Thesis on Probiotic, Part 1, Abadin

8/17/2019 Thesis on Probiotic, Part 1, Abadin

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ISOLATION, CHARACTERIZATION,

DETERMINATION OF PROBIOTIC PROPERTIES

OF LAB FROM BUFFALO MILK

A Thesis Submitted to

the De!"tme#t o$ Food E#%i#ee"i#% & Te'h#o(o%) o$

St!te U#i*e"sit) o$ B!#%(!deshi# P!"ti!( Fu($i((me#t o$ the Re+ui"eme#ts $o" the De%"ee o$

MS' Food E#%i#ee"i#% & Te'h#o(o%)

Submitted B)

Md Nu"u( Ab!di# Cho-dhu")

Stude#t ID. P/012132452116

D!te. 7!#u!") 3, 0146

Det o$ Food E#%i#ee"i#% & Te'h#o(o%)

St!te U#i*e"sit) o$ B!#%(!desh


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8ISOLATION, CHARACTERIZATION,

DETERMINATION OF PROBIOTIC PROPERTIES

OF LAB FROM BUFFALO MILK9

A Thesis Submitted to the De!"tme#t o$ Food E#%i#ee"i#% & Te'h#o(o%) o$ St!te

U#i*e"sit) o$ B!#%(!desh i# P!"ti!( Fu($i((me#t o$ the Re+ui"eme#ts $o" the De%"ee

o$ MS' Food E#%i#ee"i#% & Te'h#o(o%),

Submitted B)

Md Nu"u( Ab!di# Cho-dhu")Stude#t ID. P/012132452116

Sue"*iso"

::::::::::::

P"o$esso" A#is A(!m Siddi+uiHe!d



St!te U#i*e"sit) o$ B!#%(!deshD!te. De'embe", 0146


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CERTIFICATION

This is certify that this thesis entitled “Isolation, Characterization,

Determination of Probiotic Properties of Lab from Buffalo Milk submitted

by Md! "urul #badin Cho$dhury, student ID% P&'()(*)+)((-, Department

of .ood /n0ineerin0 1 Technolo0y, 2tate 3ni4ersity of Ban0ladesh, has been

carried out under my super4isor! This is further to certify that this thesis $ork

is carried out as partial re5uirement for fulfillment of the M!2c de0ree in .ood

/n0ineerin0 1 Technolo0y!

Sue"*iso"

::::::::::::

Professor #nis #lam 2iddi5ui

6ead

Dept! of .ood /n0ineerin0 1 Technolo0y

2tate 3ni4ersity of Ban0ladesh


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Dedicated to My Parents

&

All Friends


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ACKNO;LED/MENTS

.irstly, I $ould like to thank to my super4isor Prof! #nis #lam 2iddi5ui for all his kind

support, patience and encoura0ement and also thanks to his bein0 an ideal model for

his students! I $ant to thank for his kind support, help for completin0 this thesis $ork!

I $ould also like to thank to Mr! Talha and Cho$dhury Dairy .arm for h i s help to 0et

b u f fa l o milk samples! Thanks for ori0inal and pure buffalo milk!

I $ant to e7press my thanks to my friends8 Md! Mo9ibul 6o5ue, Md! /asdani, Md!

6abibur :ahman due to sharin0 all kinds of e7perience $ith me! #lso I $ould like to thank

Md! ;amruzzaman for her special help and encoura0ement!

.inally I am 0rateful to my family members for their endless support, encoura0ements and

lo4e!

#uthor


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ABSTRACT

In this study, isolates $ere identified by biochemical and molecular

characterization and also probiotic properties of lactic acid bacteria, isolated from

buffalo milk $ere in4esti0ated! Three of the isolates $ere obser4ed as potential

probiotic ! T$o of them are bacilli and the other is cocci! These isolates sho$ed

resistance to stomach p6 , tolerance a0ainst (!=? bile concentration and

antimicrobial acti4ity a0ainst Salmonella thyphimurium CCM --, Escherichia coli

@+-A%6A "CTC +'((( and Escherichia coli "::L B)=((! #fter in4esti0ation the

probiotic properties of these isolates, they $ere identified by standard cultural,

morpholo0ical and biochemical characterization techni5ues! T$o lactobacilli $ere

identified as Lactobacillus oris and Lactobacillus fermentum! In the li0ht of this study, it

is obser4ed that, buffalo milk is a source of potential probiotic strains!


