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Page 4 of 15 Spus et al. Microbiome Res Rep 2023;2:33 https://dx.doi.org/10.20517/mrr.2023.20

The blend design included 24 strains representing all eight genetic lineages of the Ur starter: eleven strains
belonging to L. cremoris lineages 1 & 5, three strains belonging to L. lactis lineages 2 & 4, two strains of
L. cremoris lineage 3, three of L. cremoris lineage 6, two of L. cremoris lineage 7 and finally three belonging
to Lc. mesenteroides lineage 8 [Figure 1]. To prepare the strains for the blend preparation, frozen cultures of
L. lactis and L. cremoris were streaked on M17 agar (OXOID, Basingstoke, UK) plates supplemented with
0.5% (wt/vol) lactose (LM17) and Lc. mesenteroides strains were streaked on MRS agar plates (1.5% agar,
Merck KGaA, Darmstadt, Germany) supplemented with vancomycin (final concentration of 30 μg/mL).
Next, single colonies were picked and transferred to LM17 broth (in cases of L. lactis and L. cremoris strains)
and MRS broth (in cases of Lc. mesenteroides strains). Cultures of L. lactis and L. cremoris strains were
incubated at 30 °C for 18 h (overnight) and cultures of Lc. mesenteroides were incubated at 25 °C for 18 h
(overnight). Cells were washed twice by removing the supernatant after centrifugation at 2,500 × g for 30
min and exchanging it with peptone physiological saline (PPS). The optical density of washed cell
preparations was determined at 600 nm (OD ) (Novaspec II, Pharmacia Biotech, Pharmacia LKB,
600
Montreal, Canada). The cell suspensions were diluted to OD = 0.4 (st. dev. ± 0.02) and 100 µL of each
600
suspension was then mixed to obtain the blend to inoculate milk for the propagation experiment. After the
blend preparation, the relative abundance of each genetic lineage was determined using a qPCR approach
(described below). The initial ratio of the genetic lineages was determined to be as follows: 49.5% of
L. cremoris lineage 1 & 5, 12.5% of L. lactis lineage 2 & 4, 8% of L. cremoris lineage 3, 15.5% of L. cremoris
lineage 6, 12.5% of L. cremoris lineage 7 and 2% of Lc. mesenteroides lineage 8. Strains used to create blends
[5]
were previously characterized in terms of their bacteriophage resistance . The levels of resistance to three
bacteriophages [Lactococcus phage ϕTIFN1 and Lactococcus phage ϕTIFN7 - both P335 lytic phages and
Lactococcus phage ϕTIFN5 (a 936 lytic phage)] used in the cocktail to challenge the blends are indicated in
Figure 1. Eleven strains included in the blend showed diverse susceptibility to at least one of the three
phages and ten out of these eleven were ascribed to L. cremoris lineage 1 & 5 and one to L. cremoris lineage 6
(strain 2MS47). In total, four blends (two sets of two biological replicates) were prepared: A-1 and A-2;
control, no phage cocktail added; B-1 and B-2; treatment, cocktail of three phages added (see Graphical
Abstract of the paper). At the onset of the propagation experiment, a cocktail of three phages, ϕTIFN1,
ϕTIFN5, and ϕTIFN7, was added to two (treatments) out of four replicates (final concentration: 10 pfu/mL
8
[3]
per phage type). As previously reported based on sequence analysis , phages ϕTIFN1 and ϕTIFN7 belong to
p335-type lactococcal bacteriophages, and phage ϕTIFN5 belongs to 936-type. Both lactococcal
bacteriophages are strictly lytic [24,25] .

Propagation regime of the blends
The blends were cultivated in skim milk (“Friesche Vlag Lang Lekker”, non-fat, UHT, FrieslandCampina,
the Netherlands) for 24 h. After 24 h, 1% (0.1 mL) of the culture was transferred to fresh milk (9.9 mL).
Consequently, each transfer cycle corresponds to approximately 6.64 generations. The experiment was
performed continuously for 81 days, which approximately corresponds to 538 generations. Samples of
propagated blends were collected after one day (6.64 generations) and next after every 20 days (each 132.8
generations).

Unique-gene based qPCR for monitoring the relative abundance of genetic lineages
[5]
[3]
The unique-gene based qPCR method developed by Erkus et al. and described in detail in was used to
monitor the relative abundance of the genetic lineages. Each sample was measured in duplicate, and for
each measurement, a melting curve was monitored to check for unspecific product amplification. The
relative abundances of L. cremoris lineages 1 & 5, 3, 6, 7, L. lactis lineages 2 & 4 and Lc. mesenteroides lineage
[26]
8 were determined after 6.6, 139, 272, 405 and 538 generations. The Shannon diversity index at the level of
genetic lineages was calculated using the following equation: -Ʃ[n/N × ln(n/N)], with n - the abundance of
i
i
i
i
lineage i and N - the total lineages abundance. In this case, a higher Shannon diversity index indicates a
more equal distribution of the genetic lineages.