Ipaprint Na 'To!!!

CENTRO ESCOLAR UNIVERSITY College of Medical
Technology PAGE 1
ACKNOWLEDGEMENT
We would like to extend our gratitude to the following who never fail to
support us in this research:
Mr. Romeo Ramos and Sir Drandreb Bermude! our laborator"
technicians! for helping us whenever we need something regarding the process
of our research#
Mr. Rogelio $ru and Dr. Romina Barcarse! for consultations and
accommodations in regards with our stud"#
Ms. M"rna Biscocho RM%! our Research & adviser! for the guidance#
Ms. 'n Margarete %an RM%! the thesis adviser! for her dedication and
never(failing supervision#
Ms. )rika *a"le +ipana RM%! Mr. Roderick Balce RM% and Mr. Mark
Rodrigo Mendros RM%! for all the helpful advices and for giving us the concept
for our research topic#
,or undeviating moral and support of our famil" and friends#
,or all the researchers! who put their time and effort in making this
research possible.
,or the 'lmight" ,ather! for -is divine guidance in providence in this
academic achievement.
The Researchers
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THESIS ABSTRACT
+actic acid bacteria have shown to pla" an important ph"siological role in
the human gastrointestinal .*l/ tract. 0t functions as a microbial barrier against
microbial pathogens together with the cells lining the gastrointestinal epithelium
b" preventing unwanted intrusions of microorganisms into the gastrointestinal
microbiota! and against the penetration of harmful microorganisms which
arrogate the cellular molecules and signaling pathwa"s of the host to become
pathogenic. Due to ver" low p- .1(2/ of +actic acid bacteria other pathogens
cannot withstand the acidit" of the environment. +actic acid bacteria are gram(
positive! non(sporulating! and catalase negative rods or cocci that produce lactic
acid as a ma3or or sole product of their fermentative metabolism. +actic acid
bacteria were isolated from chicken intestines and separated from other
organisms through the use of a selective medium ( MRS Broth and MRS 'gar!
incubated anaerobicall" for 24 hours at 15
5
$ ( 16
5
$ using 'naero*en .'75518'!
9xoid/. %hree subcultures were made ever" 24 hours to obtain pure cultures of
the isolates. $arboh"drate fermentation patterns were determined using BB+
$r"stal 0dentification S"stem .BD ( Diagnostic S"stems/. %he species were
identified using the software version from BD ( Diagnostic S"stems. Standardied
cultures grown at MRS broth were used to test for antagonistic activit" against
indicator pathogens Staphylococcus aureus and Escherichia coli b" an agar well
diffusion method.
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TABLE OF CONTENTS
Page
'cknowledgement:::::::::::::::::::::..:::.:.&
'bstract::::::::::::.::::::::::::::...:::..;
%able of $ontents:::::::::::::::::::::...:.:.::1
+ist of %ables::::::.::::::::::::::::...:::::.<
+ist of ,igures:::::::::..::::::::::::::::::..6
Chapter
&. =roblem and 0ts Background
&.& 0ntroduction:::::::::::::::::::::..4
&.; Background of the Stud":::::::::::::::..&5
&.1 Statement of the =roblem::::::::::::::.:&&
&.2 9b3ectives of the Stud"::::::::::.......................&&
&.8 -"potheses of the Stud"::::::::::.:::::..&&
&.< Scope! +imitations and Delimitations::::::::::. &;
&.6 Significance of the Stud":::::::::::::::..&;
&.4 Settings of the Stud"::::::::::::::::..:&1
&.> $onceptual ,ramework::::::::::::::::.&2
&.&5 Definition of %erms:::::::::::::::...::&<
;. Review of Related +iterature and Studies
;.& +ocal +iterature:::::::::::::::::::.&4
;.; ,oreign +iterature:::::::::::::::::.:&>
;.1 +ocal Studies::::::::::::::::::::.12
;.2 ,oreign Studies:::::::::::::::::::12
Chapter
1. Methods and =rocedures
1.& Method and Materials::::::::::::::::25
i. Standards! Reagents! and $hemicals::::..:...25
1.; Sampling =rocedures::::::::::::::::25
ii. 0solation of +actic 'cid Bacteria and
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$ulture $onditions::::::::::::....2&
iii. $haracteriation =rocedures of +actic 'cid
Bacteria:::::::::::::::::..2;
iv. 0dentification of lactic acid bacteria using
BB+ $r"stal 0dentification S"stem::::::.2;
v. Screening of lactic acid bacteria for
'ntimicrobial activit"::::::::::::21
vi. Detection of antimicrobial activit" b" agar well
diffusion method.................................................21
vii. $onfirmator" procedures about the inhibition activit" of
+actic 'cid Bacteria ?sing 9ther
Methods........22
1.1 Statistical 'nal"sis:::::::::..:::::::.22
2. 'nal"sis! =resentation and 0nterpretation of Data:::::...:2<
8. Summar"! $onclusions! and Recommendations
8.& Summar" of ,indings:::::::::::::::...8>
8.; $onclusions:::..::::::::::::::::.<5
8.1 Recommendations::::::.:::.:::::::.<&
<. Bibliograph"::::::::::::::::::.:..::..:<;
6. 'ppendices:::::::::::::::::::::.....:<<
'. $ertificate of BB+ $r"stal 0D @it::::.::.:...:::<<
B. Media =reparation:::::::.::............................<6
$. -omogeniation and Standardiation::::::......::<4
D. $olonial $haracteriation and Morpholog" 0dentification.....<>
). =reparation for Media ?sed::::::::::........::65
,. $omputation of 0nhibition 0ndex::::::...:....:::..62
*. BB+ $r"stal $olor $hart:::::::::...::...::..66
-. BB+ $r"stal @its:::::::..::::......:::::.64
0. ?A 'nal"ing +amp:::::::::::....:...:::.6>
B. '=0 @it:::::::::::::::::......::::45
@. Spectrophotometer::::::::::.....::::..:4&
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+. Results of 'ntagonistic =ropert"::::::.....::...:4;
M. )xpenses:::::::::::::::.....::::..48
7. %imetable:::::::::::::::....::::..46
$urriculum Aitae::::::::::::::: ..:.....::::...44
LIST OF TABLES
Table Page
&. $haracteristic of Bacteria from all plated
$olonies::::::::::::::::::::........2<
;. $haracteriation and 0dentification of
$oded 9rganisms::::::::::::::::........2<
1. ,irst Sub($ulture on MRS 'gar:::::::::::::..24
2. ,irst Sub(culture on $olumbia 'gar::::::::..::.....2>
8. Second Sun(culture on MRS 'gar and
$olumbia 'gar =lates::::::::::::.::.........85
<. 0dentification of +actic 'cid Bacteria using
BB+ $r"stal *ram =ositive 0D S"stem:::::..:...........81
6. 9ptical Densit" at <55nm::::::::::::::::.82
4. Serial Dilution::::::::::::::::..::::...88
>. )valuation of the antagonistic activit"
using C(D inhibition Method::::..:.::::::........8<
&5. Measurement of the Eones of inhibition
?sing Well(Diffusion Method::::::::::........:.86
&&. 'ntagonistic 'ctivit" of +actic 'cid Bacteria ?sing
'gar Well Diffusion Method .in mm/:::::::.........:86
&;. 0nterpretation of Results::::::::..........................:.8>
LIST OF FIGRES
F!g"re Page
&. Bustillos Market:::::::::::::::..::::..........&1
;. Map of $entro )scolar ?niversit":::::::::::..........:&1
1. $entro )scolar ?niversit"::::::::::::::..........:.&1
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CHAPTER #
The Pr$ble% a&' Its Bac(gr$"&'
#)#I&tr$'"ct!$&
%he gastrointestinal tract .*0%/! either from humans or animals! is a complex
structure within the living organism where certain residents of other kind reside
within the surface of its epithelial wall expressing specific metabolic activities.
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%he microbiota differs Fuantitativel" and Fualitativel" at different points along the
gastrointestinal tract. %he stomach has few resident microorganisms# however
lactobacilli have been isolated from the microbiota resident in the stomach. %he
microbial profile of the digestive tract is t"pified b" the absence of anaerobic
microorganisms in the stomach and conversel" their overwhelming
predominance in the distal colon. Moreover! different microbial communities ma"
be located in the intestinal lumen! in the mucus covering the epithelium! in the
cr"pt spaces or in the different cells lining the epithelium! and in addition! some
species adhere whereas others do not. %he number of bacterial species in the
intestinal microbiota has been estimated to be about 255. 'erobic! facultative and
anaerobic bacteria all inhabit the gastrointestinal microbiota. .Servin! ;552/
+actic acid bacteria have shown to pla" an important ph"siological role in the
human gastrointestinal .*l/ tract. Microbial interactions represent the main force
in the homeostasis of the bacteria flora in the *l tract. $ombined with the host
this microflora form a uniFue ecos"stem in which the complex interactions can
either be s"nergistic to or antagonistic depending on their strain identif"!
population levels and metabolic activit". %he health" survival of the host is
determined b" these interactions. %he ecos"stem is destabilied as a result of
gastrointestinal disorders and other endogenous components such as en"mes!
hormones and immunoglobulins can influence it. .*uo et al.! ;55</ Due to ver"
low p- .1(2/ of +actic acid bacteria other pathogens cannot withstand the acidit"
of the environment.
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+actic acid bacteria .+'B/ are ma3or commensal bacteria of the intestinal tract
of mammals! including humans! in whom the" are used as probiotics. =robiotics
that are characteried as bio(preparations that contain living cells or metabolites
of stabilied autochthonous microorganisms that optimie the coloniation and
composite of gut microbiota in both animals and humans and have a stimulator"
effect on the digestive process and immunit" of the host.
=robiotic bacteria are non(pathogenic microorganisms that! when ingested in
proficient viable number! perform a positive influence on the host to upgrade the
intestinal microbiota b" inhibiting the growth andGor activit" of a pathogen
bacteria. .Aerschuere et al.! ;555/.
%he ob3ective of this research is to determine the antagonistic activities of
specific lactic acid bacteria against other pathogens within a chickenHs intestine.
't least 8 lactic acid bacteria were screened for its antagonistic activities against
pathogens in their same habitat. Some of these pathogens were Escherichia coli,
Salmonella, and Campylobacter spp.
#)*) Bac(gr$"&' $+ the St"',
+actic acid bacteria are mostl" gram(positive! non(sporulating! and
catalase negative rods or cocci that produce lactic acid as a ma3or or sole
product of their fermentative metabolism.
Most of these lactic acid bacteria! as mentioned before! are known to be
probiotics. $hicken intestines compared to intestines of some animals are said to
Technology PAGE 9
have more of these +'B# however! it also inhabits other pathogens. 'nd the role
of these probiotics within a chickenHs bod" is mainl" to inhibit the growth of these
pathogens to prevent disease(causing microbes to spread to the animalHs bod"
which ma" have an effect to those who will eat it and even to the eggs it will la".
%he most common pathogens that ma" also be found! and most are
common! inside its colon are Salmonella spp.! Enterococcus spp.! and the like.
0n order for the +'B to perform its role! ph"sical and chemical barriers are
created b" the gastrointestinal epithelium of the host to protect itself from the
attack of potentiall" harmful microbial microorganisms. %he gastrointestinal cells
that make up the epithelium provide a ph"sical barrier that protects the host
against the unwanted intrusions of microorganisms into the gastrointestinal
microbiota! and against the penetration of harmful microorganisms which
arrogate the cellular molecules and signaling pathwa"s of the host to become
pathogenic. 9ne of the basic ph"siological functions of the
resident microbiota is that it functions as a microbial barrier against microbial
pathogens.
