Selection of a Chicken Lactobacillus Strain With Probiotic Properties and Its Application in Poultry Production

8/12/2019 Selection of a Chicken Lactobacillus Strain With Probiotic Properties and Its Application in Poultry Production

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S E L E C T I O N O FACHICKEN LACTOBACILLUS STRAINWITHPROBIOTICP R O P E R T I E S A N D ITSAPPLICATION IN PO UL T R Y PRODUCT ION

by

S O U M E L ASAWIDOU

A thesis submi t ted to theUnivers i tyof P l ymouth


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Copyright Statement


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S E L E C T I O N OF A CHICKEN LACTOBACILLUS STRAINWITHPROBIOTICP R O P E R T I E S A N D ITSAPPLICATION INP O U L T R Y P R O D U C T IO N

by

S O U M E L ASAWIDOU

A thes is submi t tedto theU nivers i tyofP lymou thin partial fulf i lment forthe de greeof


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AbstractS O U M E L ASAWIDOU

Selection of a chicken Lactobacillus strainwithprobioticproperties andits application inpoultryproduction

O n e hundred and eleven lact ic acid bacteria, of chicl


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Frequently used abbreviationsF R E Q U E N T L Y U S E DABBREVIATIONS

A M F Ac id i f i ed M ois t F e e dC F U ml"'' or C F U g"^ C olo ny forming unit pe rmillilitre or per gram, a

me asu re o f v iab le bac ter ia l num bersC O N C ont ro l groupD N A Deox yr ibonuc le i c ac idF L F , F M F Fer m en t ed liq u id f eed , Fe r m en t ed m o i st F e e dG l Gast ro intest inalH P L C H igh Per form anc e L iqu id C hrom atograph yL A B Lac t i c A c id Ba c ter iaLact. Lactobacillusm mo l L'"" M il imo le pe rlitre


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LISTOF CONTENTSListof contents

Copyr ightStatement.. 1Title P a g e IIA bs t r ac t IllFrequent l y u s e dAbbreviations IVL is tof C o n t en ts VL is tof T a b l e s . . , IXL is tof F igures XIL is tof A p p e n d i c e s XIIA c k n o w l e d g e m e n t s XIVAuthor 'sDeclaration XVI

Chapter 1


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Listof contents1.5.1 Administration of LAB tfirougti water. '. 391.5.2 Administration of LAB tfirougii fermented feed 40

1.6 S E L E C T I O N C R I T E R I A F O R P R O B I O T I C S 511.6.1 Aggregation experiments 541.6.2 Antagonistic activity , 551.6.3 Acidic pH andbilesaltresistance '. 561.6.4 Adhesion, of LAB to chicken intestinal epithelial cells , 571.6.5 Hydrophobicity 611.6.6 Homo/heterofermentation test 62

1.7 P R O B L E M SOFP R O B IO T I C D E V E L O P M E N T A N D U S A G E 6 21.8 R A T I O N A L E F O R T H E S T U D Y 6 4

Chapter 2 67Selection, characterisation and screening of lactic acid bacteria ofchicken origin thathaveprobiotic properties 67

2.1 I N T R O D U C T I O N 6 7


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List of contents3.2.2 Study 2. Fermentation abilityof selected organisms 114

3.3 R E S U L T S A N D D I S C US S I O N 1183.3.7 Study 1. Sun/ival of selected organisms inwater. 7783.3.2 Study 2. Fermentation ability of selected organisms 779

3.4 C O N C L U S I O N S 126Chapter 4 127Wloist feed, fermented with Lactobacillus salivarius Salivarius NCIWIB41606,reduces susceptibility of one day old chicks to SalmonellaentericaserovarTyphimuriumSal 1344 nar, during their40 daydevelopment....127

4.1 I N T R O D U C T I O N i274.2 M A T E R I A L S A N D M E T H O D S 128

4.2.1 Ethical approval 7284.2.2 Experimental design and treatments 7284.2.3 Strains and culture conditions 729


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Listof contents4.3.8 Intestinal mucosa and liver histology 7 6 5

4 . 4 D I S C U S S I O N 167Chapters 187Conclusions '. 187References 197Appendices 221


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List of tablesLIST O F T A B L E SChapter 1.Literature reviewT able 1. S afe microb ia l products for use as feed addi t ives inpoultry production

(Sc ient i fic C omm it tee on A nim al Nutr i tion, 2003) 7T able 2 . Perfo rma nce of prob iot ic organ isms use d in ch ic ken product ion 31Ta ble 3 . Des irab le Probiot ic C harac ter is t ics and Func t ions 52