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*

TABLE OF CONTENTS

LI2T @. .I&3:/2 !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 4iii

LI2T @. T#BL/2 !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! i7

C6#PT/: +! I"T:@D3CTI@"!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +

+!+! The 6istory and the Definition of Probiotics!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +

+!'! The /ffects of Probiotics on 6ealth!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

+!'!+! Lactose Intolerance !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

+!'!'! Immune 2ystem and Probiotics!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! -

+!'!=! Diarrhea !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! *

+!'!! Cancer!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! A

+!'!-! Cholesterol :eduction !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

+!=! Mechanism of Probiotics !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

+!! 2election Criteria for Probiotics !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +(

+!!+! #cid and Bile Tolerance!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ++

+!!'! #ntimicrobial #cti4ity !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +=

+!!=! 2afety #spects of Probiotics !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +-

+!-! Molecular Identification of Probiotic 2trains !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +*

+!*! Buffalo Milk # 2ource of Potential Probiotic 2train !!!!!!!!!!!!!!!!!!!!!! +

C6#PT/: '! M#T/:I#L2 #"D M/T6@D2 !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ''

'!+! Isolation of Lactic #cid Bacteria from Buffalo Milk !!!!!!!!!!!!!!!!!!!!!!!!!! ''

'!+!+! &ram 2tainin0 !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ''

'!+!'! Catalase Test !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '=

'!+!=! Lon0 Term Preser4ation of Isolates !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '=

'!'! Probiotic Properties of Isolates !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '=

'!'!+! :esistance to Lo$ p6 !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '

'!'!'! Tolerance a0ainst Bile !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '

'!'!=! #ntimicrobial #cti4ity !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '

'!=! Physiolo0ical and Biochemical Characterization !!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '-

'!=!+! &as Production from &lucose !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '-

'!=!'! &ro$th at Different Temperatures !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '-


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4ii

'!=!=! &ro$th at Different "acl Concentrations !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '-

'!=!! #r0inine 6ydrolysis Test !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '*

'!=!-! Carbohydrate .ermentations !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '*

C6#PT/: =! :/23LT2 #"D DI2C322I@" !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ===!+! Isolation of lactic acid bacteria !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ==

=!'! Probiotic Properties !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ==

=!'!+! :esistance to Lo$ p6 !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! ==

=!'!'! Tolerance a0ainst Bile !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! =-

=!'!=! #ntimicrobial #cti4ity !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! =*

=!=! Physiolo0ical and Biochemical Characterization !!!!!!!!!!!!!!!!!!!!!!!!!!!!!! =A

=!!! 2e5uencin0 of Isolates!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! A

C6#PT/: ! C@"CL32I@" #"D .3T3:/ P/:2P/CTIE/ !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

:/./:/"C/2 !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!


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#PP/"DIC/2

#PP/"DIF #! Chemicals for Microbiolo0ical /7periments and Molecular

Characterization !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! -

#PP/"DIF B! :ecipes for Culture Media and Biochemical Tests !!!!!!!!!!!!!!!!!!!!!!!!!!!!! -*#PP/"DIF C! Carbohydrates 3sed for Carbohydrate .ermentation Tests!!!!!!!!!!!!!!!!! *'

#PP/"DIF D! Buffers and 2tock 2olutions for Molecular Characterizaton !!!!!!!!!!!!!! *=

#PP/"DIF &! 2e5uences of Isolates !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! *


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+(

LIST OF FI/URES

Fi%u"e P!%e

.i0ure =!+! 2ur4i4al in p6 =!( @D*'( 4alues!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! =

.i0ure =!'! 2ur4i4al in p6 =!( cfu 4alues !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! =

.i0ure =!=! Tolerance a0ainst (!=? bile @D*'( 4alues!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! =-

.i0ure =!! Tolerance a0ainst (!=? bile cfu 4alues !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! =*

LIST OF TABLES

T!b(e P!%e

Table +!+! Microor0anisms applied in probiotic products !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! =

Table +!'! 2election criteria for probiotics!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +(Table+!=! Contents of Buffalo milk!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +

Table '!+! Indicator microor0anisms !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! '-

Table =!+! Diameter of inhibition zones!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! =*

Table =!'! Biochemical Test :esults of Isolates !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! =

Table =!=! Literature Information of Biochemical Test :esults !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! +


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LIST OF ABBRE


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+

CHAPTER 4

INTRODUCTION

44 The Histo") !#d the De$i#itio# o$ P"obioti's

The $ord Jprobiotic comes from &reek lan0ua0e Jpro bios $hich means Jfor

life opposed to Jantibiotics $hich means Ja0ainst life! The history of probiotics be0an

$ith the history of man by consumin0 fermented foods that is $ell kno$n &reek and

:omans consume 4ery much


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') J# microbial dietary ad9u4ant that beneficially affects the host physiolo0y by

modulatin0 mucosal and systemic immunity, as $ell as impro4in0 nutritional and

microbial balance in the intestinal tract by "aidu et al!, +!

=) J# li4e microbial food in0redient that is beneficial to health by 2alminen et

al! +!