#)-) State%e&t $+ the Pr$ble%
%his stud" aims to determine the antagonistic activities of lactic acid
bacteria isolated against other pathogens from a chickenHs .Gallus Gallus
domesticus/ intestine.
#).) Ob/ect!0es $+ the St"',
Specificall"! it sought to answer the following Fuestion:
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What are the identified +actic 'cid Bacteria isolated from a chickenHs
intestineI
What are the factors that can affect the antagonistic activities exhibited b"
a specific lactic acid bacteriumI
What other pathogens are being inhibited b" the isolated +actic 'cid
BacteriaI
#)1) H,p$theses $+ the St"',
%he +actic 'cid Bacteria isolated from the samples can show
antagonistic activit" against certain pathogens.
%he isolates were Lactobacillus fermentum, and Lactobacillus johnsonii.
%he +actic 'cid Bacteria prevented the growth of Escherichia coli and
Staphylococcus aureus! organisms that ma" affect the chickenHs
digestion.
#)2) Sc$pes3 L!%!tat!$&s a&' Del!%!tat!$&s
0n this stud"! 8 chicken intestine samples were collected from a stall from
Bustillos Market in Sampaloc! Manila.
%he MRS 'gar and MRS Broth were used for the isolation of the +actic
'cid Bacteria! with $olumbia 'gar Base as a preliminar" medium for the
detection and confirmator" test of anaerobicall" growing bacteria. %wo out of
fourteen isolates were confirmed to be +actic 'cid producing bacteria.
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Escherichia coli and Staphylococcus aureus were tested for inhibitor" activit"
against the isolated lactic acid bacteria using three different methods: 'gar well
diffusion method! Well diffusion method! and the C(D inhibition method. ,our pure
colonies of Lactobacillus# Lactobacillus acidophilus, Lactobacillus casei,
Lactobacillu paracasei, Lactobacillus plantarum, were also tested for inhibition.
%hese isolates were from ?niversit" of the =hilippines! +os BaJos in +aguna.
#)4) S!g&!+!ca&ce $+ the St"',
%his stud" is important for:
%he possibilit" of using the +actic 'cid Bacteria with the highest inhibition
as a food supplement and vitamins for chickens.
%he identification of specific chicken gastro(enteric pathogens which
affects digestion of chickens.
%he information it ma" give to our local poultr" farmers.
%he isolated +actic 'cid Bacteria and the pure colonies that are positive
in antagoniing E.coli and S.aureus ma" be used and added to probable
medicines in curing certain diseases and implications caused b" E.coli and
S.aureus.
#)5) Sett!&gs $+ the St"',
%his stud" is conducted in $entro )scolar ?niversit"! Manila $ampus for
the fulfillment of the Research program of the $ollege of Medical %echnolog".
%he samples were gathered from a stall in Bustillos Market Sampaloc! Manila.
%he experiment was performed inside the $entennial Research +aborator"
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located at the 1
rd
floor of the $'- building in $entro )scolar ?niversit" and at
Room 1&5 of the *D+S$ building of the same institution.
#)6) C$&cept"al Fra%e7$r(
Isolation of lactic acid bacteria from chicken small
intestine.
Isolation of lactic acid bacteria from chicken small
intestine.
Identification/ Characterization of isolates using BBL
Crystal Identification System.
Identification/ Characterization of isolates using BBL
Crystal Identification System.
Antagonistic activity against chicken
pathogens.
Antagonistic activity against chicken
pathogens.
Bustillos Maket! "a#paloc Manila
Map o$ %ento &scola
'nivesity
%ento &scola 'nivesity
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?pon the cleansing and homogeniation of the chicken intestines!
approximatel" &5 grams of the collected surface mucus were streaked in two
wa"s: a./ Direct inoculation without the addition of the =hosphate Buffer Solution!
and b./ 0ndirect inoculation where =hosphate Buffer Solution was added before
streaked into MRS 'gar! $olumbia 'gar! and inoculated into MRS broth.
'fter the growth of the suspected +actic 'cid Bacteria! specific colonies
were sub(cultured thrice on both MRS 'gar and $olumbia 'gar! except on the
third and last subculture where the colonies where streaked on MRS 'gar onl".
'lso part of the subculture process! organisms that grew on the MRS broth were
transferred to the agar plates and colonies from the agar plates were transferred
on the broth.
B" the end of the subculture process! expected pure colonies of +actic
'cid Bacteria from MRS 'gar were identified using the BB+ 'naerobic
0dentification $r"stal. )ach colon" was placed on one s"stem and was read after
&4(;2 hours of incubation. %he reading process was made through the help of
software known as KBD ( Diagnostic S"stems ;5&2(52L. %he identified +actic 'cid
Bacteria were the ones used to test the antagonistic activit" against Escherichia
coli and Staphylococcus aureus.
%he antagonistic activit" was tested through agar well diffusion method
wherein the stock culture of the E.coli was streaked on Mac$onke" 'gar! and
S.aureus was streaked on 7utrient 'gar. %he identified +actic 'cid Bacteria
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diluted &:&5 up to &:&55 were aspirated on the agar plates where known cultures!
E.coli and S. aureus,were streaked. 'gar wells were made b" making bores on
the agar plates using a cork borer. %he aspirated +actic 'cid Bacteria where
transferred on the agar wells. %his method is known as the agar well diffusion
method. ?pon incubation of the agar wells for about ;2 hours! the ones of
inhibition from the wells were measured using a vernier caliper. %he diameter
determines the antagonistic activit" of isolated +actic 'cid Bacteria from chicken
intestines against enteric pathogens! E.coli and S. aureus.
#)#8) De+!&!t!$& $+ Ter%s
A&aer$b!c) 0t is an organism or tissue! living in the absence of air or free ox"gen.
A&tag$&!st!c) 0t is showing or feeling active opposition or hostilit" toward
someone or something.
A&t!b!$t!c) 0t is an agent that either kills or inhibits the growth of a
microorganism.
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A"t$chth$&$"s %!cr$$rga&!s%s) %he" are indigenous form of soil
microorganisms! responsible for chemical processes that occur in the soil under
normal conditions.
Catalase) 0t is an en"me found in most plant and animal cells that functions as
an oxidative catal"st# decomposes h"drogen peroxide into ox"gen and water.
C$%%!&gl!&g) 0t is the proximit" of animals to one another.
G"t %!cr$b!$ta) %he" are the microorganisms that t"picall" inhabit a bodil"
organ or part# flora.
H!ghl, t$lera&t) %he abilit" to endure the action of a drug! poison etc.
Lact$bac!ll"s) 0t is a rod shaped gram(positive bacterium. 0t can also live in
fermenting products such as "oghurt.
M!cr$aer$ph!l!c) %he characteristic that reFuires ox"gen for their metabolism!
but cannot survive at atmospheric concentration.
N$&9path$ge&!c) 0t is an organism that is not known to cause disease in plants
or one that is not pathogenic. ' pathogen on the other hand is a biological agent
that causes diseases or illness to its host.
N$&9sp$re +$r%!&g) =athogenic characteristic that have low pathogenicit" when
the" occur in the digestive tract of an insect! but ma" be ver" pathogenic.
Ph,l$ge&et!cs) 0t refers to the stud" of the relationships among various species
based upon their similarities and differences in their or genetic characteristics. 0t
is relating their evolutionar" development or histor".
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Pr$b!$t!cs) %he" are the bacteria that help maintain the natural balance of
organisms in the intestines
Pr$+!c!e&t) %he characteristic of being competent andGor skilled in doing
something.
Pr$ph,la:!s) 0t is the action taken to prevent disease! especiall" specified
means or against a specified disease.
CHAPTER *
Re0!e7 $+ Relate' L!terat"re
%he review of the related literature for this stud" focuses on the various
properties of lactic acid bacteria such as its use as a probiotics! antimicrobial
Technology PAGE 17
propert"! and antagonistic activit" against other pathogens. %his stud" also
focuses on the diseases that can cause illness to the chicken.
*)# L$cal L!terat"re
C$%%$& D!arrhea9ge&!c Path$ge&s
0n an article made b" Bunga" ;558! she stated that a developing countr"
such as the =hilippines! as"mptomatic and possibl" multiple infections ma"
occur! so much so that there is a need to conduct surveillance studies for the
presence of this pathogen. %here is probabl" a lack of awareness regarding the
significance of this pathogen because there are few studies conducted so far.
%he focus of most studies in the countr" is still on more common diarrhea(genic
organisms such as Escherichia coli! Vibrio cholerae! and Salmonella. Since the
organism is a microaerophilic thermophile! there is difficult" in isolation and
identification of the organism.
%here was one stud" conducted in ;555 at the ?niversit" of the
=hilippines 7ational 0nstitutes of -ealth .?=(70-/ on the prevalence of
Campylobacter spp. from =hilippine foods! which utilied conventional methods
of isolation and identification .Bunga" et al. unpublished/
*)* F$re!g& L!terat"re
Lact!c Ac!' Bacter!a
+actic acid bacteria .+'B/ are group of *ram(positive bacteria that are
devoid of c"tochromes and preferring anaerobic conditions! fastidious! acid(
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tolerant and strictl" fermentative. %he" are usuall" non(motile and non(
sporulating bacteria that produce lactic acid. %his bacterial group contains both
rods .Lactobacilli and Carnobacteria/ and cocci .Streptococci/. Different species
of lactic acid bacteria .such as Streptococcus, Leuconostoc, Pediococcus,
Aerococcus, Enterococcus, Vagococcus, Lactobacillus, Carnobacterium/ have
adapted to grow under widel" different environmental conditions. 'lthough lactic
acid bacteria are not dominant in the normal intestinal microbiota! several trials
have been undertaken to induce an artificial dominance of lactic acid bacteria
.Aerschuere et al.! ;555/. Based on their carboh"drate metabolism! +actic 'cid
Bacteria are divided into two distinct groups. %he homo(fermentative group
utilies the )mbden(Me"erhof(=arnas .gl"col"tic/ pathwa" to transform a carbon
source chiefl" into lactic acid. -etero(fermentative bacteria produce eFuimolar
amounts of lactate! $9;! ethanol or acetate from glucose exploiting =hospho(
@etolase pathwa". -omo(fermentative group consist of Lactococcus!
Pediococcus, Enterococcus, Streptococcus. -etero(fermentative group include
Leuconostoc, eisella .Aasil3evikM Shah! ;554/.
%he term +actic 'cid Bacteria .+'B/ was graduall" accepted in the
beginning of the ;5th centur" .$arol et. al.! ;5&5/. 9ther terms as Kmilk souringL
and Klactic acid producingL bacteria had previousl" been used for the same
bacteria causing a slight confusion. $lassification of +'B genera was based on
morpholog"! mode of glucose fermentation! growth at certain temperatures! and
range of sugar utiliation. )ven though the taxonom" has been revised since
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then! characters used b" 9rla(Bensen are still ver" important in current
classification of +'B. ,or identification of +'B! phenot"pic methods have been
most commonl" used .$orsetti et. al.! ;55&/. More recentl"! genetic techniFues!
such as &<S rD7' seFuencing have been developed which allows a more
consistent and accurate identification of individual strains .Buddhiman et al.!
;554/.
Ph,s!$l$g, a&' M$rph$l$g,
9rla(Bensen used a few characters as classification basis: morpholog"
.cocci or rods! tetrad formation/! mode of glucose fermentation .homo( or hetero(
fermentation/! growth at certain KcardinalL temperatures .e.g.! &5N$ and 28N$/!
and form of lactic acid produced .D! +! or both/ .@en3i et al.! ;55>/.