Chapter2.Selection,characterisationand screening of lactjcacidbacteriaofchickenoriginthathaveprobiotic properties 67Table 4. The number o f s t ra ins subjected to each test dur ing the scre ening

process and the number o f iso la tes processed to the next stag e 70Tab le 5 . Identification, autoaggregat ion of lactobaci l l i , and their coaggregat ion

with seve ra l pathogen s 80Tab le 6 . 16S r R N A gene sequence ident i f icat ion and Nat ional Col lect ion

Nu m b e r o f Lactobacillus N os 3 , 6 , 1 6 and 18 84Table 7 . Pathogenic bacter ia used for the antagonis t ic activity ass ay 87


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List of tablesT a b l e 15. pH and concentration (mmol L' ') of Maltose, Glucose and Fructose,

Lac t i c acid and Acetic acid of Barley fermented with Lactobacillus strainNo3 and N0I6 and Red Sorgum fermented with Lactobacillus strain No3andN0I6after 24 and 48h* 122

Chapter 4. Moist feed, fermentedwith Lactobacillus salivarius SaiivariusNCIMB41606, reduces susceptibility of one day old chicks to Salmonellaenterica serovar Typhimurium Sal 1344 nal"", during their 40 daydevelopment : 127T a b l e 16. Declared analysis and composition of basal diet* 132T a b l e 17. Concentrations (mmol L *) of maltose, glucose, fructose, lactic and

acet i cacid and pH ofA M F and FMF when delivered to chickens* 147T a b l e 18. Average feed intake (grams/bird/day dry feed basis) during the 40

d a y sof the experiment for the four groups: C O N ,WAT, F M F , A M F * 149T a b l e 19. Average weight gain (grams/bird/day) during the 40 days of the

experiment for the four groups: C O N ,WAT, F M F , A M F * 149


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List of figuresLISTOF F IGURESChapter 2. Selection, characterisationand screening oflacticacidbacteria

of chicken originthathaveprobioticpropertiesFigure 1 . F lo w chart o f the sc ree ning and selec t ion pro ce ss 69Figure 2 . Au toagg rega t ion test 74Figure 3 . Aggregat ion o f two Lactobacillus s t ra ins. A . Au toggre gat ion of

Lactobacillus spp. No 3. B. Normal ly aggregat ing Lactobacillus spp.. C .Rapid lyagg regat ing Lactobacillus spp 7 7

Figure 4 . Aggregat ion of two Lactobacillus strainswith Salm. entefitidis showihgstrands of LAB aggregat ing prote in adher ing to Salm. enteritidis. A .Lactobacillus sp p. sh ow ed par t ia l aggreg at ion B. Lactobacillus spp (No 3)s h o w e d ma ximu m aggreg at ion '. 78Figure 5 . An tago nis t ic act iv i ty test 87

Figure 6 . The level o f mucus b inding of 9 Lactobacillus strains 91Figure 7 . A d h e s i o n , s c o r e d a s " v e r y g o o d " , o fLact.S a l i va r ius (No16) to ch icken

ep i the l ia l ce l l s (Sc ann ing e lec t ron m ic rosco py - S E M ) 94


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List of figuresFigure 13 . N of Salmonella negat ive b irds per ten tagge d an d c loa ca l ly

s w a b b e d b i rds per group 153Figure 1 4 . Th e m e a n Salm. Typ h imur ium counts o f the c loac a l sw ab s o f ten

t a g g e d b i rd s f o r e a c h g r o u p C O N ( X ) , W A T (0) , F M F ( ) a n d A M F ( A ). . .1 5 4Figure 1 5 .Salmonella cou nts of env i ronm enta l sa m ple s at day 32 158Figure 16. Photo of h igh resolut ion gels conta in ing samples obta ined from

c a e c u m and i leumfrom chicl


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Listof appendicesLISTOF APPENDICEST a b l e 1.Total results of the screening programme 2 2 1


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A C K N O W L E D G E M E N T SAcknowledgements

1 wo uld l ike to exp res s my gratitude to a l l those who have inf luenced thepreparat ion and the complet ion of my thesis.

I am d eep ly indebted to my sup erv i sor P r . Pete r B roo ks wh ose he lp , s t imula t ingsuggest ions , exce l len t adv i ce and encouragement , he lped me in a l l t he time ofresearch for andwriting of this thesis.

I am very gratefu l to my superv isor Dr . J a n e B e a l , who he lped and suppor tedme in my researchwork and for shar ing her knowledge in microbio logy.