) J# preparation of or a product containin0 4iable, defined microor0anisms in

sufficient numbers, $hich alter the microflora in a

compartment of the host and by that e7ert beneficial health effects in this host by

2chrezenmeir and de Erese, '((+!

-) JLi4e microor0anisms $hich $hen administered in ade5uate amounts confer a

health benefit on the host is accepted by .#@GH6@ !

Probiotics are also challin0in0 fo r the industrial applications ! The probiotic

concept is open to lots of different applications in a lar0e 4ariety of fields rele4ant for

human and animal health! Probiotic products consist of different enzymes, 4itamins,

capsules or tablets and some fermented foods contain microor0anisms $hich ha4e

beneficial effects on the health of host! They can contain one or se4eral species of

probiotic bacteria! Most of products $hich destine human consumption are produced in

fermented milk or 0i4en in po$ders or tablets! These capsules and tablets do not used

for medicial applications! They are 9ust used as health supportin0 products! The oral

consumption of probiotic microor0anisms produces a protecti4e effect on the 0ut flora!

Lots of studies su00est that probiotics ha4e beneficial effects on microbial disorders of

the 0ut, but it is really difficult to sho$ the clinical effects of such products! The

probiotic preperations use for tra4ellers diarrhoea, antibiotic associated diarrhoea and

acute diarrhoea $hich is sho$ned that they ha4e positi4e therapeutic effect ! To ha4e a healthy intestine the

balance of the bacteria must be maintained but this is difficult as the lifestyles chan0e!

Lots of factors may chan0e the balance a$ay from potentially beneficial or health


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promotin0 bacteria like lactobacilli and bifidobacteria to potentially harmfull or

patho0enic microor0anisms like clostridia, sulphate reducers and Bacteroides species. It

makes the host more susceptible to the illnesses! In this case the pre4alence of the

beneficial bacteria must be supported! 3sin0 of probiotics help to protect the host from

4arious intestinal diseases and disorders $hile increasin0 the number of beneficial

bacteria and make the balance steady a0ain ! Probiotics ar e

su00ested as food to pro4ide for the balance of intestinal flora !

Probiotics are used for lon0 times in food in0redients for human and also to feed

the animals $ithout any side effects! #lso probiotics are acceptable because of bein0

naturaly in intestinal tract of healthy human and in foods ! The probiotics $hich are use to feed both man and animals are sho$n in the

Table +!+!

Table +!+! Microor0anisms applied in probiotic products

Lactobacillus se'ies Bifidobacterium

se'ies

Othe"s

L. acidophilus

L. rhamnosus

L. gasseri

L. casei

L. reuteri

L. delbruecii subsp. bulgaricus

L. crispatus

L. plantarum

L. sali!arus

L. "ohnsonii

L. gallinarum

L.plantarum

L. fermentum

L. hel!eticus

B. bifidum

B. animalis

B. bre!e

B. infantis

B. longum

B. lactis

B. adolascentis

Enterococcus faecalis

Enterococcus faecium

Streptococcus sali!arus subsp!

thermophilus

Lactococcus lactis subsp! lactis

Lactococcus lactis subsp! cremoris

Propionibacterium freudenreichii

Pediococcus acidilactici

Saccharomyces boulardii

Leuoconostoc mesenteroides


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40 The E$$e'ts o$ P"obioti's o# He!(th

There are lots of studies on searchin0 the health benefits of fermented foods and

probiotics! 6o$e4er, in most of these studies researchers did not use sufficient test

sub9ects or they use microor0anisms $ere not identified definitely !

) Mana0in0 lactose intolerance!

) Impro4in0 immune system!

) Pre4ention of colon cancer!

) :eduction of cholesterol and triacyl0lycerol plasma concentrations !

) Lo$erin0 blood pressure!

) :educin0 inflammation!

) :eduction of aller0ic symptoms!

) Beneficial effe cts on mineral metabolism, par tic ula rl y bo ne density and

stability!

) :eduction of #elicobacter pylori infection!

) 2uppression of patho0enic microor0anisms !

) Pre4ention of osteoporosis!

) Pre4ention of uro0enital infections!

404 L!'tose I#to(e"!#'e

Most of human commonly non)Caucasians bec ome lactose intolerant aft er

$eanin0! These lactose intolerant people cannot metabolize lactose due to the lack of

essential enzyme )0alactosidase! Hhen they consume milk or lactose)containin0

products, symptoms includin0 abdominal pain, bloatin0, flatulence, crampin0 and

diarrhoea ensue! If lactose passes throu0h from the small intestine, it is con4erted to 0as

and acid in the lar0e intestine by the colonic microflora! #lso the presence of breath

hydro0en is a si0nal for lactose maldi0estion! The studies pro4ide that the addition of


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certain starter cultures to milk products, allo$s the lactose intolerant people to consume

those products $ithout the usual rise of breath hydro0en or associated symptoms

!