9rla(Bensen regarded lactic acid bacteria as a great natural group!
indicating a belief that the bacteria included were ph"logeneticall" related and
separated from other groups. 't that time! onl" phenot"pe characters could be
examined and evaluated ph"logenetic markers. %oda"! we have means to
examine! in detail! macro(molecules of the cell! believed to be more accurate in
defining relationships and ph"logenetic positions. %hese are! of course! the
nucleic acids. ,ortunatel"! nature has provided us with different kind of nucleic
acids for different kind of taxonomic studies.
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%he ph"siolog" of lactic acid bacteria has been of interest ever since it
was recognied that these bacteria involved in the acidification of food and feed
products. 0ncreased knowledge of lactic acid bacteria ph"siolog"! such as
metabolism and nutrient utiliation has been one wa" to achieve more controlled
processes. %oda"! modern genetic techniFues are considered to be promising in
this regard. -owever! effort in this direction will not be fruitful unless there is a
sound understanding of the ph"siolog" of these bacteria. %he designation +actic
'cid Bacteria perhaps implies that these bacteria have a somewhat KsimpleL
metabolism .Delphine et al.! ;5&&/! resulting in one or few fermentation end
products. %his ma" also be the case in laborator" environment that we often
impose to them. -owever! it is clear that lactic acid bacteria have a ver" diverse
metabolic capacit"! which enables them to adapt to a variet" of conditions.
Pr$b!$t!cs
=robiotics bacteria were first studied b" )lie Metchnikoff! a 7oble laureate
of &>54 in the field of medicine. -e theoried that proteol"tic microbes in the
colon produce toxic substances responsible for the aging process and proposed
that consumption of fermented milk would coat the colon with +'Bs! decreasing
intestinal p-! suppressing proteol"tic bacteria and thus leading to slowing of the
aging process .*ordon! ;554/. %o Fualif" as a probiotic! certain criteria need to
be met b" a bacterium: a bacterial strain must be identified completel"! be
harmless for ingestion! adhere to mucosal membrane! able to colonie the gut
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epithelium! stable when stored! must survive the acid and bile salt concentration
persisting in upper *0 tract .Aerna and +ucak! ;5&5/.
A&!%al G"t M!cr$b!$ta
%he animal gut is host to an abundant and diverse microbiota that pla"s
an important role in the health and nutrition of the animal. Most of these
organisms are considered commensal or s"mbiotic but the gastrointestinal
microbiota can also have detrimental effects on host health and nutrition. %he
relationship between the host animal and its gut microbiota can therefore be
viewed as a balance between mutualism and pathogenicit" .Dumonceaux et. al.
;55</.
%he vertebrate *0%! including that of humans! is home to a vast collection
of microbial! mostl" bacterial! species! which is referred to as the gut microbiota.
$omparisons of the characteristics of germ(free animals and those of
conventional animals have clearl" demonstrated that the gut microbiota has
considerable influence on host biochemistr"! ph"siolog"! immunolog"! and low(
level resistance to gut infections. Because of the variations in ph"sical and
chemical properties in the different compartments of the *0%! specific microbial
communities exist in the stomach! small intestine! and large intestine. 0n
monogastric animals! the largest numbers of bacteria reside in the distal gut
.colon/! reaching densities of around &5 microbes per gram of luminal contents.
%he carbon and energ" reFuirements of the enormous numbers of
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microbes residing in the colon are met b" two sources: b" complex
carboh"drates! proteins! and fats that have escaped digestion in the small bowel
and b" the components of host secretions .mucins/ and sloughed epithelial cells.
'lthough nutrient availabilit" is highest proximal to sites of absorption .e.g.! the
stomach and the first two(thirds of the small bowel/! these sites contain relativel"
small numbers of microbes in humans. Microbial numbers are restricted in these
areas because of the p- of the stomach contents .as low as p- ;/! the toxicit" of
bile salts! and the relativel" swift flow of the digesta. 0n contrast to humans!
however! some animal species have relativel" large numbers of bacteria .mainl"
Lactobacilli/ in the proximal gut .e.g.! the fore(stomachs of rodents! the crops of
chickens! and the parsoesophageas of pigs/. %he reason for this special foregut
association is likel" due to the adherence of Lactobacilli to the surface of the
nonsecretor" epithelium lining of these sites! which enables the bacteria to form
a biofilm(like structure that provides a bacterial inoculum of the digesta .Walter!
;554/.
0n health" chicken! the composition of intestinal microbiota remains stable.
-owever! this stabilit" ma" be disrupted b" various factors such as pathogen
invasion! antibiotic administration and environmental stress .e.g.! overcrowding!
poor feeding! extremel" high or low temperature! and transportation/. Doung
animals under stressful conditions suffer from changes in the composition and
activit" of the gut microbiota. =revious studies reported that disturbances of the
intestinal microbiota dela"s growth! weakens resistance! and leads to various
Technology PAGE 23
infectious diseases demonstrated that heat stress resulted in a marked change of
bacterial composition in chicken intestine! which was subseFuentl" associated
with depression of bod"(weight gain. While the cecal microbiota functions to
protect chickens against bacterial infection! the normal microbiota present in the
small intestine contributes significantl" to small intestinal function! including
digestion and nutrient absorption! which significantl" determine the growth rate of
the animal .Sakamoto! ;552/.
Salanitro et al. enumerated anaerobic bacteria from the ilea and ceca of
&2(da"(old chicks and showed that the predominant cultured flora of the ileum
included Lactobacillus .11.4 to 8>O/! Streptococcus .4.> to &<.4O/! E. coli .&2.6
to 11O/! and eubacteria .> to ;2.1O/! while eubacteria .<5.<O/ and !acteroides
.&;.4O/ dominated the flora of the cecum.
'ndrews .;5&1/ stated the high rates of intestinal bacteria found in
chicken intestines. Enterococcus .on 45 O of samples/! E. coli .<8 O/!
Campylobacter .21 O/! "lebsiella .&2 O/! Salmonella .&& O/ and Staphylococcus
aureus .> O/.What is important to note! however! is that onl" some of the
bacteria present at those levels would pose a food poisoning risk if ingested. ,or
example! Enterococcus does not cause food poisoning! and all of the food
poisoning maladies associated with E. coli came from a few select strains! while
most are harmless to humans P and some are even beneficial.
Technology PAGE 24
But while Enterococcus and most E. coli ma" not pose a food poisoning
threat! the" do indicate widespread fecal contamination of chicken meat. %he"
are also capable of causing infections of the skin! blood and urinar" tract if
presented an opportunit". %wo of the other bacteria P Campylobacter and
Salmonella P are more likel" to cause diarrhea and vomiting! the classic
s"mptoms of foodborne illness. 7earl" &6.8 O of the E. coli strains detected are
a t"pe known to more likel" cause urinar" tract infections! known as )x=)$! or
extra(pathogenic E. coli .Rangan! ;5&1/
H"%a& G"t M!cr$b!$ta
'ccording to the review article b" Scaldaferriet. al.! the human microflora!
known as KmicrobiotaL! includes bacteria! fungi! bacteriophages! and viruses and
acts as an organ s"nergisticall" with the host! creating an ecos"stem. 0t is able to
colonie skin! the genitourinar" s"stem! the respirator" s"stem! and! above all!
the gut. *ut microbiota includes around a thousand different species and more
than &8!555 different strains of bacteria! for a total weight of about & kg. Stomach
and small intestine are relativel" poor of bacteria! whilst the colon hosts about
&5&; microorganisms! mainl" belonging to the ,irmicutes and
Bacteroidetesph"la. 9ther domains represented are those of 'rchaea and
)ukar"a! plus man" viruses and bacteriophages. Several "east families! whose
role in gastrointestinal ph"siolog" as well as in diseases still remains unclear!
harbor gut microbiota.
Technology PAGE 25
Since most species seem to be refractor" to cultivation with usual
methods! culture(independent molecular techniFues! such as &<S rD7'
genot"ping! are used to characterie the gut microflora from both fecal samples
and bowel biopsies. 't birth! the human gut is sterile! and the first coloniation
occurs during childbirth and the first feed. SubseFuentl"! the microbiota changes
under the influence of age! sex! state of immune maturation! and environmental
factors. 'fter the first two "ears! the microbiota becomes more stable! although a
stable endogen flora could be differentiated b" a transient one! which is! on the
contrar"! more sensitive to external stimuli! as the gut mucosa is the first line of
communication with exogenous agents.
Bacter!al Path$ge&s
Bacterial pathogens can be classified according to their location relative to
cells in the host organism. )xtracellular bacteria usuall" colonie surfaces of
mucosal tissue or blood! while facultative intracellular bacteria have developed
the abilit" to survive and grow within professional phagoc"tes and other cell
t"pes. Some other bacteria that exhibit specific features# including massive
reduction in genome sie and a biased nucleotide base composition! have
become obligate(intracellular parasites .Moran! ;55;/. 9wing to the loss of
genes involved in essential metabolic pathwa"s! these microorganisms are
unable to multipl" outside of a host cell where the" find nutrients and metabolites
reFuired for their survival. Such associations can be beneficial to the host! as it is
Technology PAGE 26
the case for the insect s"mbionts !uchnera aphidicola and iggles#orthia
glossinidia! which provide the host with vitamins and cofactors .Dale M Moran!
;55</. 0nfectious diseases are a ma3or health threat because the" still represent
the main cause of mortalit" worldwide .@indhauser! ;551/.
F$$'b$r&e Path$ge&s +r$% A&!%al Or!g!&
Bacterial enteric pathogens are estimated to cause approximatel" 8 million
illnesses! 2<!555 hospitaliations! and &284 deaths in the ?nited States each
"ear. ,ood(producing animals .e.g.! cattle! chickens! pigs! and turke"s/ are the
ma3or reservoirs for man" of these organisms. ,ood producing animals acFuire
these pathogens b" ingestion. $ontamination of animal feed before arrival at and
while on the farm contributes to infection and coloniation of food( producing
animals with these pathogens. =athogens can then be transmitted through the
food chain to humans and cause human foodborne illness. $oncern about the
contribution of contaminated animal feed to human foodborne illness has been
heightened b" the recent emergence of variant $reutfeldt(Bakob disease in
humans in the ?nited @ingdom. Aariant $reutfeldt(Bakob disease is a prion
disease thought to be associated with the feeding of cattle with meat and bone
meal .$rump et. al. ;55;/
Sillankorva et.al stated that the four main foodborne pathogens from
animal origin are accounted to be E. coli! Campylobacter, Salmonella, and
Technology PAGE 27
Listeria. %hese bacteria are all common contaminants of ruminants! poultr"! and
swine and are usuall" carried in their gastrointestinal tract as"mptomaticall".
Escherichia coli
Escherichia coli is a gram(negative bacterium. Serot"pe 5&86 -! classified
as Shiga toxin(producing E. coli! is a well(known food poisoning pathogen. 0ts
ma3or reservoir comprises ruminants and! as it can survive well under intestinal
conditions! if proper care is not taken during slaughter! the contents of the
intestines! fecal material! or dust on the hide ma" contaminate meats. %he most
common route of E. coli transmission to humans is via undercooked
contaminated food! while water and raw milk are assumed to be related to cross(
contamination events! b" direct or indirect contact with feces. %his microorganism
is highl" virulent and a public health threat because ingestion of a concentration
as low as &5 cells is able to cause infection.
