recorded every 6 min, using pH Multiscan software (v.5.1, HNH Consult Aps, 9530 Støvring, Denmark). After 18-h static incubation at 30°C, most samples in the plate had a pH of 4.3–4.5, where the fresh milk was converted to fermented milk gel. The plates were kept at 4°C overnight, and TADM (Total Aspiration Dispense Monitoring) pressure curves were obtained by aspirating the samples using Hamilton liquid robot.
A Hamilton MicroLab Star liquid handling device (Hamilton, Bonaduz, Switzerland) was used to collect pressure versus time data using TADM software of the Hamilton (Camenisch 2001 ). Aspiration pressure curves were used to identify samples with elevated texture. A volume of 500 μl was aspirated (350 μl/s) using wide-bore tips (Hamilton Robotics). Pressure versus time data (TADM) were expressed as a single number by either recording pressure at a particular time point (e.g. 1 s) or as TADM curve area.
Three different tools for carbohydrate active enzyme annotation in dbCAN2 (http://cys.bios.niu.edu/dbCAN2) were combined to classify glucosyltransferases: HMMER search against the dbCAN HMM (hidden Markov model) database, DIAMOND search against the CAZy pre-annotated CAZyme sequence database and Hotpep search against the conserved CAZyme short peptide database (Zhang, Yohe and Huang 2018 (link)).
Lactococcus lactis subsp. lactis strain Lll3 (CHCC11848) was deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 21 August 2014 under the accession no. DSM 29291.