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Author's declaration

Poster presented at conference s:S . S a v y i d o u , J . D . B e a l , R . M . L a R ag i one , M . J . W ood w ar d , P . H . B r ook s ( 2009).Mois t feed fermented with Lactobacillus salivarius N C I M B 4 1 6 0 6 r e d u c e sshedd ing o f Salmonella in ch i cke ns. S um m er C onfe renc e o f the Soc ie ty fo rApp l ied M ic rob io logy , Manchester Met ropo l i tan Un ivers i t y , Manchester , UK, 6-9Ju l y 2009S . S a w i d o u , J . D .B e a l , P .H . Bro oks (2007) . Isolat ion of lac t ic ac id bac ter ia fromc h i c k e n s that dem onst rate probiotic proper t ies of autoag gregat ion andcoaggregat ionwith S .enteritidis. International Y a k u lt S y m p o s i u m , V e r o n a ,Italy,22- 23 N ov em ber 2007S . S a v v i d o u , J . D .B e a l , P .H . Bro oks (2007 ) . Isolation of lac t ic ac id bacter iafromc h i c k e n s that dem onst rate probiotic proper t ies of autoag gregat ion andcoaggregat ion with S . enteritidis. 58 th Annua l Meet ing o f the EuropeanAssoc ia t ion fo r An imal P roduc t ion ( E A A P ) , Dub l in, I re land 2 6-2 9, Au gu st 200 7

Word count of main body of thesis: 44,535


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Chapter 1 Literature review

Chapter1

Literature review1.1 Problems o fmodern poultry productionM o d e r n poul t ry husbandry requi res a system of h igh ly in tens ive product ion, inwhich b i rds are hatched ar t i f i c ia l ly in incubators , which are kept c lean anddis in fec ted. T hu s, newly ch ick s, wh ich n ormal ly acq ui re the i r in test ina lmicrobiota f rom the fowl and sur face of the eggshel ls , hatch in a s ter i leenv i ronm ent a nd ma y delay or fa i l to develop m icrobia l eco sys tem of thegastro intest ina l tract with des i rab le m ic roorgan isms (S te rzo e t a l . 2005) . Gutcolonisat ion in the beginning of l i fe with des i rab le m ic robes cou ld ac t as a


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Chap ter 1 Literature reviewf rom the invas ion of pathogenic organisms or f rom the cont inu ing changes inthe number and the composi t ion of the in test ina l microf lora. The in test ina lmicrof lora is regarded as s table in the heal thy b i rd, a l though factors such asc h a n g e s in the bird i tsel f , deprivat ion of food or water, t ransportat ion,administrat ion of ant ibiot ics and radiat ion may result in instabi l i ty of the gutmicrof lora.

In 2007 the European Food Safe ty Au thor i t y ( E F S A ) publ ished a survey of thelevels of Salmonella detec ted in bro i ler f locks (ch icke ns reared for meat) ac ro ssthe European Un ion in 2005-6 . I t was repor ted that one in four broi ler f lockss u r v e y e d b e t we e n O c t o b e r 2 0 0 5 a n d S e p t e m b e r 2 0 0 6 w e r e Salmonella-


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Chapter 1 Literature review1983) or a mild clinical infection (including colonisation) (Kaldhusdal andH of s l i agen 1992), and liver d i s e a s e ,as well as the more commonly recognisedfulminant infection that can result in disease outbreaI


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Chapter 1 Literature reviewpromoters throughout the European Union in 2000 and the potent ia l o f a ban inthe Uni ted Sta tes . De spi te the ban in the use on ant ib io t ics as growthpromoters , repor ts sugges tthat th e total amount o f an t ib io t i cs used inc reaseda s a resul t o f inc rea sed therape ut ic u se of ant ib io t ics (K ob ash i e t a l . 20 08 ). Inorder to red uc e ant ib io t ic use there ha s bee n an inc rea se d co mm erc ia l in terest

in deve lopin g a l ternat ives to ant ib io t ics for poul t ry prod uct ion . A s a result thereh as been increased in terest in , and use of , d ie tary ac ids and probiot ics .

1.2 Definition of Pro b io t icsMetchnikof f (1907) was the first to p rov ide some ev idence that intest inalbacter ia have an important ro le in the maintenance of heal th when he noted the


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Chapter 1 Literature reviewef fect on the host an im al by improv ing i ts in test ina l microbia l ba lan ce .H a v e n a a r and Huis In ' t Veld (1992) expanded Ful ler 's (1989) def in i t ion, whichw a s rest r ic ted to feed sup plem ents , an im als and the ir in test ina l tract, andredef ined 'probiot ic ' as a mono or mixed cu l ture of l iv ing microorganisms whichapp l ied to anim al or m an , benef ic ia l ly a f fects the host by improv ing thepropert ies of the ind igenous microf lora.