'vian pathogenic Escherichia coli .'=)$/! the causal organism of E. coli
infections of poultr"! are responsible for significant morbidit" and mortalit" in the
poultr" industr" worldwide. 0nfection with '=)$ generall" begins as a localied
infection of the air sacs commonl" referred to as air sacculitis or the air sac
disease! which in turn ma" spread! to other internal organs resulting in s"stemic
infection. %his initial infection generall" occurs in 2P>( week(old broiler chickens
and in la"ing hens at the peak of egg production that takes place around week
15. Birds simultaneousl" infected with various combinations of 0nfectious
Technology PAGE 28
Bronchitis virus .0BA/ and other viruses inevitabl" suffer from a damaged
respirator" tract! causing them to become increasingl" susceptible to invasion b"
'=)$ .Do"le! ;55</.
t!l!;at!$& $+ A&t!b!$t!cs !& P$"ltr, I&'"str,
'ntibiotics have been used in food animals .primaril" cattle! swine! and
poultr"/ for more than 85 "ears to treat! prevent! or control infectious disease! or
to improve efficienc" of feed utiliation and weight gain. 'dministration of these
veterinar" drugs to food animals is a critical component of an overall
management s"stem that food animal producers use to secure the health and
welfare of the animals and ensure the safet" of the products that enter the food
chain.
*iven the close proximit" of the animals to one another .commingling/!
ph"siological and environ mental stressors! and immature immune s"stems! an"
underl"ing viral infections! or bacterial respirator" or enteric diseases that ma"
occur in a few animals can spread to others! including en( tire herds or flocks.
Within the limits of the production s"stem! and depending on the nature of the
disease! the producer andGor veterinarian ma" intervene in such situations b"
medicating the entire group via the feed or water rather than treating each
affected animal. ,eed medication is more efficient for long(term proph"laxis!
whereas medication of water is more effective for treating disease outbreaks due
to its rapid intake and clinical response elicitation. Medicated water is also a
Technology PAGE 29
more effective means for treating sick animals! which often continue to drink
despite not continuing to eat. 'dministration of medication via water also allows
large numbers of animals to be treated in an efficient manner! and avoids worker
safet" issues associated with in3ecting large numbers of animals .Do"le et al.!
;55</.
Starter and grower rations ma" contain up to three drugsQa proph"lactic
coccidiostat! an antibiotic growth promoter! and an arsenical compound having
both anticoccidiostat and growth promoting properties. Several antibiotics!
administered as feed additives! are approved for treating intestinal infections!
such as necrotic enteritis .caused b" $lostridium perfringens/ and coccidiosis .a
common parasitic poultr" disease caused b" )imeria species/.
Res!sta&ce t$ A&t!b!$t!cs
%he term antibiotic is used in this report to refer to drugs used to treat
infectious disease in humans! animals! or plants! b" inhibiting the growth of or
destro"ing microorganisms# such substances ma" be naturall" occurring!
semis"nthetic! or s"nthetic. 'ntibiotics are also used in food animals to prevent
infectious disease and improve the efficienc" of feed utiliation. 'ntibiotics are
legall" classified as such onl" when used in humans. %he" are classified as
Kveterinar" antimicrobial drugsL when used in animals and as KpesticidesL when
used in plants.
Technology PAGE 30
'ccording to Do"le et al. the availabilit" of antibiotics to treat infectious
diseases has radicall" improved human and animal well being! and to a minor
degree! plant health. =aradoxicall"! this ver" success threatens the future utilit"
of antibiotics. %he discover" of penicillin in &>25 ushered in the era of Kmodern
medicine.L 7umerous antimicrobials! including most structural classes of
antibiotics were discovered during &>;5 to &>65. $hemical modification of man"
of these compounds led to new entities with superior activities. Because of the
great success in antibiotic discover"! b" the late &>65s! man" proclaimed that the
war on infectious diseases had been won! leading ultimatel" to de(emphasis of
antibiotic discover" during the &>45s and a decline in the &>>5s.
's pointed out b" $ourvalin .;558/! resistance to antimicrobial drugs is an
unavoidable aspect of the general evolution of bacteria that occurs b" chance.
Mechanisms for emergence of bacterial resistance are Fuite diverse as are the
modes of action of antimicrobials! which ma" include inhibition of various steps of
D7' replication! transcription! and translation! or action at the level of the cell
wall or cell membrane. ,urther! bacteria can resist the effects of antimicrobials b"
en"maticall" degrading the drug before it reaches its target site! altering the
protein.s/ within the bacterium that serve as receptors for the antimicrobials! and
changing their membrane permeabilit" to the antibiotics .$loete! ;551/
Class!+!cat!$& $+ Res!sta&ce
I&&ate $r I&tr!&s!c Res!sta&ce
Technology PAGE 31
0nnate resistance is related to the general ph"siolog" or anatom" of a
microorganism and stems from pre(existing mechanisms or properties. %his t"pe
of resistance is most likel" responsible for differences in resistance observed
among different t"pes! genera! species! and strains of microorganisms in
identical environmental conditions and concentrations. 0nnate resistance ma"
stem from the complexit" of the cell wall! efflux mechanisms or en"matic
inactivation of the antimicrobial .Russell! ;55&/.
%here are certain circumstances in which antimicrobial agents do not
adversel" affect bacteria that are generall" susceptible to the particular agent.
Because the efficac" of most food antimicrobials and sanitiers is dependent
upon and influenced b" the conditions of the application! some situations ma"
permit bacterial resistance that would not have occurred otherwise .0,%! ;55;/.
)xposure conditions! such as the environmental conditions .temperature! p-!
and food composition/ of the antimicrobial application! or interaction of the
antimicrobial with components of the suspension medium or food product can
influence the efficac" of the antimicrobial agent .Davidson! ;55&/. -owever!
microorganisms that are generall" susceptible to antibiotics can themselves also
become temporaril" resistant to an antimicrobial through activation of silent!
resident gene.s/ that confer this resistance.
Ac<"!re' $r E:tr!&s!c res!sta&ce
'cFuired resistance! the most common t"pe of antibiotic resistance! has
Technology PAGE 32
been well studied for antibiotics! but has not been well studied for food
antimicrobial agents and sanitiers. 'lthough acFuired resistance is of concern in
the use of food antimicrobial agents and sanitiers! occurrence of such
resistance appears to be rare.
L$cal St"'!es
Gastr$!&test!&al Gr$7th a&' Pr$b!$t!c M$'!+!cat!$&
'dministration of a probiotic culture ma" modif" the ecolog" of the
gastrointestinal tract during the first da"s of a chickHs life! a time that is
considered an open window for the establishment of pathogens! such as
Salmonella. 7eonates are born with an almost a sterile gastrointestinal tract! but
microorganisms present in the environment after birth rapidl" begin colonie .8/.
'lthough lactic acid bacteria are normal inhabitants of the gastrointestinal
tract! their presence follows a succession with Lactobacillus delbruec$ii as the
ma3or species at da" 1! Lactobacillus acidophilus and eissella spp. dominating
at da" 6! and Lactobacillus crispatus predominating from da"s &2P2>. +astl"! L.
sali%arius appeared at da" 2>. 0n addition! *uan et al observed that L.
acidophilus and L. sali%arius appeared in developmental succession! whereas
other species! such as Lactobacillus reuteri and Lactobacillus johnsonii were
consistentl" detected .Aicente et. al. ;556/
F$re!g& St"'!es
Pr$b!$t!c P$te&t!al $+ Lact!c Ac!' Bacter!a
Technology PAGE 33
'ccording to Madi! et al! it is well(known to have a number of beneficial
health effects in human and animals. %he" pla" an important role in the
protection of the host against harmful microorganisms and also strengthen the
immune s"stem. Some probiotics have also been found to improve feed
digestibilit" and reduce metabolic disorders. %he" must be safe! acid and bile
tolerant! able to adhere and colonie the intestinal tract. -uman enteroc"te(like
$aco(; cell have often been used for in vitro studies on the mechanisms of
cellular adhesion of nonpathogenic Lactobacilli.
.'nother and s"nergistic mechanism b" which probiotics exert their
beneficial effects is the enhancement of the intestinal barrier function. 0t has been
found that treatment with probiotic bacteria ma" prevent or reverse increased
permeabilit" of the epithelium induced b" pathogens. %hese results were
essentiall" obtained b" measurement of the transepithelial electrical resistance
.%))R/ which appears as a reliable method to evaluate in vitro the epithelial
permeabilit" and compare the probiotic activit" of bacterial species .Manai! et.al
;5&;/.
Based on $onnilHs et al. stud"! Lactobacillus sali%arius has gained
attention as a promising probiotic species that influences c"tokine profiles and
modulates cellular responses to pathogeninc challenge. 0n a recent stud"! the"
isolated L. sali%arius SMCD8& from the cecum of a %unisian poultr". %his strain
was found to produce a bacteriocin(like substance active against Campylobacter
jejuni! a bacterium known as a food borne pathogens in humans and a common
Technology PAGE 34
commensal of poultr". 0n addition to Campylobacter, salivaracin SMCD8& showed
inhibition against a number of food borne pathogens! such as Listeria
monocytogenes, Staphylococcus aureus and Salmonella.
0n parallel! pharmaceutical preparations of probiotics contain selected
living or heat(killed! l"ophilied! Lactobacilli and !ifidobacteria are used as
biotherapeutic agents. %hese pharmaceutical preparations have the advantage of
providing a stable and reproducible preparation of bacteria! which correspond to
pharmacologicall" established dose(efficac" of Lactobacilli and !ifidobacteria.
's pertinentl" underlined b" Boriello et al.! although lactobacilli are
extremel" rare causes of infection in humans! strains used for new probiotics
should be chosen from the commensall" flora of humans and should not carr"
intrinsic resistance to antibiotics that would prevent treatment of a rare probiotic
infection and these strains should be sent to reference centers for molecular
characteriation and confirmation of safet".
0n recent "ears! worldwide interest in the use of probiotic bacteria for
health promotion and disease prevention has increased significantl" in scientific
communit"! consumers and food producers. %his interest is based on the
knowledge that the targeted use of microorganisms with suitable properties ma"
have beneficial effect on animal and human health. Bacterial strains most
commonl" used as probiotics are Lactobacillus, Lactococcus, Enterococcus and
!ifidobacterium which belong to the lactic acid bacteria .Bu3nakova et al.! ;5&1/.
Technology PAGE 35
A&t!%!cr$b!al Pr$pert, $+ Lact!c Ac!' Bacter!a
9n the basis of evidence from the in vitro and in vivo studies discussed
above it would appear that the antimicrobial activit" of Lactobacilli and
!ifidobacteria is a strain(specific propert"! and cannot be extrapolated to other
strains. ,ollowing on from these in vitro and in vivo experimental studies! well(
designed! double(blind! placebo(controlled clinical trials have been conducted in
order to demonstrate that a selected Lactobacilli or !ifidobacteria strain does
actuall" displa" these properties in humans developing the diseases.
Reid et al. have demonstrated experimentall" that selected +actobacillus
strains of urovaginal origin have adhesive properties that enable them to inhibit
andGor prevent the coloniation of uroepithelial cells b" uropathogens. $omplete
or partial inhibition of the adherence of *ram(negative uropathogens has been
obtained b" pre(incubation of the uroepithelial cells with bacterial cell wall
fragments isolated from the Lactobacillus strains *R(&! *R(;! *R(1! and '(<5.
