340 Modern Food Microbiology
cloned and expressed in E. coli. When tested against 48 strains of C. perfringens, the phage endolysin
destroyed all by its lytic activity when applied exogenously.
224 Clostridium fallax was the only other
species that was lysed, and it has a peptidoglycan structure similar to that of C. perfringens.
Endolysins from L. monocytogenes phages have been introduced into a lactic starter culture, enabling
the phage enzyme to reduce or eliminate the pathogen during cheese ripening. In order to optimize the
release of the intracellularly synthesized endolysin from the bacterial cells onto the cheese surface, the
endolysin encoding gene was modified to carry a signal peptide. When this construct was introduced
into a dairy starter culture of Lactococcus lactis, a clone was identified, which expressed a strong
lytic activity that was quantitatively exported from the lactococcal cells into the surrounding medium
where it caused rapid lysis of L. monocytogenes cells.
64 The vector was also introduced into a lactoseutilizing strain of L. lactis, where a functional enzyme was produced and the vector was shown to be
compatible with native lactococcal plasmids.
64 These recombinants were also used in preliminary dairy
fermentation experiments to control L. monocytogenes, and a 95% reduction of the pathogen at the end
of a camembert cheese ripening period was demonstrated (unpublished results: M.J. Loessner et al.).
A broad-spectrum endolysin from a Lactobacillus helveticus phage was shown to lyse different species of lactobacilli and also some lactococci, pediococci, Enterococcus faecium, and some
other Gram-positive bacteria.
45 Listeria innocua, Streptococcus salivarius subsp. thermophilis, three
species/strains of propionibacteria as well as Escherichia coli, Pseudomonas fluorescens, and
Salmonella Abortus-ovis were not affected by the construct.
BACTERIOPHAGES AS BIOCONTROL AGENTS
Lytic phages specific for given bacterial species and strains are known to be effective in destroying
their host cells, and this is the basis of phage typing, which is described in Chapter 11. To control
pathogens and spoilage bacteria on foods, the question is whether phages can destroy their specific
host cells in this environment. In other words, do food substrates prevent phage attachment to host
cells and if not, do other factors come into play to prevent cell lysis? Dating back to the 1960s, research
has been published on the efficacy of bacteriophages to destroy their host cells in a number of settings
including meats, poultry, and certain human illnesses; and some of these have been reviewed.
6,70,75
Phages were shown in the late 1960s to lyse their host cells recovered from fish, meats, and skim
milk but these early studies employed phages and their host bacteria in broth and meat extract cultures.
It appears that the first study of phages directly added to meat to control meat spoilage bacteria was
that of Greer.
75 In this study, rib–eye steaks, a Pseudomonas sp. previously isolated from spoiled beef,
and a homologous phage at a level of 10
8 pfu/ml were used. Four days after adding the phage to the
steaks (held at 7
◦ C) that were surface inoculated with the host bacterium, a 1–2-log reduction of the
bacterium and a 2-log increase in phage numbers were noted. Adding 10
8 pfu/ml of phage caused
steak case-life to increase from 1.6 to 2.9 days.
75 Overall, surface discoloration and retail acceptance
were improved by the phage treatments.
The reduction of S. Enteritidis and Campylobacter jejuni by specific phages on excised chicken
skin was investigated.
70 On S. Enteritidis, phages increased from an initial of 1.0 to 3.49 pfu/cm
2
after 48 hours while those on untreated skin decreased. These investigators employed a multiplicity
of infection (MOI) factor, which relates the relative numbers of phages to their host bacteria. At an
MOI of 1, phages increased in numbers and reduced the two pathogens by <1 log/cm
2 . At an MOI of
100–1,000, the numbers of bacteria were reduced rapidly by up to 2 logs over 48 hours. No salmonellae
were recovered with MOI of 10
7 .
70
cloned and expressed in E. coli. When tested against 48 strains of C. perfringens, the phage endolysin
destroyed all by its lytic activity when applied exogenously.
224 Clostridium fallax was the only other
species that was lysed, and it has a peptidoglycan structure similar to that of C. perfringens.
Endolysins from L. monocytogenes phages have been introduced into a lactic starter culture, enabling
the phage enzyme to reduce or eliminate the pathogen during cheese ripening. In order to optimize the
release of the intracellularly synthesized endolysin from the bacterial cells onto the cheese surface, the
endolysin encoding gene was modified to carry a signal peptide. When this construct was introduced
into a dairy starter culture of Lactococcus lactis, a clone was identified, which expressed a strong
lytic activity that was quantitatively exported from the lactococcal cells into the surrounding medium
where it caused rapid lysis of L. monocytogenes cells.
64 The vector was also introduced into a lactoseutilizing strain of L. lactis, where a functional enzyme was produced and the vector was shown to be
compatible with native lactococcal plasmids.
64 These recombinants were also used in preliminary dairy
fermentation experiments to control L. monocytogenes, and a 95% reduction of the pathogen at the end
of a camembert cheese ripening period was demonstrated (unpublished results: M.J. Loessner et al.).
A broad-spectrum endolysin from a Lactobacillus helveticus phage was shown to lyse different species of lactobacilli and also some lactococci, pediococci, Enterococcus faecium, and some
other Gram-positive bacteria.
45 Listeria innocua, Streptococcus salivarius subsp. thermophilis, three
species/strains of propionibacteria as well as Escherichia coli, Pseudomonas fluorescens, and
Salmonella Abortus-ovis were not affected by the construct.
BACTERIOPHAGES AS BIOCONTROL AGENTS
Lytic phages specific for given bacterial species and strains are known to be effective in destroying
their host cells, and this is the basis of phage typing, which is described in Chapter 11. To control
pathogens and spoilage bacteria on foods, the question is whether phages can destroy their specific
host cells in this environment. In other words, do food substrates prevent phage attachment to host
cells and if not, do other factors come into play to prevent cell lysis? Dating back to the 1960s, research
has been published on the efficacy of bacteriophages to destroy their host cells in a number of settings
including meats, poultry, and certain human illnesses; and some of these have been reviewed.
6,70,75
Phages were shown in the late 1960s to lyse their host cells recovered from fish, meats, and skim
milk but these early studies employed phages and their host bacteria in broth and meat extract cultures.
It appears that the first study of phages directly added to meat to control meat spoilage bacteria was
that of Greer.
75 In this study, rib–eye steaks, a Pseudomonas sp. previously isolated from spoiled beef,
and a homologous phage at a level of 10
8 pfu/ml were used. Four days after adding the phage to the
steaks (held at 7
◦ C) that were surface inoculated with the host bacterium, a 1–2-log reduction of the
bacterium and a 2-log increase in phage numbers were noted. Adding 10
8 pfu/ml of phage caused
steak case-life to increase from 1.6 to 2.9 days.
75 Overall, surface discoloration and retail acceptance
were improved by the phage treatments.
The reduction of S. Enteritidis and Campylobacter jejuni by specific phages on excised chicken
skin was investigated.
70 On S. Enteritidis, phages increased from an initial of 1.0 to 3.49 pfu/cm
2
after 48 hours while those on untreated skin decreased. These investigators employed a multiplicity
of infection (MOI) factor, which relates the relative numbers of phages to their host bacteria. At an
MOI of 1, phages increased in numbers and reduced the two pathogens by <1 log/cm
2 . At an MOI of
100–1,000, the numbers of bacteria were reduced rapidly by up to 2 logs over 48 hours. No salmonellae
were recovered with MOI of 10
7 .
70