Development and Diversity of Lactococcus lactis and Leuconostoc Bacteriophages in Dairies Using Undefined Mesophilic DL-Starter Cultures: A Metagenomic Approach
Research output: Book/Report › Ph.D. thesis
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Development and Diversity of Lactococcus lactis and Leuconostoc Bacteriophages in Dairies Using Undefined Mesophilic DL-Starter Cultures : A Metagenomic Approach. / Muhammed, Musemma Kedir.
Department of Food Science, Faculty of Science, University of Copenhagen, 2016. 242 p.Research output: Book/Report › Ph.D. thesis
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TY - BOOK
T1 - Development and Diversity of Lactococcus lactis and Leuconostoc Bacteriophages in Dairies Using Undefined Mesophilic DL-Starter Cultures
T2 - A Metagenomic Approach
AU - Muhammed, Musemma Kedir
PY - 2016
Y1 - 2016
N2 - Bacteriophages (phages) attacking strains of Lactococcus (Lc.) lactis and Leuconostoc species,used as starter cultures in mesophilic dairy productions, produce huge problems through waste ofingredients, increased processing time, reduced product quality, consistency and safety, andoccasionally complete loss of fermentation. Dairy phages have for long time been studied usingtraditional culture-dependent methods but not using metagenomic approaches.Part of this project was devoted to develop a method for dairy metavirome extraction andanalysis. Several whey mixtures derived by defined and undefined starters were used to test thehypothesis that Lc. lactis 936 (now SK1virus), P335 and c2 (now C2virus) species phages (orderCaudovirales, family Siphoviridae) are the most frequently encountered phages in the dairyenvironment. Our results indicated the dominance of Lc. lactis 936 phages in dairies usingundefined starters and of mostly Lc. lactis c2 phages in dairies using defined cultures. Certainevidence indicating possible co-induction of temperate P335 phages and smaller Lc. lactissatellite phages was obtained.Also addressed was the issue of accurate and simultaneous quantification of dairy phages. Ahigh-throughput qPCR system was developed for Lc. lactis 936, P335, c2 and Leuconostocspecies phages. The system consisted of assays with high quantification efficiency (96% to100%) 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 showedthe presence of 936 and P335 phages already in the mother culture stage and, consequently, inthe bulk starter and in the cheese vats but not in the cheese milk.We further dealt with temperate phages associated with undefined mesophilic starters throughinduction of whole cultures. Results obtained from metavirome analysis presented evidence thatthe Lc. lactis P335 group of phages were the most frequently induced. Other Lc. lactis phagessuch as 936, 949, 1706, c2 and Leuconostoc phages were also detected in the cultures. In aspontaneously induced culture, however, 936 phages was found to dominate over P335, whichsuggested the presence of phages in the culture in a carrier state.We finally investigated the abundance and fluctuation in diversity of Lc. lactis and Leuconostocphages in a dairy using undefined mesophilic starter over a certain period of time. Lc. lactis 936phages appeared to be the most frequent in the bulk starters and wheys from the first and the lastproductions (average 108–1010, 104–108 and 105–108 phage genomes mL-1, respectively). A buildup of phages was observed across productions. On the basis of receptor binding protein-encodinggene (rbp) analysis, several of the 936-phage groups were predicted to be able to attack strains ofLc. lactis subsp. cremoris. An overpressure in the processing room and curd pressing roomsresulted in an increase in the instability of subgroups of 936 phages during the first productionsbut not during the last productions. Several evidences showing possible co-induction oftemperate P335 phages and satellite phages were also observed.
AB - Bacteriophages (phages) attacking strains of Lactococcus (Lc.) lactis and Leuconostoc species,used as starter cultures in mesophilic dairy productions, produce huge problems through waste ofingredients, increased processing time, reduced product quality, consistency and safety, andoccasionally complete loss of fermentation. Dairy phages have for long time been studied usingtraditional culture-dependent methods but not using metagenomic approaches.Part of this project was devoted to develop a method for dairy metavirome extraction andanalysis. Several whey mixtures derived by defined and undefined starters were used to test thehypothesis that Lc. lactis 936 (now SK1virus), P335 and c2 (now C2virus) species phages (orderCaudovirales, family Siphoviridae) are the most frequently encountered phages in the dairyenvironment. Our results indicated the dominance of Lc. lactis 936 phages in dairies usingundefined starters and of mostly Lc. lactis c2 phages in dairies using defined cultures. Certainevidence indicating possible co-induction of temperate P335 phages and smaller Lc. lactissatellite phages was obtained.Also addressed was the issue of accurate and simultaneous quantification of dairy phages. Ahigh-throughput qPCR system was developed for Lc. lactis 936, P335, c2 and Leuconostocspecies phages. The system consisted of assays with high quantification efficiency (96% to100%) 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 showedthe presence of 936 and P335 phages already in the mother culture stage and, consequently, inthe bulk starter and in the cheese vats but not in the cheese milk.We further dealt with temperate phages associated with undefined mesophilic starters throughinduction of whole cultures. Results obtained from metavirome analysis presented evidence thatthe Lc. lactis P335 group of phages were the most frequently induced. Other Lc. lactis phagessuch as 936, 949, 1706, c2 and Leuconostoc phages were also detected in the cultures. In aspontaneously induced culture, however, 936 phages was found to dominate over P335, whichsuggested the presence of phages in the culture in a carrier state.We finally investigated the abundance and fluctuation in diversity of Lc. lactis and Leuconostocphages in a dairy using undefined mesophilic starter over a certain period of time. Lc. lactis 936phages appeared to be the most frequent in the bulk starters and wheys from the first and the lastproductions (average 108–1010, 104–108 and 105–108 phage genomes mL-1, respectively). A buildup of phages was observed across productions. On the basis of receptor binding protein-encodinggene (rbp) analysis, several of the 936-phage groups were predicted to be able to attack strains ofLc. lactis subsp. cremoris. An overpressure in the processing room and curd pressing roomsresulted in an increase in the instability of subgroups of 936 phages during the first productionsbut not during the last productions. Several evidences showing possible co-induction oftemperate P335 phages and satellite phages were also observed.
UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122542031105763
M3 - Ph.D. thesis
BT - Development and Diversity of Lactococcus lactis and Leuconostoc Bacteriophages in Dairies Using Undefined Mesophilic DL-Starter Cultures
PB - Department of Food Science, Faculty of Science, University of Copenhagen
ER -
ID: 169360185