الفهرس 
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Abstract
SUMMARY
Pathogenic bacteria cause diseases in human and plants, and
directly affect the supply and quality of the food chain. Numerous
bacteria cause food-borne illnesses throughout the world every year.
Salmonella is one of the pathogenic bacteria and it is enteric bacteria
commonly found in animals and its presence in human is usually
indication of an infection. Gastroenteritis, including diarrhea, fever
and abdominal cramps is a common symptom of infection resulting
from ingestion of contaminated foods such as poultry or dairy
products. In recent years it has become widely recognized that,
bacteriophages have several potential applications in the food industry
they have been proposed as alternatives to antibiotics in animal health
as bio preservatives in food and as tools for detecting pathogenic
bacteria through the food. Moreover, biotechnology companies began
developing bacteriophage products to kill bacteria that cause foodborne
illness in human.
This study aimed to investigate the presence of pathogenic bacteria
in some food products and also isolation and characterization of some
bacteriophages specific for these bacteria and using bacteriophages as
a food additive to kill pathogens or eliminate microbes.
1- Four samples of sewage water were obtained from Shoubra ELKheima
(Qalubia) and El-Aiat (Giza) and were examined for the
presence of bacteriophages specific for some pathogenic bacteria.
The obtained results shows that, by using the spot test technique,
there are specific phages for E. coli strains NRRL3008 on sample
No. 1; E. coli strains (B and H1B1D1), Salmonlla typhimurium
strains (ATCC25566 and MM11) and Staphylococcus aureus
(ATCC3565 and MM46T) on samples No. 2 and 3; and E. coli
Abeer A. Feisal, Ph.D., 2013
H1B1D1, S. typhimurium strains (ATCC25566 and MM11) and
Staph. aureus (ATCC3565 and MM46T) on sample No. 4.
2- The virulent bacteriophages specific for S. typhimurium
ATCC25566 were isolated from sewage water sample No. 4 by
spot test and then were biologically purified by single plaque assay.
The pure isolated S. typhimurium phage was propagated in liquid
culture of its host to obtain large amount of the particles before the
chemical purification by PEG, 6,000, Salmonella phage titer was
2.3 X 1012 pfu /ml, and phage titer was 8.9 X 109 pfu/ml after
purification by PEG, 8,000.
3- The effect of thermal properties of the phage was studied by using
spot test technique after exposure Salmonella bacteriophage to
different degrees of temperature (from 30 to 98 ºC) for ten min.
The virus lost its ability to lyses its host (S. typhimurium ) at 80 ºC.
The thermal inactivation point was exactly determined when, the
virus was exposed to temperatures to be 70 to 80 ºC (two degree
intervals) for ten min and the thermal inactivation point were found
at 76 ºC, and the viral concentration decreased with increasing the
temperature degrees. The Salmonella phage has high stability at
different temperature ranged from -20 ºC to 42 ºC for several days
4- Salmonella bacteriophage infectivity was tested against different
pH values from 4 to 12 and it was found that, the phage has activity
and able to lyse its host at pH 7, 8 and 9 while inhibited completely
at pH 4, 5, 6, 10, 11 and12.
5- The phage stability for UV radiation was tested at constant
wavelength and different distances from the UV lamp. The
particles of Salmonella phage were exposed to UV at distance of
35, 53 and 70 cm from the UV source for 10 to 60 min. At a
distance of 70 and 53 cm, the phage particles has still active from
10 to 50 min and lost thier activity after 60 min.
SUMMARY
Abeer A. Feisal, Ph.D., 2013
146
6- The effect of some food preservatives and additives on the phage
activity was detected using spot test. The effect of different
concentrations of sodium chloride (5, 10, 15, 20, 25, 30, 35 and 40
%) on phage was tested and it was found that, phage was active at
concentrations of 5, 10 and 15 % and then, lost its activity at 20 %.
7- The virus concentration and infectivity was affected negatively by
mixing with different concentrations of sodium benzoate,
potassium sorbate and citric acid (0.05, 0.1, 0.5 and 1.0%).
