Abstract
Bacteriophages (phages) attacking strains of Lactococcus (Lc.) lactis and Leuconostoc species,
used as starter cultures in mesophilic dairy productions, produce huge problems through waste of
ingredients, increased processing time, reduced product quality, consistency and safety, and
occasionally complete loss of fermentation. Dairy phages have for long time been studied using
traditional culture-dependent methods but not using metagenomic approaches.
Part of this project was devoted to develop a method for dairy metavirome extraction and
analysis. Several whey mixtures derived by defined and undefined starters were used to test the
hypothesis that Lc. lactis 936 (now SK1virus), P335 and c2 (now C2virus) species phages (order
Caudovirales, family Siphoviridae) are the most frequently encountered phages in the dairy
environment. Our results indicated the dominance of Lc. lactis 936 phages in dairies using
undefined starters and of mostly Lc. lactis c2 phages in dairies using defined cultures. Certain
evidence indicating possible co-induction of temperate P335 phages and smaller Lc. lactis
satellite phages was obtained.
Also addressed was the issue of accurate and simultaneous quantification of dairy phages. A
high-throughput qPCR system was developed for Lc. lactis 936, P335, c2 and Leuconostoc
species phages. The system consisted of assays with high quantification efficiency (96% to
100%) and comparable dynamic ranges of detection (~9 x 103 to ~9 x 107 phage genomes mL-1).
Analysis of test samples from a dairy using a traditional mother-bulk-cheese vat system showed
the presence of 936 and P335 phages already in the mother culture stage and, consequently, in
the bulk starter and in the cheese vats but not in the cheese milk.
We further dealt with temperate phages associated with undefined mesophilic starters through
induction of whole cultures. Results obtained from metavirome analysis presented evidence that
the Lc. lactis P335 group of phages were the most frequently induced. Other Lc. lactis phages
such as 936, 949, 1706, c2 and Leuconostoc phages were also detected in the cultures. In a
spontaneously induced culture, however, 936 phages was found to dominate over P335, which
suggested the presence of phages in the culture in a carrier state.
We finally investigated the abundance and fluctuation in diversity of Lc. lactis and Leuconostoc
phages in a dairy using undefined mesophilic starter over a certain period of time. Lc. lactis 936
phages appeared to be the most frequent in the bulk starters and wheys from the first and the last
productions (average 108–1010, 104–108 and 105–108 phage genomes mL-1, respectively). A build
up of phages was observed across productions. On the basis of receptor binding protein-encoding
gene (rbp) analysis, several of the 936-phage groups were predicted to be able to attack strains of
Lc. lactis subsp. cremoris. An overpressure in the processing room and curd pressing rooms
resulted in an increase in the instability of subgroups of 936 phages during the first productions
but not during the last productions. Several evidences showing possible co-induction of
temperate P335 phages and satellite phages were also observed.
used as starter cultures in mesophilic dairy productions, produce huge problems through waste of
ingredients, increased processing time, reduced product quality, consistency and safety, and
occasionally complete loss of fermentation. Dairy phages have for long time been studied using
traditional culture-dependent methods but not using metagenomic approaches.
Part of this project was devoted to develop a method for dairy metavirome extraction and
analysis. Several whey mixtures derived by defined and undefined starters were used to test the
hypothesis that Lc. lactis 936 (now SK1virus), P335 and c2 (now C2virus) species phages (order
Caudovirales, family Siphoviridae) are the most frequently encountered phages in the dairy
environment. Our results indicated the dominance of Lc. lactis 936 phages in dairies using
undefined starters and of mostly Lc. lactis c2 phages in dairies using defined cultures. Certain
evidence indicating possible co-induction of temperate P335 phages and smaller Lc. lactis
satellite phages was obtained.
Also addressed was the issue of accurate and simultaneous quantification of dairy phages. A
high-throughput qPCR system was developed for Lc. lactis 936, P335, c2 and Leuconostoc
species phages. The system consisted of assays with high quantification efficiency (96% to
100%) and comparable dynamic ranges of detection (~9 x 103 to ~9 x 107 phage genomes mL-1).
Analysis of test samples from a dairy using a traditional mother-bulk-cheese vat system showed
the presence of 936 and P335 phages already in the mother culture stage and, consequently, in
the bulk starter and in the cheese vats but not in the cheese milk.
We further dealt with temperate phages associated with undefined mesophilic starters through
induction of whole cultures. Results obtained from metavirome analysis presented evidence that
the Lc. lactis P335 group of phages were the most frequently induced. Other Lc. lactis phages
such as 936, 949, 1706, c2 and Leuconostoc phages were also detected in the cultures. In a
spontaneously induced culture, however, 936 phages was found to dominate over P335, which
suggested the presence of phages in the culture in a carrier state.
We finally investigated the abundance and fluctuation in diversity of Lc. lactis and Leuconostoc
phages in a dairy using undefined mesophilic starter over a certain period of time. Lc. lactis 936
phages appeared to be the most frequent in the bulk starters and wheys from the first and the last
productions (average 108–1010, 104–108 and 105–108 phage genomes mL-1, respectively). A build
up of phages was observed across productions. On the basis of receptor binding protein-encoding
gene (rbp) analysis, several of the 936-phage groups were predicted to be able to attack strains of
Lc. lactis subsp. cremoris. An overpressure in the processing room and curd pressing rooms
resulted in an increase in the instability of subgroups of 936 phages during the first productions
but not during the last productions. Several evidences showing possible co-induction of
temperate P335 phages and satellite phages were also observed.
| Originalsprog | Engelsk |
|---|
| Forlag | Department of Food Science, Faculty of Science, University of Copenhagen |
|---|---|
| Antal sider | 242 |
| Status | Udgivet - 2016 |