$ompetitive exclusion was most effective with whole viable cells! but
Lactobacillus cell(wall preparations suggested that lipoteichoic acid was
responsible for the adherence of the Lactobacillus cells to the uroepithelial cell!
but that steric hindrance was the main factor in preventing the adherence of
uropathogens.
Man" mechanisms have been postulated b" which Lactobacilli and
!ifidobacteria could produce antimicrobial activit". 0n addition to their competitive
inhibition of the epithelial and mucosal adherence of pathogens and inhibition of
Technology PAGE 36
epithelial invasion b" pathogens! Lactobacilli and !ifidobacteria also show
antimicrobial activit" b" producing antimicrobial substances andGor stimulating
mucosal immunit" .Servin! ;552/.
'ntimicrobial compounds obtained from lactic acid bacteria ma" help to
inhibit $. perfringens throughout the food chain! b" themselves or in combination
with other treatments. Reuterin is a broad spectrum antimicrobial compound
produced and secreted b" the food grade lactic acid bacterium Lactobacillus
reuteri. Reuterin is produced as a b"product of gl"cerol fermentation and consists
of h"drated! non(h"drated! and dimeric forms of 1(h"drox"propionaldeh"de
.*arde et al.! ;5&2/.
'ccording to the stud" of %orres et al.! the antimicrobial activit" of reuterin
against a variet" of both gram(negative and gram(positive pathogens has been
evaluated b" different research groups. -owever! the" noticed that there is no
information on the antimicrobial activit" of reuterin against C. perfringens. %he
bacteriocin nicin! a pol"peptide produced b" Lactococcus lactis! exerts its
antimicrobial properties against several gram(positive organisms. 7icin is
approved as a food preservative b" the )? .);12/! as well as b" the World
-ealth 9rganiation .W-9/ and the ?S ,ood and Drug 'dministration. -owever
few studies have been carried out to test the antimicrobial activit" of nicin against
C. perfringens.
0n addition to lactic acid bacteria antimicrobials! food andGor feed additives
ma" control C. perfringens growth. %hus! nitrite is used as an antimicrobial agent
Technology PAGE 37
primaril" to prevent the growth of Clostridium botulinum in meat products.
+"so"me is mainl" added to prevent cheese late blowing caused b" Clostridium
tyrobutyricum! but it has also been tested as a feed additive to control C.
perfringens(associated disease in poultr" .'llaart et al.! ;5&1/.
A&tag$&!st!c Act!0!t, $+ LAB aga!&st $ther path$ge&s
%he adhesion of probiotic strains to intestinal mucus is considered a
prereFuisite for successful coloniation and an important factor for antagonistic
activit" against enteropathogens. %he two selected +'B isolates were! therefore!
submitted to further assa"s investigating their adhesion capacities. %he target
cells used in this stud" were chicken enteroc"tes collected from duodenum!
3e3unum! and ileum. %he results showed that two isolates! namel" %7& and %74!
were highl" capable of adhering to these poultr" intestinal epithelial cells
compared to =. acidilactici M' &4G 8M used as a control. %he strain isolated from
the giard .%74/ was noted to displa" better adhesion to enteroc"tes than that
isolated from the ileum .%7&/. %he adhesion of several strains that were
eliminated from the screening .&5 strains with less acid production and less
resistance to bile and acid growth conditions/ was also investigated. %he results
show that these strains presented low Oages of adhesion! var"ing from ;6 to
14O. %he value of the in vitro screening process is! therefore! demonstrated
.Salah et al.! ;5&;/.
Technology PAGE 38
CHAPTER -
The Research Meth$'s
Technology PAGE 39
%his chapter presents the methods and procedures used! the instruments
in collecting data and statistical treatment applied.
-)# Meth$'s a&' Mater!als
%he experimental method of research was used in this stud".
)xperimental research was an attempt b" the researchers to maintain control
over all factors that ma" affect the result of an experiment.
-)#)# Sta&'ar's3 Reage&ts3 a&' Che%!cals
%he stud" used of the following materials: >5 m+ of phosphate buffered
saline .=BS/! $olumbia 'gar! MRS 'gar! MRS Broth! 'naerobic 3ars with
'naerobic packets! *ram stain! -"drogen peroxide! Mineral 9il! &5O skim milk
and 1.5 Mc,arland Standard with sterilied 5.48O 7a$l solution.
-)#)* Sa%pl!&g Pr$ce'"re
-)#)*A Is$lat!$& $+ Lact!c Ac!' Bacter!a a&' C"lt"re C$&'!t!$&s
%he stud" includes &5 grams of homogenied chicken intestine. %he
samples were collected from a stall in Bustillos Market in Sampaloc! Manila! kept
on ice! and immediatel" transferred to the laborator" for bacterial isolation. 'fter
dissection! surface mucus was removed from the tissue to obtain adhering
bacterial isolates. %en grams of epithelial tissue were scraped with a sterile
blade! re(suspended in >5 ml of phosphate buffered saline .=BS/ and vigorousl"
Technology PAGE 40
shaken using a vortex mixer for & min. Serial dilutions were spread on plates of
MRS 'gar and $olumbia 'gar and incubated at 15(16R$ for 24 hours. 'nother &5
grams of the sample were directl" streaked onto the MRS 'gar and $olumbia
'gar plates! under the same incubation time and period as the homogenied
samples.
%he grown isolates were transferred to MRS Broth! incubated
anaerobicall" for 24 hrs at 15 R$ using 'naerobic packets. 'fter incubation! the
isolates from the broth were streaked to another MRS 'gar and incubated again
for the same time and period. 'fter incubation for ; da"s at 15 R$ under
anaerobic conditions in anaerobic packets! onl" acid producing bacterial colonies
were selected. $olonies with different morpholog" were counted! selected and
purified b" transferring from agar to broth and re(streaking broth to agar. %his
process was repeated 1 times! except for the third subculture! where isolates
were streaked onl" on MRS 'gar and not on $olumbia 'gar.
$ell morpholog"! *ram staining and $atalase test were performed as a
preliminar" screening tests for lactic acid bacteria. *ram(positive! non(spore
forming and catalase(negative strains were selected for further studies.
Well ( isolated colonies with t"pical characteristics namel" pure white! small .;(
1 mm diameter/ with entire margins were picked from each plate and were grown
in MRS medium. %he selected lactic acid bacteria were maintained as stock
cultures at Refrigerator temperature in MRS broth.
Technology PAGE 41
0ndicator pathogens! Escherichia coli and Staphylococcus aureuswere
grown in %r"ptic So" Broth .%SB! 9xoid/ and 7utrient 'gar.-iMedia/ and
incubated at 15 R$.
-)#)*B Character!;at!$& Pr$ce'"res +$r Lact!c Ac!' Bacter!a
%he cultures were identified according to their morphological! cultural!
ph"siological and biochemical characteristics. %hree subcultures were made
ever" 24 hours. %he tests were: *ram reaction# production of catalase using
h"drogen peroxide and growth at 15
o
$ to 16
o
$ ever" 24 hours. 'long the
procedure! certain plated isolates were removed due to disFualifications to the
characteristics of a +actic 'cid Bacteria.
-)#)*C I'e&t!+!cat!$& $+ lact!c ac!' bacter!a "s!&g BBL Cr,stal
I'e&t!+!cat!$& S,ste%
$arboh"drate fermentation patterns were determined using BB+ $r"stal
0dentification S"stem .BD ( Diagnostic S"stems ;5&;(52/. %he species were
identified using the software version from BD ( Diagnostic S"stems. ,our other
pure cultures were also identified and confirmed using the same s"stem. %hese
organisms were Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus
paracasei, and Lactobacillus plantarum.
-)#)*D Scree&!&g $+ lact!c ac!' bacter!a +$r a&t!%!cr$b!al act!0!t,
Technology PAGE 42
'n overnight culture of each isolate grown in MRS broth at 15 R$ was
standardied to an optical densit" of 5.8 S 5.58 at a wavelength of <55 nm. 9ne
percent of standardied culture was used to inoculate MRS broth. 'fter
incubation at 16 R$ for ;2 hrs! cells were removed b" centrifugation at &5!555 x g
for &8 min. %wo sets of the standardied cultures were re3uvenated. 9ne is for
non(ad3usted p- cultures! and the other is for the p- ad3usted cultures. %he p-
of one set of cultures was ad3usted to 6.5 b" adding distilled water to acidif" or
=hosphate Buffered Saline to alkalie! if needed. %o evaluate antimicrobial
activit"! the agar well diffusion method was used.
-)#)*E Detect!$& $+ a&t!%!cr$b!al act!0!t, b, agar 7ell '!++"s!$&
%eth$'
'n agar well diffusion method as described b" Barefoot and @laenhammer
was used with some modifications. 'n overnight culture of pathogens including
E. coli, and S. aureus grown in %SB medium at 15 R$ was diluted to a turbidit"
eFuivalent to that of a 1.5 Mc,arland standard .bioMerieux! ,rance/ with a
sterilied 5.48O 7a$l solution. ' lawn of an indicator strain was made b"
spreading the cell suspension over the surface of %S' plates with a cotton swab.
%he plates were allowed to dr" and a sterile cork borer of diameter 6.5 mm was
used to cut uniform wells in the agar plates. )ach well was filled with
approximatel" 65 Tl of filterPsterilied supernatant obtained from culture grown in
MRS medium. 'll the assa"s were carried out in triplicate. 'fter incubation at
Technology PAGE 43
16R$ for ;2 hrs! the diameter .mm/ of the inhibition one around the well was
measured. =ositive results were recorded when the one of inhibition of at least &
mm around the wells was observed.
-)#)*F) C$&+!r%at$r, pr$ce'"res ab$"t the !&h!b!t!$& act!0!t, $+ Lact!c
Ac!' Bacter!a "s!&g $ther %eth$'s
0n cases of negative results in the agar well diffusion method! other
methods were performed. %hese methods are Durham %ube Method! C(D
inhibition Method! and Well Diffusion Method.
0n the Durham Method! the procedure is the same with the agar well
diffusion method! but instead of using a cork borer! Durham %ube with a bore sie
of 8 mm! bigger than the cork borer was used. Same as the procedure in the
agar well diffusion method! approximatel" 45 Ul! instead of 65Ul of isolates were
placed on the wells. 'fter incubation for &4(;2 hrs! the ones of inhibition around
the wells were measured.
0n the C(D inhibition Method! the pathogens! E.coli and S.aureus were
streaked on a 7utrient 'gar separatel" on a D(axis! while the isolates and pure
cultures were streaked on an C(axis intersecting with the streaked pathogens.
%he agar plates were then incubated overnight at 15
o
$ and then examined for
the ones of inhibition on the point of intersection.
Technology PAGE 44
0n the Well(Diffusion Method! pre(poured MRS agar plates were overlaid
with 6 ml MRS soft agar containing 5.; ml of indicator cultures. Wells sied 8 mm
in diameter were cut into off the agar plate using a sterile Durham tube and &55
Ul of the culture supernatant fluid was placed into each well. 'fter overnight
incubation at 15
o
$! the plates were examined for ones of inhibition around the
wells containing the isolates and pure cultures. .Aol.88. ,asc. 2 Mourad et al.!
;552/
,or Well(diffusion and Durham method! an inhibition of greater than & mm
denotes a positive inhibition! while an inhibition of less than & mm showed a
negative inhibition.
-)* Stat!st!cs
%he samples! directl" taken from the epithelium of chicken intestine and
from with =BS! were plated on both $olumbia 'gar and MRS 'gar. )ach colon"
on ever" plate were tested for gram stain and catalase test.