The beneficial effects of probiotics on lactose intolerance are e7plained by t$o

$ays! @ne of them is lo$er lactose concentration in the fermented foods due to the hi0h

lactase acti4ity of bacterial preparations used in the production! The other one is8

increased lactase acti4e lactase enzyme enters the small intestine $ith the fermented

product or $ith the 4iable probiotic bacteria !

400 Immu#e S)stem !#d P"obioti's

The effects of immune system are promisin0! 6o$e4er, the mechanism is not

$ell understood! 6uman studies ha4e sho$n that probiotic bacteria can ha4e positi4e

effects on the immune system of their hosts


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concentrations


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researchers like &uandalini et al. !

@ne of types of dierrhea is tra4ellers diarrhea $hich affects the healthy

tra4ellers not only in de4elopin0 countries but also in /urope! Probiotics ha4e beneficial

effects in pre4entin0 some forms of TD! @ksanen et al! e4aluated the efficacy of

Lactobacillus && in pre4entin0 diarrhea in '( people tra4ellin0 from .inland to

Turkey! In a double)blind study by Black et al. lyophylised bacteria

$ere 0i4en to -* Danish

tourists on a ')$eek trip to /0ypt! The occurence of diarrhea in the 0roup recei4in0 the

lactic acid bacteria $as =? $hile it $as A+? in the placebo 0roup !

#ntibiotic therapy causes mild and se4ere outbreaks of diarrhea! The normal

microflora may be suppresed durin0 the microbial therapy and resultin0 $ith fillin0

$ith patho0enic strains! The chan0es of microflora may also ancoura0e the resistant

strains at least %lostridium difficile $hich is the reasan of antibiotic associated diarrhea

! 2e4eral clinical trails ha4e

used Saccharomyces boulardii$ Lactobacillus spp! and Bifidobacterium spp! in #DD!

Probiotics $hich are able to restore and replace the normal flora should be used! #lso

they shoukd be used in hi0h risk patients such as old, hospitalised or immuno)

compromised! 2tudies $ith Saccharomyces boulardii pro4ed that %lostridium difficile

concentraiton is decreased in the presence of Saccharomyces boulardii !

405 C!#'e"

/pidemiolo0ical studies point out that if the consumption of saturated fats

increases in the diet, the occurrence of colon cancer increases in Hestern Horld!

Bacterial enzymes < )0lucornidase, nitroreductase and azoreductase> con4ert

precarcino0ens to acti4e carcino0ens in the colon! It is thou0ht that probiotics could

reduce the risk of cancer by decreasin0 the bacterial enzymes acti4ity! #lthou0h the

e7act mechanism for the anti tumor action is not kno$n, some su00estions ha4e been

proposed by McIntosh as follo$s %


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+! Carcino0enGprocarcino0en are suppressed by bindin0, blockin0 or remo4al!

'! 2uppressin0 the 0ro$th of bacteria $ith enzyme acti4ities that may con4ert

the procarcino0ens to carcino0ens!

=! Chan0in0 the intestinal p6 thus alterin0 microflora acti4ity and bile solubility!

! #lterin0 colonic transit time to remo4e fecal muta0ens more efficiently!

-! 2timulatin0 the immune system!

There are in 4itro and in 4i4o e4idences not only from animal studies but also

from human studies that probiotics ha4e beneficial effects on suppression of cancer!

@ral administration of lactic acid bacteria has been sho$n to reduce D"# dama0e

caused by chemical carcino0ens, in 0astric and colonic mucosa in rats! The

consumption of lactobacilli by healthy 4olunteers has been demonstrated to reduce the

muta0enicity of urine and feaces associated $ith the in0estion of carcino0ens in cooked

meat! Hhen it comes to epidemiolo0ical studies, they sho$ an association bet$een

fermented dairy products and colorectal cancer! The consumption of a lar0e 5uantity of

dairy products especially fermented foods like yo0urt and fermented milk $ith

containin0 Lactobacillus or Bifidobacterium may be related to a lo$er occurrence of

colon cancer ! # number of studies ha4e

sho$n that predisposin0 factors are altered positi4ely by consumption of certain probiotics

!

# study on the reduction of cholesterol $as sho$ed that Lactobacillus reuteri

C:L +( decreased total cholesterol by =? $hen it is 0i4en to mice for A days in the


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rate of +(

cellsGday! This dose of Lactobacillus reuteri caused a (? reduction in

tri0lycerides and a '(? increase in the ratio of hi0h density lipoprotein to lo$ density

lipoprotein $ithout bacterial translocation of the nati4e microflora into the spleen and

li4er !