8- The effect of different concentrations of the mixed spices
consisting of Black pepper, (30.0 g); Nut Meg (8.0 g) on phage was
tested. All spices (15.0 g); Red pepper (8.0 g); Cloves (8.0 g);
Cinnamon (15.0 g); Ginger (8.0 g) and coriander (8.0 g) which
added used to be to some processed meat products (Luncheon
meat, Berger and Sausage) on phage activity were studied at
concentrations of 0.5, 1.0, 1.5 and 2 % and the phage remained
active at all concentrations.
9- The effect of some detergent such as sodium dodecyl sulfate and
sodium hypochlorite at concentrations of 1, 2, 3, 4 and 5 % was
recorded for phage stability, and it was revealed that, the phage
activity decreased by increasing in the detergent concentrations.
10- Host specificity of the isolated bacteriophage was determined
and tested by the spot test method and it was found that the isolated
S. typhimurium bacteriophage had very limited host range.
11- Electron microscopy of S. typhimurium bacteriophage showed
particles with long non-contractile tail and isometric head 69.6 nm
in diameter length 243.5 and 17.4 nm.
12- The total protein content of purified suspension particles was 0.
142 mg/ml purified phage.
SUMMARY
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147
13- The molecular weight of the isolated S. typhimurium phage
genome was successfully detected to be 18 kbp in 1 % agarose gel
electrophoresis.
14- The immunogenicity of purified Salmonella bacteriophage was
detected after raising their specific polyclonal antibodies by 4
injection doses in different injection types in rabbits. The
serological sensitivity reaction for purified phage against specific
produced antiserum was detected by Ouchterlony double diffusion
test.
Application of lytic Salmonella bacteriophage to biocontrol
food borne Salmonella in some food products:
In this study, a number of experiments have been performed to
evaluate the potential of the isolated Salmonella bacteriophage for the
reduction of S. typhimurium in different ready-to-eat foods of both
plant and animal origin and it was found that:
1- Application of Salmonella bacteriophage on vegetable and fruits:
Salmonella bacteriophage specific for S. typhimurium was isolated
through the study and used for application on vegetable and fruits and
other food products.
In this study 14 samples of fresh salads were obtained from
different local markets and analyzed microbiologically to assay their
bacteriological load and presence or absence of the pathogenic
bacteria before application of the bacteriophage to remove or/and
reduce the Salmonella viable cells. Results of the microbiological
analysis showed that 85.7 % of samples (12 out of 14) were
contaminated with coliform and only 2 samples were found to be un
contaminated. It was also found that all samples contained Salmonella
bacteria, 78.5 % from the samples (11 from 14 samples) contained
SUMMARY
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Staphylococcus and 64.3 % from the samples (9 from 14 samples)
contained Listeria cells.
Microbiological analysis of the fourteen green salads revealed also
that, the green salads contained the bacteriophage specific for S.
typhimurium ATCC25566 and S. typhimurium MM11.
The obtained results are logic because the green salads are handmade
and its row materials from lettuce, tomato, cucumber and so on
are exposed to contamination with different microbes. On the other
hand presence of bacteriophage specific for Salmonella is also very
logic because of the presence of its specific bacterial host.
Application of S. typhimurium bacteriophage to eliminate or
reduced the total count of Salmonella cells in green salads in the study
revealed that, the bacteriophage reduce the total counts of Salmonella
cells significantly from 7.64 log10 units to 4.38 log10 cfu /g units with
rate of 42.67 % through 24 hours of incubation at 4°C.
Salmonella bacteriophage and Salmonella cells interactions on
fresh cut apples were examined in this study. Virulent bacteriophage
specific for S. typhimurium was used to eradicate or reduce
Salmonella as a food borne pathogen in green and red apples, and it
was found that, spraying of both green and red apples with virulent
Salmonella bacteriophage solution led to reduce the total count of
viable Salmonella cells significantly from 5.36 log10 units to 3.0 log10
cfu /g in case of the green apple and from 5.27 log10 units to 2.2 log10
cfu /g in case of red apples after incubation for 24 hours at room
temperature. The bacteriophage reduced also the total counts of viable
Salmonella cells in the treated apple slices after incubation at 4°C
from 6.08 log10 units to 2.98 log10 cfu /g.