%he statistical method used was through the use of a freFuenc"
distribution table to determine the O rate of colonies taken from the MRS 'gar
and $olumbia 'gar.
Technology PAGE 45
CHAPTER .
A&al,s!s3 Prese&tat!$&3 a&' I&terpretat!$& $+ Res"lts
%his chapter includes the anal"sis! presentation and interpretation of the
data gathered throughout the stud".
Based on the data gathered! the following tables are presented.
Table #
Character!st!c $+ Bacter!a +r$% all plate' c$l$&!es
M$rph$l$g, Fre<"e&c, =age
$occi onl" ; &2.;>O
Bacilli 1 ;&.21O
$occi and Bacilli > <2.;>O
%able & shows that &2.;>O of all kinds of colonies are cocci! ;&.21O are
bacilli! and <2.;>O is a mixture of both cocci and bacilli.
Table *
Character!;at!$& a&' I'e&t!+!cat!$& $+ C$'e' Orga&!s%s
C$'e Gra% Sta!& M$rph$l$g, Catalase Test
MD55& =ositive Mixed 7egative
MD55; =ositive
$occi and Bacilli in
chains and clusters
7egative
MD551 =ositive
$occi in chains and
clusters
7egative
Technology PAGE 46
$D552 =ositive Bacilli 7egative
$D558 =ositive Bacilli 7egative
$D55< =ositive Bacilli! round! clear 7egative
$D556 =ositive
Bacilli! round!
cream"
7egative
M=55& =ositive Mixed! small 7egative
M=55; =ositive
$occi in chains and
clusters
7egative
M=551 =ositive
Bacilli in chains
and clusters
7egative
$=552 =ositive Bacilli 7egative
$=55< =ositive Bacilli 7egative
%able ; shows the *ram stain! morpholog"! and catalase test results of
organisms coded as MD55&! MD55;! MD551! $D552! $D558! $D55<! $D556!
M=55&! M=55;! M=551! $=552! $=558! $=55<! and $=556. 'll of the
organisms were *ram positive and $atalase test negative. Some of the
organisms were identified as cocci! bacilli and some are mixed.
Table -
F!rst S"b9c"lt"re $& MRS Agar
Technology PAGE 47
C$'e Gra% Sta!& M$rph$l$g,
MRS =& =ositive $occi
MRS =; =ositive $occo(bacilli
MRS =1 =ositive Bacilli
MRS =8 =ositive Mixed
MRS =< =ositive Mixed
MRS $1 =ositive $occi
MRS $6 =ositive Bacilli
MRS $< =ositive Bacilli
MRS $&5 =ositive Bacilli
MRS $> =ositive $occi
%able 1 shows the *ram stain result and morpholog" of organisms
isolated from MRS agar coded as MRS =&! MRS =;! MRS =1! MRS =8! MRS
$1! MRS $6! MRS $<! MRS $&5! '7D MRS $>. 'll were gram positive and
there were cocci! bacilli! coco(bacilli! and mixed colonies isolated.
Table .
F!rst S"bc"lt"re $& C$l"%b!a Agar
Technology PAGE 48
C$'e Gra% Sta!& M$rph$l$g,
$$< =ositive Bacilli
$$6 =ositive Bacilli
$$4 =ositive Bacilli
$$&5 =ositive Bacilli
$=8 =ositive Bacilli
$=; =ositive Bacilli
7o tag =ositive Bacilli
$=1 =ositive Mixed
%able 2 shows the *ram stain result and morpholog" of
organisms isolated from $olumbia agar coded as $$<! $$6! $$4! $$&5! $=8!
$=;! $=1! and one without a tag. 'll were gram positive and were bacilli. 9nl"
$=1 has a mix of cocci and bacilli.
Table 1
Sec$&' S"b9c"lt"re $& MRS Agar a&' C$l"%b!a Agar Plates
Technology PAGE 49
MRS Agar
Gra% react!$& M$rph$l$g, Arra&ge%e&t Catalase Test
MD55& =ositive
$occi
$hains and
clusters
7egative
MD55; =ositive
$occi =airs 7egative
MD551 =ositive
$D552 =ositive
$occi
$hains and
clusters
7egative
$D558 =ositive
$occi
$hains and
clusters
7egative
$D55< =ositive
Mixed $lusters 7egative
$D556 =ositive
Bacilli $lusters =ositive
M=55& =ositive
M=55; =ositive
M=551 =ositive
$=552 =ositive
Bacilli
$hains and
pairs
=ositive
$=558 =ositive
$occi
$hains and
pairs
7egative
$=55< =ositive
Short bacilli $hains =ositive
$=556 =ositive
$occi
$lusters and
pairs
7egative
C$l"%b!a Agar
Gra%
react!$&
M$rph$l$g, Arra&ge%e&t Catalase Test
MD55& =ositive
Mixed
$hains and
clusters
7egative
Technology PAGE 50
MD55; =ositive
Mixed
$hains and
clusters
=ositive
MD551 =ositive
$occi
$hains and
clusters
=ositive
$D552 =ositive
Mixed $lusters =ositive
$D558 =ositive
Bacilli $hains 7egative
$D55< =ositive
Bacilli
$hains and
clusters
=ositive
$D556 =ositive
Bacilli $lusters =ositive
M=55& =ositive
$occi
=airs and
$lusters
7egative
M=55; =ositive
$occi
=airs and
$lusters
7egative
M=551 =ositive
$occi
$hains and
pairs
7egative
$=552 7egative
$=558 =ositive
$=55< =ositive
Mixed
$hains and
=airs
=ositive
$=556 =ositive
Mixed $hains =ositive
%able 8 shows the morphological characteristics and arrangement of
coded organisms using MRS and $olumbia 'gar =lates. Some organisms! without
identification were removed due to invalidation and disFualification with the
characteristics of a +actic 'cid Bacteria.
Table 2
I'e&t!+!cat!$& $+ Lact!c Ac!' Bacter!a "s!&g BBL Cr,stal Gra% P$s!t!0e ID
S,ste%
CODE BBL CR>STAL Gra% P$s!t!0e
Technology PAGE 51
ID S,ste%
CAP * Lactobacillus johnsonii
CAP - Lactobacillus fermentum
MRS C4 Lactobacillus fermentum
MRS P* Lactobacillus fermentum
MRS P. Lactobacillus fermentum
MRS P1 Lactobacillus fermentum
MRS C#8 Lactobacillus fermentum
CAC5 Lactobacillus fermentum
%able < shows the different lactic acid bacteria isolated and identified using
BB+ $r"stal *ram =ositive 0D S"stem. %he identified organisms were Lactobacillus
johnsonii and Lactobacillus fermentum.Most of the identified species
wereLactobacillus fermentum. %he BB+ $r"stal *ram =ositive 0D S"stem were not
able to identif" them as a famil" of +actobacillus spp.
Table 4
Opt!cal De&s!t, at 288&%
Sta&'ar'!;e' C$l$&, Pre0!$"s OD A'/"ste' OD
$'= & 5.42> 5.26&
'= ; 5.6>< 5.8;<
Technology PAGE 52
$'= 1 5.2>8 7ot ad3usted
$'= 2 5.4&< 5.2>&
$'= 8 5.<&< 5.2>8
$'$ &5 5.2>; 7ot ad3usted
$'$ 6 5.212 5.2>6
$'$ 4 5.2>4 7ot ad3usted
MRS $6 5.8&4 7ot ad3usted
MRS =& 5.856 7ot ad3usted
MRS =; 5.2&1 5.2>5
MRS =2 5.2>> 7ot ad3usted
MRS =8 5.82; 5.856
%able 6 shows the optical densit" of the standardied organisms. 0ts
9ptical densit" was ad3usted within the range of 5.28( 5.885. 0n order to rise its
optical densit"! a portion of the organism was added! and a portion of MRS broth
was added to decrease its optical densit" to fit through the range. ,or organisms
whose optical densit" is within the range! it is noted as it is.
Table 5
Ser!al D!l"t!$&
D!l"t!$& C4 CAP*
&:&5 ( (
&:;5 5 &&&
&:15 25 84
&:25 &85 0nvalid
&:85 &46 ;2&
&:<5 ;15 >1
&:65 1 >;
&:45 & 64
Technology PAGE 53
&:>5 1< 66
&:&55 &56 &;
%able 4 shows the colon" count from organisms $6 and $'=; which were
diluted from &:&5 to &:&55. ,or organism $6! it reached the highest colon" count
of ;15 in &:<5 dilution. While organism $'=; reached its highest colon" count of
;2& in &:85 dilution. ,or the &:&5 dilution! both samples were disregarded due to
turbidit".
Table 6
E0al"at!$& $+ the a&tag$&!st!c act!0!t, "s!&g ?9> I&h!b!t!$& Meth$'
SPECIES
TRIAL # TRIAL * TRIAL -
S.aureus
E.coli
S.aureus
E.coli
S.aureus
E.coli
Lactobacillus
fermentum
=ositive
7egative
7egative
7egative
7egative
=ositive
Lactobacillus
johnsonii
=ositive
7egative
7egative
7egative
=ositive
=ositive
Lactobacillus
acidophilus
7egative
=ositive
=ositive
=ositive
7egative
7egative
Lactobacillus
paracasei
=ositive
7egative
7egative
=ositive
=ositive
=ositive
Technology PAGE 54
Lactobacillus
casei
7egative
=ositive
=ositive
=ositive
7egative
7egative
Lactobacillus
plantarum
7egative
7egative
7egative
=ositive
=ositive
=ositive
%able > shows three trials of the antagonistic activit" of the isolates and
pure cultures using the C(D inhibition method. 0t showed that on ever" trial! some
organisms were positive onl" on one pathogen! some were positive on both! and
some were negative on both.
Table #8
Meas"re%e&t $+ the @$&es $+ I&h!b!t!$& s!&g Well9D!++"s!$& Meth$'
SPECIES
Meas"re%e&t
S.aureus
E.coli
Lactobacillus fermentum (
(
Lactobacillus johnsonii (
(
Lactobacillus acidophilus (
(
Lactobacillus paracasei &.1; mm
(
Lactobacillus casei (
&.14 mm
Lactobacillus plantarum (
&.18 mm
Technology PAGE 55
%able &5 shows the ones of inhibition of ever" isolate and pure culture
using the Well(Diffusion Method. Most of the organisms refuse to show an"
inhibition on both E.coli and S.aureus. L.paracasei was the onl" organism that
inhibits S.aureus, while L.casei and L. Plantarum inhibited E.coli.
Table ##
A&tag$&!st!c Act!0!t, $+ Lact!c Ac!' Bacter!a "s!&g Agar Well D!++"s!$&
Meth$' A!& %%B
Spec!es Tr!al # Tr!al * Tr!al -
S.
aureus
E. coli S.
aureus
E. coli S.
aureus
E. coli
L. johnsonii 8.6 5 8.6 5 <.< 5
L. fermentum 6.8 5 6.; 8.5 4.1 8.;
L. casei &;.; >.5 &&.4 &5.5 &5.; >.5
L. paracasei &;.1 6.4 &&.> 6.8 >.& 6.<
L. acidophilus &5.& 5 4.8 <.1 6.< 6.6
L. plantarum &;.; 4.; &5.> 6.6 4.6 4.<
%able && shows the antagonistic activit" of lactic acid bacteria using agar
well diffusion method. 'll the species were able to inhibit the growth of
Staphylococcus aureus on the first trial. Both of the isolated organisms were not
able to inhibit the growth of Escherichia coli but the pure stock cultures were able
to inhibit the growth of E. coli on the first trial. 9n the second trial! all the species
were also able to inhibit the growth of Staphylococcus aureus. 'll except
Technology PAGE 56
+actobacillus 3ohnsonii were able to inhibit the growth of Escherichia coli on the
second trial. ,inall"! on the third trial! all the species were able to inhibit the
growth of Staphylococcus aureus. 'll except Lactobacillus johnsonii were able to
inhibit the growth of Escherichia coli on the third trial.