4= Me'h!#ism o$ P"obioti's

Probiotic microor0anisms are considered to support the host health! 6o$e4er,

the support mechanisms ha4e not been e7plained ! There are

studies on ho$ probiotics $ork! 2o, many mechanisms from these studies are tryin0 to

e7plain ho$ probiotics could protect the host from the intestinal disorders! These

mechanisms listed belo$ briefly !

+! Production of inhibitory substances% Production of some or0anic acids ,

hydro0en pero7ide and bacteriocins $hich are inhibitory to both 0ram)positi4e and

0ram)ne0ati4e bacteria!

'! Blockin0 of adhesion sites% Probiotics and patho0enic bacteria are in a

competition! Probiotics inhibit the patho0ens by adherin0 to the intestinal epithelial

surfaces by blockin0 the adhesion sites!

=! Competition for nutrients% Despite of the lack of studies in 4i4o, probiotics

inhibit the patho0ens by consumin0 the nutrients $hich patho0ens need!

! 2timulatin0 of immunity% 2timulatin0 of specific and nonspecific immunity

may be one possible mechanism of probiotics to protect the host from intestinal disease!

This mechanism is not $ell documented, but it is thou0ht that specific cell $all

components or cell layers may act as ad9u4ants and increase humoral immune response!

-! De0radation of to7in receptor% Because of the de0redation of to7in receptor

on the intestinal mucosa, it $as sho$n that S. boulardii protects the host a0ainst %.

difficile intestinal disease!

2ome other offered mechanisms are suppression of to7in production, reduction

of 0ut p6, attenuation of 4irulence !


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The selection criteria can be cate0orized in four basic 0roups! #ppropriatness,

technolo0ical suitibility, competiti4eness, performance and functionality ! 2trains $hich ha4e these criteria should be used in order to 0et

effecti4e on health and functional probiotic strains! Probiotics are chosed by usin0 the

criteria in Table +!'! 2aarela et al!


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found resistant to acid at p6 =!- for ( min! at =A RC! #lso these strains $ere capable of

0ro$th in medium at p6 =!- containin0 (!'? mi7ed bile salts !

#n in4esti0ation of probiotic potential of A selected strains of Lactobacillus

spp! $ere e7amined for resistance to p6 '!- and (!=? o70all! They sho$ed hi0h

resistance to bile salts and 0ro$th $as delayed from +h to more than h for +* of these

strains e7amined and e7cept one all of these strains sur4i4ed for in such conditions

mentioned abo4e! The results obtained in 4itro e7periments8 fi4e strains


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in +h in p6 +! #lso all of them $ere tolerated (!=? bile salts concentration in h! .or

in 4i4o testin0 the most suitable strains $ere chosen, L.casei 2hirota #C#)DC *((',

L.plantarum #C#)DC +*, L.paracasei subsp! tolerans #C#)DC (=A

!

450 A#timi'"obi!( A'ti*it)

#ntimicrobial acti4ity is one of the most important selection criteria for

probiotics! #ntimicrobial acti4ity tar0ets the enteric undesirables and patho0ens

! #ntimicrobial effects of lactic acid bacteria are formed by

producin0 some substances such as or0anic acids ,

carbon dio7ide, hydro0en pero7ide, diacetyl, lo$ molecular $ei0ht antimicrobial

substances and bacteriocins ! Till today

there are some researchs on sho$in0 that different species produce different

antimicrobial substances! 6ere are some e7amples of these substances% Lactobacillus

reuterii, $hich is a member of normal microflora of human and many other animals,

produce a lo$ molecular $ei0ht antimicrobial substance reuterin8 subspecies of

Lactococcus lactis produce a class I bacteriocin, nisin #8 Enterococcus feacalis D2+*

produces a class I bacteriocin cytolysin8 Lactobacillus plantarum produces a class II

bacteriocin plantaricin 28 Lactobacillus acidophilus produces a class III bacteriocin

acidophilucin # !

Lactobacilli and Bifidobacteria isolated from human ileum $ere assayed if they

ha4e antimicrobial acti4ity a0ainst a ran0e of indicator microor0anisms, Listeria$

Bacillus$ Enterococcus$ Staphylococcus$ %lostridium$ Pseudomonas$ E. coli$


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Lactobacillus$ Streptococcus$ Bifidobacterium and Lactococcus. #ntimicrobial acti4ity

of Lactobacillus sali!arus 3CC++ $as counted a0ainst to these bacteria listed abo4e!

The study sho$ed that Lactobacillus sali!arus 3CC++ is si0nificantly capable of

inhibitin0 in 4itro 0ro$th of both some 0ram positi4e and some 0ram ne0ati4e bacteria

such as, L. fermentum ;LD $ B. longum$ B. bifidum$ Bacillus subtilus$ B. cereus$

B.thuringiensis$ E. faecalis$ E. faecium etc! althou0h it is not effecti4e a0ainst some of

Lactobacillus$ Lactococcus$ Leuconostoc$ Streptococcus etc! species !