2- Foods of animal origin samples of preserved beef Berger, beef
luncheon, beef frankfurter, minced beef, nuggets, and oriental
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sausages were obtained from local markets and evaluated for
microbial contamination and the bacteriological load. Obtained
results revealed that, all samples contain fecal coliform, fecal
Sterptococci as polluted bacterial pathogen, 92 % from samples
contain Ps. aeroginosa and 61.5 % from samples contain
Salmonella. The bacterial pathogens present in the different
samples with different densities. The obtained data are logic
because of preservation conditions some times in the markets are
bad, in addition it may be contaminated during the manufacturing
of products.
Chicken Berger was manufactured in the laboratory and treat with
Salmonella and Salmonella bacteriophage to evaluate the potential of
the specific bacteriophage for reduction of Salmonella through 15
days incubation at 4°C. Obtained results indicated that, treatment of
the manufactured chicken Berger by virulent Salmonella
bacteriophage led to reduction of the indigenous Salmonella viable
cells from 2.32 log10 units to 0.60 log10 units during 15 days incubation
at 4°C.
Data also showed that, the specific bacteriophage reduced the total
Salmonella cells in the manufactured chicken Berger treated with
Salmonella and its bacteriophage through 15 days incubation at 4°C
from 5.43 log10 units to 2.04 log10 units, on the other hand the
bacteriophage titer increased significant from 5.3 log10 units to 11.96
log10 units. Increasing of the bacteriophage particles is very logic and
due to the presence of the susceptible host with high denstiy
(indigenous and added Salmonella cells).
Application of Salmonella phage to fresh chicken cuts was done.
Fresh chicken cuts were treated by flipping, spraying and soaking and
incubated for 7 days at both 4°C and 10 °C.
Result of the experiments showed the following:
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1- Application of the specific Salmonella bacteriophage to the fresh
chicken cuts by flipping in foam plate reduced the total counts of
indigenous Salmonella from 1.48 log10 to 0.78 log10 cfu /g after 3
days and eliminated the all cells after at the fourth day from
incubation at 4°C.and reduced the total numbers of viable
Salmonella cells in the treated fresh chicken cuts from 3.75 to 1.0
log10 cfu /g and eliminated the remained cells after 7th days
incubation at 4°C.
2- Fresh chicken cuts were treated with spraying of both Salmonella
and bacteriophage. The virulent bacteriophage of Salmonella
caused an elimination of the indigenous Salmonella after one day
incubation at 4°C. On the other hand it reduced the Salmonella
from 3.32 log10 to 0.48 log10 cfu /g after 3 days, and then
eliminated the remained viable cells completely, through the
incubation at 4°C.
3- Application of the virulent Salmonella bacteriophage to fresh
chicken cuts by soaking led to reduce the total numbers of the
indigenous cells from 1.3 log10 0.84 log10 cfu /g. Through 3 days
of incubation at 4°C.it also caused a reduction from 3.11 log10 to
1.0 log10 cfu /g after 4 days from incubation at 4°C and eradicated
the Salmonella after fourth day.
4- Treatment of fresh chicken cuts by the virulent Salmonella
bacteriophage using the flipping method followed by incubation
at 10°C caused a reduction from 1.7 log10 to 0.6 log10 of the
indigenous Salmonella, then eradicated the all cells, and a
reduction from 3.83 log10 to 0.6 log10 after 7 days from incubation
at 10°C.
5- Fresh chicken cuts treated with Salmonella bacteriophage by
spraying and preserved for 7 days at 10°C showed a reduction in
the content of viable Salmonella cells because of the presence of
SUMMARY
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virulent Salmonella bacteriophage. The bacteriophage reduced the
indigenous numbers from 1.48 log10 to 0.7 log10 cfu /g after 3
days and then eradiate the cells. It reduced the cells of Salmonella
in case of addition both Salmonella and virulent bacteriophage
from 3.36 log10 to 1.0 log10 cfu /g after 4 days incubation at 10°C.
6- In case of application of virulent Salmonella bacteriophage to
fresh chicken cuts by the soaking method, it was found that, the
virulent phage reduced the numbers of indigenous Salmonella
cells from 1.95 log10 to 0.6 log10 cfu /g through four days
incubation at 10°C and from 3.2 log10 to 1.0 log10 cfu /g through
four day at 10°C.
Generally, the obtained results are premising, since
bacteriophage effectively reduced and eliminate S. typhimurium in the
contaminated food products of plant and animal origin.