Table #*
I&terpretat!$& $+ Res"lts
Source& "'()L Sci. )ech. *. Vol. + ,o.- *an.. *un. /00+
Schllinger, 1. And Luc$e, 2.". -3+3 4Antibacterial Acti%ity of
Lactobacillus sa$e (solated 2rom meat, Applied and
En%ironmental 'icrobiology5, 66 7+8, -30-.-309
0nhibition .mm/ 0nterpretation
V& =ositive
W& 7egative
Technology PAGE 57
%able &; shows that positive results were recorded when the one of
inhibition of at least & mm around the wells was observed.
CHAPTER 1
S"%%ar,3 C$&cl"s!$&s3 a&' Rec$%%e&'at!$&s
%his chapter includes the summar"! conclusions and recommendations
made b" the researchers on the stud".
S"%%ar, $+ F!&'!&gsC
Based on the results of the data gathered! the following findings are
presented:
Technology PAGE 58
&. 0solates
%he stud" showed that Lactobacillus fermentumand Lactobacillus
johnsoniiwere the +actic 'cid Bacteria that were isolated from the
chicken intestines after a three times of subculture. ,or ever" colon"
that grew on MRS 'gar! was transferred to MRS broth for further
inoculation and subculture. While! inoculums on MRS broth were
transferred to MRS 'gar. 'fter getting the organismsH optical densit"!
their p- were ad3usted up to 6.5 S 5.58 before placing it to the agar
wells. 'fter incubation for ;2 hours! no inhibitions were seen! so a
Durham tube was instead used to bore in holes into the 7utrient 'gar
plates. 'fter incubation for ;2 hours at 15( 16
o
$! inhibitions were seen!
measured and then evaluated.
;. Methods
%his stud" showed a lot methods performed in order to reach the
most accurate and most precise result. %he methods used were the
'gar Well Diffusion Method .Durham %ube/! Well Diffusion Method!
and the C(D inhibition method. %hese methods showed that most
organisms inhibit S.aureusthrough the agar(well diffusion method using
Durham tube! but through the use of the Well(diffusion method! most
organisms inhibit E.coli.9n the first agar well diffusion method using a
cork borer! no inhibition was seen! but on the second agar well
Technology PAGE 59
diffusion method using the Durham tube! which has bigger bore sie
than the cork borer! inhibitions were seen.
C$&cl"s!$&s
Based on the results! the lactic acid bacteria isolated from chicken
intestine! Lactobacillus fermentumand Lactobacillus johnsinii,are effective in
inhibiting common chicken intestinal flora such Escherichia coli and
Staphylococcus aureus. %he ones of inhibition on ever" method was measured
and evaluated in order to which +actic 'cid Bacteria inhibited these pathogens.
Rec$%%e&'at!$&s
Based on the findings of the stud"! the researchers recommend the
following for the improvement and development of the stud":
-. %he use of bacteria such as Salmonella typhi! :elicobacter pylori! Shigella
dysenteriae! Clostridium perfringens and Vibrio cholera for antagonistic
activit" testing instead of Escherichia coli and Staphylococcus aureus.
/. ?se of =$R for the ph"logen" of Lactobacillus fermentum and
Lactobacillus johnsonii with Molecular testing.,urther researches and
actual testing on test animals! to determine its inhibitor" effect.
Technology PAGE 60
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and pork.L Li%estoc$ Production Science C9! p. ;&8(;;;
Eapata! 'na et al. .;5&1/ K'ntimicrobial 'ctivities of +actic 'cid Bacteria Strains
0solated from 7ile %ilapia 0ntestine .?reochromis niloticus8L *ournal of
Technology PAGE 64
!iology and Life Science! vol. 2! no.&! p. &<2(&6&
ARTICLES
Bunga"! '.'! De los Re"es! $.S. and )stacio! M.B. K%he Eoonotic =otential of
$amp"lobacteriosis and its 0mplications to -uman -ealthL ?niversit" of the
=hilippines! 7ational 0nstitutes of -ealth! ;558
Delphine! + .;5&&/ K?sing heme as an energ" boost for lactic acid bacteriaL
Current ?pinion in !iotechnology //7/8,p. &21(&2>Y
Do"le! M.=. et al. .;55</ K'ntimicrobial Resistance: 0mplications for the ,ood
S"stemL, Comprehensi%e ;e%ie#s in 2ood Science and 2ood Safety! vol. <!
pages 6&(45
Aerna! ).$ and +ucak! S .;5&5/ K?se of probiotics in gastrointestinal disorders:
what to recommendIL )herapeutic Ad%ances in Gastroenterolgy! p. 156 (
1&>
APPENDIX A
Certifcate of BBL
Technology PAGE 65
APPENDIX B
Media Preparation
Technology PAGE 66
Anaerobic Gas Pack MRS Broth

MRS Agar Plates
APPENDIX C
Homogenization and Standardization
Technology PAGE 67
Chicken Intestines in Vortex Mixer
Technology PAGE 68
APPENDIX D
Colonial Characterization and Morphological Identifcation
Catalase Test
Technology PAGE 69
Gram Staining
APPENDIX E
Preparation of Media Used
1. Columbia Agar Base
Formula:
Ingredients g/liter
Casein pancreatic digest 10.0
Meat peptic digest 5.0
Yeast extract 5.0
Sodium 5.0
Heart pancreatic digest 3.0
Com starch 1.0
Bacteriological Agar 13.5
Technology PAGE 70
Direction:
Suspend 42.5 g of the medium in one liter distilled water. Mix well. Heat with
frequent agitation and boil for one minute. Distribute into appropriate containers
and autoclave at 121C for 15 minutes.pH fnal: 7.3 0.2 a 25C
2. MRS Agar
Formula:
Ingredients g/liter
Dextrose 20.0
Bacteriological Peptone 10.0
Beef Extract 8.0
Sodium Acetate 5.0
Yeast Extract 4.0
Dipotassium Phosphate 2.0
Technology PAGE 71
Ammonium Citrate 2.0
Tween 80 1.0
Magnesium Sulfate 0.2
Manganase Sulfate 0.05
Bacteriological Agar 10.0
Direction:
Suspend 62 grams of the medium in one liter of distilled water. Mix well and
dissolve by heating with frequent agitation. Boil for one minute until complete
dissolution. Sterilize in autoclave at 121C for 12 minutes. Cool to 45-50C, mix
well and dispense into plates.
3. MRS Broth
Formula:
Ingredients g/liter
Dextrose 20.0
Technology PAGE 72
Bacteriological Peptone 10.0
Beef Extract 8.0
Sodium Acetate 5.0
Yeast Extract 4.0
Dipotassium Phosphate 2.0
Ammonium Citrate 2.0
Tween 80 1.0
Magnesium Sulfate 0.2
Mangenese Sulfate 0.05
Direction:
Suspend 52.25 grams of the medium in one liter of distilled water. Mix well
and dissolve by heating with frequent agitation. Boil for one minute until complete
Technology PAGE 73
dissolution. Dispense into appropriate containers and sterilize in autoclave at
121C for 12 minutes.
4. Nutrient Agar
Formula:
Ingredients g/liter
Peptic digest of animal tissue 5.00
Sodium chloride 5.00
Beef extract 1.50
Yeast extract 1.50
Agar 15.00
Direction:
Suspend 28.0 grams in 100 ml distilled water. Heat to boiling to dissolved
the medium completely. Sterilized by autoclaving at 15 lbs pressure (121 C) for
15 minutes. If desired, the medium can be enriched with 5-10% blood or other
biological fuids. Mix well and pour into sterile Petri plates.
Technology PAGE 74
APPENDIX F
Computation of Inhibition Index
Formula: measurement of inhibition measurement of Durham tube (5mm)
Against Staphylococcus aureus
Control Organisms
L. casei T1: 17.2 mm 5 mm = 12.2mm
L. casei T2: 16.8 mm 5mm = 11.8 mm
L. casei T3: 15.2 mm 5mm = 10.2 mm
L. plantarum T1: 17.2 mm 5mm = 12.2 mm
L. plantarum T2: 15.9 mm 5mm = 10.9mm
L. acidophilus T1: 15.1 mm 5mm = 10.1 mm
L. acidophilus T2: 13.5mm 5mm= 8.5 mm
L. paracasei T1: 17.3 mm 5mm = 12.3 mm
Technology PAGE 75
L. paracasei T3: 14.1 mm 5mm = 9.1mm
Isolated Organisms
L. johnsonii T1: 10.7 mm 5mm = 5.7mm
L. johnsonii T2: 10.7 mm 5 mm = 5.7mm
L. johnsonii T3: 11.6 mm 5mm = 6.6 mm
L. fermentum T1: 12.5 mm 5mm = 7.5 mm
L. fermentum T2: 12.2 mm 5 mm= 7.2 mm
L. fermentumT3: 13.3 mm 5mm = 8.3 mm
Against Escherichia coli
Control Organisms
L. casei T1: 14.0mm 5mm = 9.0 mm
L. casei T2: 15.0 mm 5mm = 10mm
L. casei T3: 14.0 mmm 5mm = 9mm
Technology PAGE 76
L. plantarum T2: 12.7 mm 5 mm = 7.7 mm
L. acidophilus T1: 0.00
L. acidophilus T3: 12.7 mm 5mm = 7.7mm
L. paracasei T1: 12.8 mm 5mm = 7.8mm
Isolated Organisms
L. johnsonii T1: 0.00
L. fermentum T1: 0.00
L. fermentum T2: 10.0mm 5mm = 5mm
L. fermentumT3: 10.2mm 5mm = 5.2mm
Technology PAGE 77
APPENDIX G
BBL Crystal
Color Chart
Technology PAGE 78
APPENDIX H
Technology PAGE 79
BBL Crystal Kits
APPENDIX I
Technology PAGE 80
UV Analyzing Lamp
BBL Crystal Kit in UV Light
Technology PAGE 81
APPENDIX J
API Kit
Technology PAGE 82
APPENDIX K
Spectrophotometer used in Optical Density
Technology PAGE 83
APPENDIX L
Staphylococcus aureus non adjusted pH (left) and adjusted pH (right)
in Control Organism
Staphylococcus aureus non adjusted pH (left) and adjusted pH (right)
in Isolated Organism
Technology PAGE 84
Escherichia coli non adjusted pH (left) and adjusted pH (right)
in Control Organism
Escherichia coli non adjusted pH (left) and adjusted pH (right)
in Control Organism
Staphylococcus aureus punctured using Durham Tube.
Control Organism (left) and Isolated Organism (right)
Technology PAGE 85
Escherichia coli punctured using Durham Tube.