2ome milk products $ere used to isolate potential probiotic bacteria and

determination of their possible antimicrobial acti4ities! Staphylococcus aureus$

Escherichia coli$ Pseudomonas aeruginosa$ Salmonella typhi$ Serratia marcescens and

%andida albicans $ere used as indicator microor0anisms! #fter the study, the results

sho$ed that, Uakult and 2ki D Lite probiotics inhibited all of the test indicator

microor0anisms, "estle yo0urt probiotics $ere bactericidal for S.aureus and P.

aeruginosa but inhibitory for S. typhi , "eslac probiotics killed /! coli and S. typhi $hile

they $ere only inhibitory for S.aureus and % ! albicans$ &ain probiotics inhibited %.

albicans


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ten different Lactobacillus species an also S.milleri, P ! !ulgaris, P ! aeroginosa, E. coli,

E. cloacae and '. !aginalis. But none of isolated strains sho$ed efficiency on test

or0anisms S. aureus and %. albicans. #lso some characteristics of bacteriocins $ere

obtained from the research !

In another research, potential probiotic lactobacilli strains , used as additi4es in pelleted

feedin0, $ere e7amined accordin0 to their antibacterial acti4ity a0ainst to Salmonella

typhimurium #TCC 'A+*, E. coli, %. perfringens ''&, S. aureus #TCC '-'=, B.

megaterium .*, L. innocua D2M '(* and B. hyodysenteriae #TCC 'A+*! &enerally

the cell free e7tracts of lactobacilli $ere able to inhibit all potential patho0ens e7cept B.

hyodysenteriae #TCC 'A+*! The study sho$ed that, neutralization and treatment $ith

catalase affect the antibacterial acti4ity a little !

45= S!$et) Ase'ts o$ P"obioti's

Today, there are e4idences that probiotic strains used as commercial bacteria are

safe to use in applications! The safety of the probiotic products is appraised $ith the

phenotypic and 0enotypic characteristics and the statistics of used microor0anisms


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=! They ha4e to be non)patho0enic!

! They ha4e to no history of relationship $ith diseases like, infecti4e

endocarditis or 0astrointestinal tract disorders!

-! They do not decon9u0ate bile salts!

*! They should not carry transmissible antibiotic resistance 0enes ! Comparison bet$een these methods, the most po$erful and accurate one

is se5uencin0


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In one study, the PC:)#:D:# techni5ue $as used to identify potential

probiotic Lactobacillus species isolated from bo4ine 4a0ina! +*2 r:"# 0ene $as

amplified by PC: and products $ere di0ested $ith four restriction enzymes ! Most of the di0estion profiles obtained from the amplified

+*2 rD"# 0ene of these strains a0reed $ith the theoretical profile matchin0 $ith

Lactobacillus fermentum! #mon0 all strains, four homofermentati4e lactobacilli sho$ed

a restriction profile that matched $ith Lactobacillus gasseri and a facultati4e

heterofermentati4e strain $as identified as Lactobacillus rhamnosus


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In another study potential probiotic Lactobacillus strains isolated from human,

animal and food $ere identified by +*2)'=2 r:"# restriction profilin0 at species le4el!

.irstly PC: amplification of +*2)'=2 r:"# inter0enic spacers $as done by usin0

uni4ersal primers! It is follo$ed by di0estion of PC: products by ++ restriction

enzymes $ith *bp specificities! 2ome of the enzymes $ere Sfu I, #ind III, Dra I,

Eco:I, Eco:E etc! the study $as concluded that identification could be done by D"#

fin0erprints 0enerated by restriction endonucleases! The amplified ribosomal D"#

restriction analysis $as an easier, faster and more accurate method ! It is estimated that an infant in0ests


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+7+(- +7+(

Acommensal bacteria $hile sucklin0 if the infant comsumes appro7imately

(( ml milk per day !

The composition of the 0ut microflora is thorou0hly influenced by the diet of the

infant! Thus, the presence of a fe$ predominant &ram)positi4e species in milk may

be a reason e7plainin0 $hy microbiota of composed of a narro$ spectrum of

species, and a more di4erse microbiota de4elops after $eanin0 ! #lthou0h there

are limited kno$led0e about the commensal or probiotic bacteria that milk contain,

bacteria commonly isolated from this biolo0ical fluid include staphylococci,

streptococci, micrococci, lactobacilli and enterococci ! Bacteria from these 0enera can be

easily isolated from fresh milk of healthy Buffalo! 2o, these 0roups of bacteria should be

considered the natural microbiota of human milk rather than mere contaminant bacteria

!

There are surprisin0ly not so much studies on the isolation and analysis of

commensal or potential probiotic bacteria from Buffalo milk ! These isolated bacteria

$ould fulfill some of the main criteria like bein0 human ori0in, adaptation to dairy

substrates and a histoy of lon0 duration and safeintake by infants ! 6ence, Buffalo milk, a natural source of potentially probiotic or

biotherapeutic L#B, protects infants a0ainst infectios diseases


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There are lots of studies on the effect of Buffalo milk on the health of infants and

the infant diseases but surprisin0ly lack of studies on the microbiolo0y of Buffalo

milk! .rom the fe$ studies, it is found that Buffalo milk is an attracti4e source for

potential probiotic strains! #s, the bacteria implement some of the main criteria for

bein0 probiotic strains such as, Buffalo ori0in, sur4i4al in the 0astrointestinal conditions

and particularly lo$ p6 and bile, production antimicrobial compounds, adhesion to the

intestinal mucosa !

Martin et al! studied on three lactobacilli strains isolated from Buffalo milk

$hether they $ere potential probiotic bacteria! They performed some assays to

in4esti0ate some criteria need to be used as probiotic bacteria such as8 sur4i4al to

conditions simulatin0 in the 0astrointestinal tract, production of antimicrobial

compounds, adherence to intestinal cells, production of bio0enic amines, de0radation of

mucin, enzymatic profile and pattern of antibiotic resistance! ' Lactobacillus gasseri

and + Lactobacillus fermentum strains $ere e4aluated and the results sho$ed that the

probiotic potential of lactobacilli isolated from Buffalo milk is similar to strains

commonly used in commercial probiotic products!

6eikkilW and 2aris


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Staphylococcus aureus. They concluded that the commensal bacteria in Buffalo milk

may ha4e a role in protectin0 the infant and mother a0ainst Staphylococcus aureus.

#lso the results supported that the commensal staphylococci and streptococci are

predominant bacterial species in Buffalo milk! The other isolated bacteria Lactobacillus

rhamnosus had :#PD profile identical to the commercial strain Lactobacillus

rhamnosus &&, $hich is a commonly used probiotic strain in milk products in .inland!

@li4ares et al!


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CHAPTER 0

MATERIALS AND METHODS

04 Iso(!tio# o$ L!'ti' A'id B!'te"i! $"om Bu$$!(o Mi(>

The isolation material $as Buffalo milk obtained from +- healthy Buffalo

from Cho$dhury Dairy .arm! The samples $ere collected in sterile carriers and

stored on ice until deli4ery to the laboratory! @nce deli4ered to the laboratory, they

$ere taken to the procedure for isolation! Pour plate techni5ue $as used to isolate the

or0anisms! 2amples $ere used directly and also diluted to +()+

, +()'

and +()=

usin0

sterile peptone $ater! + ml ali5uot of the samples and dilutions $ere plated into M:2 a0ar


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$ater! &ram stainin0 procedure $as applied! Then, under li0ht microscopy 0ram

positi4es and purified isolates $ere determined!

040 C!t!(!se Test

Catalase is an enzyme produced by many microor0anisms that breaks do$n the

hydro0en pero7ide into $ater and o7y0en and causes 0as bubbles! The formation of 0as

bubbles indicates the presence of catalase enzyme!

'6'@' ' 6'@ X @'

Catalase test $as performed to isolates in order to see their catalase reactions!

.or this purpose, t$o methods can be applied! @4erni0ht cultures of isolates $ere

0ro$n on M:2 a0ar at suitable conditions! #fter ' h =? hydro0en pero7ide solution

$as dropped onto randomly chosen colony! #lso fresh li5uid cultures $ere used for

catalase test by droppin0 =? hydro0en pero7ide solution onto + ml of o4erni0ht

cultures! The isolates, $hich did not 0i4e 0as bubbles, $ere choosed! 2ince, L#B are

kno$n as catalase ne0ati4e!

04= Lo#% Te"m P"ese"*!tio# o$ Iso(!tes

&ram positi4e and catalase ne0ati4e isolates $ere preser4ed in M:2 broth

medium containin0 '(? 0lycerol as frozen stocks at )( RC! The 0lycerol stocks

of samples $ere prepared by mi7in0 (!- ml of acti4e cultures and (!- ml M:2 medium

includin0 (? sterile 0lycerol!

00 P"obioti' P"oe"ties o$ Iso(!tes

.or the determination of probiotic properties of isolates these ma9or selection

criteria $ere choused% resistance to lo$ p6, tolerance a0ainst bile salt and the

antimicrobial acti4ity!