Control Organism (left) and Isolated Organism (right)
Cross Section Method of Staphylococcus aureus against Control
Organism
Technology PAGE 86
Cross Section Method of Staphylococcus aureus against Isolated
Organism
APPPENDI? M
E?PENSES
Et, &!t Pr!ce A%$"&t
Reage&ts
MRS'gar 855g & =hp1!4;4.55 =hp1!4;4.55

MRS Broth
;85g
& =hp5.55 =hp5.55

$olumbia 'gar
855g
& =hp;!66;.55 =hp;!66;.55

7utrient Broth
85g
& =hp255.55 =hp255.55
=BS ; =hp;&8.55 =hp215.55
Mater!als
'=0 & =hp6;5.55 =hp6;5.55
'mber Bottle & =hp;5.55 =hp;5.55
'pplicator sticks &8 =hp8.55 =hp68.55
BB+ & =hp&2!;55.55 =hp&2!;55.55
$otton 4 =hp6.55 =hp8<.55
Disposable
=lates
;; =hp&85.55 =hp1!155.55
Technology PAGE 87
Dropper ; =hp&8.55 =hp15.55
,oil 8 =hp25.55 =hp;55.55
*aue 2 =hp25.55 =hp&<5.55
*lass slides 1 =hp65.55 =hp;&5.55
0noculating +oop &8 =hp;8.55 =hp168.55

0noculating
7eedle
&8 =hp;8.55 =hp168.55
+"sol 2 =hp<5.55 =hp;25.55
Manila =aper &5 =hp6.55 =hp65.55
Masking %ape ; =hp&8.55 =hp15.55
Match ; =hp8.55 =hp&5.55
=arafilm ;8 =hp;8.55 =hp<;8.55
Red %op & =hp1<5.55 =hp1<5.55
Scalpel Blade &; =hp<.55 =hp6;.55

Screw $ap %est
%ube
1 =hp&55.55 =hp155.55
Sponge ; =hp&5.55 =hp;5.55
Darn 1 =hp&8.55 =hp28.55
Eonrox ; =hp&5.55 =hp;5.55
Pr!&t!&g =hp455.55
Tra0el =hp1!;55.55
E"$tat!$& =hp;!222.55
F$$' +$r Pa&el
'oc$ Aefense =hp2&&.55
2inal Aefense =hp&!&55.55

TOTAL =hp1<!4>4.55
TOTAL
CONTRIBUTION
=hp28!555.55
TOTAL
E!ENSES
=hp1<!4>4.55
BALANCE =hp4!&5;.55
Technology PAGE 88
APPENDI? N
Technology PAGE 89
TIMETABLE
Apr!l *- S"b%!ss!$& $+ Chapters #9-
Apr!l *5
Prel!%!&ar,
(Media =reparation
(Scraping from chicken intestines
(Streaking in B'=
(0ncubation at 16R$n for ;2(24 hrs.
Apr!l -8
Character!;at!$& $+ Lact!c Ac!'
Bacter!a
(*ram Stain
($atalase %est
($ell Morpholog"
Is$lat!$& !& MRS br$th
Ma, 1 API
Ma, 2 Chec(!&g $+ res"lts !& API
Ma, 4
Scree&!&g +$r Lact!c Ac!' Bacter!a
(?sing spectrophotometer with 9D of
<55 nm! wavelength of 5.8.
Ma, 5
Test!&g +$r A&tag$&!st!c act!0!t, $+
Lact!c Ac!' Bacter!a
(?sing agar well diffusion method.
Ma, 6
Chec(!&g $+ res"lts !& agar 7ell
'!++"s!$& %eth$'
(Measuring of the one of inhibition
Ma, #*
N$t!&g $+ res"lts3 s"b%!ss!$& $+
chapters .91
Ma, #.
Re0!s!$&s AI+ a&,B3 preparat!$& +$r
'e+e&se
Ma, #6 Thes!s De+e&se
Ma, *# Re0!s!$&s AI+ a&,B
Ma, *5 Pass!&g $+ re0!se' Chapters #91
Technology PAGE 90
CRRICLM FITAE
PERSONAL BACKGROND
Na%eC 'pril S. $apalungan
N!c(&a%eC 'prilG=ril
AgeC &4 "ears old
Date $+ B!rthC 'pril &4! &>>8
Per%a&e&t A''ressC 25 =a"umo St.! San Ramon! Dinalupihan! Bataan
Te%p$rar, A''ressC ?nit 2;2 Dona $armen 'pts. ;! ;
nd
St. San Miguel!
Mendiola! Manila
FAMIL> BACKGROND
FatherC Dominador $. $apalungan
Occ"pat!$&C Seaman
M$therC Marilou S. $apalungan
Occ"pat!$&C 9ptometrist
EDCATIONAL BACKGROND
Ele%e&tar,C Dinalupihan )lementar" School
;555(;55<
H!ghsch$$lC Regional Science -igh School ( 000
;556(;5&&
C$llegeC $entro )scolar ?niversit"
;5&&( =resent
Technology PAGE 91
CRRICLM FITAE
PERSONAL BACKGROND
Na%eC @r"sstina $ru
N!c(&a%eC %inG %ina
AgeC &> "ears old
Date $+ B!rthC 9ctober ;8! &>>2
Per%a&e&t A''ressC &28< Metrica St. Sampaloc! Manila
Te%p$rar, A''ressC &441($ Dapitan St. $or. ). Xuintos St. Sampaloc! Manila
FAMIL> BACKGROND
FatherC 7G'
M$therC ,lorentina =. $ru
Occ"pat!$&C 9,W
Ele%e&tar,C St. %herese +earning $enter
;555(;552
Dominican School Manila
;552(;556
H!ghsch$$lC Dominican School Manila
;556(;5&&
;5&&( =resent
Technology PAGE 92
CRRICLM FITAE
PERSONAL BACKGROND
Na%eC Marix Bohn A. )vangelista
N!c(&a%eC Marix
AgeC &4 "ears old
Date $+ B!rthC September 1! &>>8
Per%a&e&t A''ressC 26 =. $orpu St.! +una! San 'ntonio! Eambales
Te%p$rar, A''ressC >64 *M %olentino St. Sampaloc! Manila
FAMIL> BACKGROND
FatherC Mar3ohn 'llan =. )vangelista
Occ"pat!$&C 9,W
M$therC Riella A. )vangelista
Occ"pat!$&C -ousewife
Ele%e&tar,C San 'ntonio $entral )lementar" School
;55&(;556
H!ghsch$$lC Regional Science -igh School 000
;556(;5&&
;5&&( =resent
Technology PAGE 93
CRRICLM FITAE
PERSONAL BACKGROND
Na%eC @laria A. *alicia
N!c(&a%eC @la
AgeC &> "ears old
Date $+ B!rthC 7ovember &4! &>>2
Per%a&e&t A''ressC +ot 1< Sampaguita Drive! 'nnal"n Subdivision!
'langilanBatangas $it"
Te%p$rar, A''ressC 265(B 'legria St. Sampaloc! Manila
FAMIL> BACKGROND
FatherC -ercules A. *alicia
Occ"pat!$&C 'A=( San Miguel $orporation
M$therC Maribel A. *alicia
Ele%e&tar,C ?niversit" of Batangas
&>>4(;556
H!gh Sch$$lC Saint Bridget $ollege
;556(;5&&
;5&&( =resent
Technology PAGE 94
CRRICLM FITAE
PERSONAL BACKGROND
Na%eC @imberl" '. *uerrero
N!c(&a%eC @im
AgeC &4 "ears old
Date $+ B!rthC Ma" ;>! &>>8
Per%a&e&t A''ressC Z;5 de Bulio Street! =oblacion 14 0nfanta! Xueon
Te%p$rar, A''ressC &;8; $astaJos Street! Sampaloc! Manila
FAMIL> BACKGROND
FatherC Bhon M. *uerrero
Occ"pat!$&C 0nstaller
M$therC 7ilda '. *uerrero
Occ"pat!$&C Businesswoman
Ele%e&tar,C Brg". %ignoan )lementar" School
;55&(;556
H!ghsch$$lC 0nfanta 7ational -igh School
;556(;5&&
;5&&( =resent
Technology PAGE 95
CRRICLM FITAE
PERSONAL BACKGROND
Na%eC 'pple $. +egaspi
N!c(&a%eC 'pple
AgeC &>
Date $+ B!rthC Oct$ber *53 #66.
C!t, A''ressC 1 Sampaguita St.! +umang Ba"an Subd.! Ma"amot! 'ntipolo $it"
FAMIL> BACKGROND
FatherC %eodorico D+. +egaspi
Occ"pat!$&C Businessman
M$therC Ma. Melissa $. +egaspi
Occ"pat!$&C -ouse wife
EDCATIONAL BACKGRONDC
Ele%e&tar,C %he =leasant Mount School
;55& ( ;556
H!gh Sch$$lC %he =leasant School
;556(;5&&
C$llegeC $entro )scolar ?niversit"( Manila
;5&&( =resent
Technology PAGE 96
CRRICLM FITAE
PERSONAL BACKGROND
Na%eC Ban =auline 7. Maralit
N!c(&a%eC =auline
AgeC &4 "ears old
Date $+ b!rthC 'ugust &<! &>>8
C!t, A''ressC +a Residencia 00! +a $onsolacion $ollege Manila
Pr$0!&c!al A''ressC <2 *en. +una Street Sabang+ipa $it"
FAMIL> BACKGROND
FatherC Banuario M. Maralit
Occ"pat!$&C Mechanical )ngineer
M$therC )lena 7. Maralit
Occ"pat!$&C -ouse wife
Ele%e&tar,C $anossa 'cadem" +ipa $it"
SD: ;555 ( ;55<
H!gh Sch$$lC De +a Salle +ipa
SD: ;55< P ;5&5
SD ;5&&( =resent
Technology PAGE 97
CRRICLM FITAE
PERSONAL BACKGROND
Na%eC Baia @leo =ascasio
N!c(&a%eC Baia
AgeC&> "ears old
Date $+ B!rthC ,ebruar" &! &>>8
Pr$0!&c!al A''ressC >; Rodolfo St. Santiago! San 'ntonio! Eambales
C!t, A''ressC ;>>5 Ramon Magsa"sa" Blvd. Sta. Mesa! Manila
FAMIL> BACKGROND
FatherC +odivino =. =ascasio
Occ"pat!$&C %eacher
M$therC Dosephine $. =ascasio
Occ"pat!$&C%eacher
Ele%e&tar,C San 'ntonio $entral )lementar" School
;55&(;556
H!ghsch$$lC Regional Science -igh School(000
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Technology PAGE 98
CRRICLM FITAE
PERSONAL BACKGROND
Na%eC $harles Danielle R. =ere
N!c(&a%eC $harles
AgeC &> "ears old
Date $+ B!rthC 7ovember ;8! &>>2
C!t, A''ressC ;<! 9val St.! =aliparan! Sto. 7iJo! Marikina $it"
FAMIL> BACKGROND
FatherC )fren S. =ere
Occ"pat!$&C Businessman
M$therC Maria Salvacion Rose Aivian R. =ere
Occ"pat!$&C Businesswoman
Ele%e&tar,C Marist School! Marikina
;555(;55<
H!ghsch$$lC Marist School! Marikina
;556(;5&&
;5&&( =resent
Technology PAGE 99
CRRICLM FITAE
PERSONAL BACKGROND
Na%eC $arrel Bo" %an"uan
N!c(&a%eC $arrel
AgeC &4 "ears old
Date $+ B!rthC Bul" 4! &>>8
Pr$0!&c!al A''ressC Mamburao! 9ccidental Mindoro
C!t, A''ressC &554 ,a3ardo )xt. Sampaloc! Manila
FAMIL> BACKGROND
FatherC Bethoven %an"uan
Occ"pat!$&C ,armer
M$therC Merial"n %an"uan
Ele%e&tar,C %alabaan )lementar" School( =roper
;55& ( ;556
H!gh Sch$$lC 9ccidental Mindoro 7ational -igh School
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