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004 Resist!#'e to Lo- H

:esistance to p6 = is often used in 4itro assays to determine the resistance to

stomach p6! Because the foods are stayin0 durin0 = hs, this time limit $as taken into

account ! .or this purpose, acti4e cultures

$ere used! Cells $ere har4ested by centrifu0ation for +( min at -((( rpm and RC!

Pellets $ere $ashed once in phosphate)saline buffer


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Table '!+! Indicator microor0anisms

I#di'!to" mi'"oo"%!#ism I#'ub!tio# 'o#ditio#s

Salmonella thyphimurium CCM -- =A RC in T2B medium

Escherichia coli @+-A%6A "CTC +'((( =A RC in T2B medium Escherichia coli "::L B)=(( =A RC in "utrient broth medium

0= Ph)sio(o%i'!( !#d Bio'hemi'!( Ch!"!'te"i@!tio#

Biochemical tests $ere run accordin0 to methods offered by Bulut, '((=!

0=4 /!s P"odu'tio# $"om /(u'ose

In order to determine the homonfermentati4e and heterofermentati4e

characterization of isolates, C@' production from 0lucose test $as applied! Citrate

lackin0 M:2 broths and in4erted Durham tubes $ere prepared and inoculated $ith +?

o4erni0ht fresh cultures! Then the test tubes $ere incubated at =A RC for - days! &as

occurrence in Durham tubes $as obser4ed durin0 - days $hich is the e4idence for C@'

production from 0lucose!

0=0 /"o-th !t Di$$e"e#t Teme"!tu"es

Temperature test media, M:2 containin0 bromecresol purple indicator, $as

prepared and transferred into tubes as - ml! Then fifty l of o4erni0ht cultures

inoculated to tubes and incubated for A days at +( RC, +- RC, - RC! Durin0 these

incubation time cells 0ro$th at any temperatures $as obser4ed by the chan0e of the

cultures, from purple to yello$!

0== /"o-th !t Di$$e"e#t N!C( Co#'e#t"!tio#s

Isolates $ere tested for their tolerance a0ainst different "aCl concentrations! .or

this purpose ? and *!-? "aCl concentrations $ere selected! Test mediums containin0

bromecresol purple indicator $ere prepared accordin0 to the appropriate concentrations

and transferred into tubes in - ml! these tubes $ere inoculated $ith +? o4erni0ht

cultures and then incubated at =A RC for A days! The chan0e of the color from purple to

yello$ $as proofed the cell 0ro$th!


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0=5 A"%i#i#e H)d"o()sis Test

#r0inine M:2 medium and "esslers rea0ent $ere used in order to see

ammonia production from ar0inine! M:2 containin0 (!=? L)ar0inine hydrocloride $as

transferred into tubes as - ml and inoculated $ith +? o4erni0ht cultures! Tubes $ere

incubated at =A RC for ' h! #fter incubation, +(( l of cultures transferred onto a $hite

back0round! The same amount of "esslers rea0ent $as pipetted on the cultures! The

chan0e in the color $as obser4ed! Bri0ht oran0e color indicated a positi4e reaction

$hile yello$ indicated the ne0ati4e reaction! # ne0ati4e control, $hich did not contain

ar0inine, $as also used as ne0ati4e control!

0=6 C!"boh)d"!te Fe"me#t!tio#s

Isolates $ere characterized accordin0 to their fermentation profiles of ability toferment +A different carbohydrates! #ll reactions $ere performed by usin0 *)$ell

microtitre plates! #cti4e cells and su0ar solutions $ere prepared separately! .or

preparation of acti4e cells8 isolates $ere acti4ated in +( ml M:2 medium and incubated

at =A RC for ' h! Then, they $ere centrifu0ed +( min at +(((( rpm! Pellets $ere

$ashed t$ice and resuspended in M:2 $ithout 0lucose and containin0 p6 indicator

bromecresol purple! /ach su0ar solutions $ere prepared at a final concentration of +(?

, only salicin $as prepared at concentration of -?! Then the solutions $ere filter

sterilized $ith filters ! #fter preparation steps the procedure$as applied! .orty l of su0ar solutions $ere pipetted into each $ell and +*( l of

suspended cells $ere added onto the su0ar solutions! Thus, '? final su0ar concentration

$as obtained! #ll the reactions $ere performed t$ice! #lso positi4e and ne0ati4e

controls $ere used to indicate any contamination! +*( l of suspended cells X ( l of

0lucose solution $ere used as positi4e control $hile '(( l of suspended cells $as used

as ne0ati4e one! #fter o4erni0ht incubation at =ARC, the turbidity and the color chan0e

from purple to yello$ $as recorded as positi4e fermentation results compared $ith the

positi4e and ne0ati4e controls! #lso results $ere compared $ith the absorbance of

samples read at *'( nm in an automated microplate reader !

Documents

Thesis on Probiotic, Part 1, Abadin