Dur ing 1989, the Uni ted States Department of Agr icu l ture ( U S D A ) p r o p o s e dthat ma nufac tu re rs us e the term 'd i rect - fed microbia l ' (DFM) instead of probiot ic(M i les and Boo twa l la 1991) . Ac co rd ing to M i les and B ootwa l la (1991) theU S F D A cha rac te r i se a D F M as a sourc e o f l ive , na tu ra ll y oc cur r ing


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Chapter 1 Literature reviewwh ich do not h ave to be def ined by ident i f icat ion of ea ch organ ism in themixture. T his defin it ion incorp orates the Nurm i co nc ept of 'comp et i t iveex c l us i on ' (C E ) . Nu rmi and R anta la 's techn ique (1973) i s con s idered as thebas i s of the compet i t ive exclusion mechanism of probibt ics in poul t ry (J in et a l .1997; M a h a j a n a n d S a h o o 1 9 9 8 ; G h a d b a n 2 0 0 2 ) . . W h e n 1 -2 -d ay o l d - c h i c k soral ly inoculated with adul t gut microf lora of heal thy bi rds, showed s igni f icantres is tance to Salm. infanti, suppl ied by feeding one day later (Nurmi andR ant a l a 1973). This study set the basis for further development of thecompet i t ive exclusion and the int roduct ion of oral administ rat ion of an undef inedmixed cu l ture f rom adul t b i rds to new ly hatched c h ic ks . Th e C E bacter iapreferent ia l ly establ ish in the gastrointest inal tract and become antagonis t i c tooppor tun i st ic pa thogens . S ev era l com me rc i a l C E p roduc ts , with def ined or


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Chapter 1 Literature reviewT a b l e 1.S a f e m ic rob ia l p roduc tsforu s easfeed add i t i ves inpoultry p rodu ct ion(Sc ien t i f ic Com mi t tee on An im a l Nu t r it ion , 20 03)Name ActiveConstituent(s) Culture collectionand

accessionnumberTarget animalcategories

Bactocell Pediococcusacidiiactici CHCM MA18/5I\/I BroilersBioplus 2 B Baciiius iicheniformisBaciiius subtilis

[in a 1/1 ratio]D SM5749D S M5750

Broilersandturkeys

Cylactin LEG Enterococcus faecium NCIMB10415 BroilersLactobacilius acidophiiusD 2 / C S L Lactobacillusacidophilus C E C T4529 Laying hensMicroferm Enterococcus faecium D SM5464 BroilersOralin Enterococcus faecium NCIMB10415 BroilersProbios PDFM Granular Enterococcus faeciumEnterococcus faecium D SM4788 /A T C C53519D S M 4 7 8 9 / A T C C

55593

Broilers


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Chapter 1 Literature reviewT h e G ltract can be subdiv ided accord ing to the ce l l type and funct ion into theepi thel ia l cel l layer, lamina propr ia, muscular is , components of the immunesys tem and the mucus layer . The first ep i the l ia l layer compr ises enterocy tes(pr imary abso rpt ive ce l ls of the intest ine), goblet c el ls (secrete m uc in andglycoprote ins) , endocr ine ce l l s (secre te hormones and neuropept ides) , M ce l l s

a n d int raepi thel ia l leukocytes ( C D S * T lymp hoc ytes an d natural k i ller ce l ls) . A l lthese ce l l s (except M ce l l s ) are ar ranged into f inger- l ike shapes known as v i l l i ,whic h co m prise the ba sic abso rpt ive structure of the gut . A t the v i llus ba se thecrypts of L ieberkuhn are formed and const i tutethe major s i te for product ion anddi f ferent iat ion of new enterocytes. The structure that l ies beneath and stabi l isesthe epi thel ium, the lamina propr ia, consists of nerve f ibre, and several immunecel ls such as p lasma ce l l s ( IgA secret ing) , T l ymphocytes (genera l l y CD4"^),


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Ch apter 1 Literature review1.3.2 Maintaining beneficial microbial population in the gastrointestinaltractand suppre ssion ofviablenumbersof specif icbacteria

1.3.2.1Developmentand microbial ecology of the chickenT h e av ian digest ive t ract inc ludes microbial populat ionsthat are variable in s izea n d co mplex i ty (Ba rnes 1979 ; B jerrum et a l . 200 6; Gab r ie l e t a l . 20 06; Kou tsosa n d Ar ias 2006; For tun-Lamothe and Boul l ie r 2007) . Broad ly , micro f lo ra o f thedigest ive t ract can be group ed into harm ful bac ter ia, wh ich m ay be invo lved inthe induction of infect ion, intest inal putrefact ion and toxin production and thec o m m e n s a l populat ions, which are involved in v i tamin product ion, s t imulat ion ofthe immune system and inhibi t ion of harmful bacter ia (Koutsos and Ar ias 2006).


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Chapter 1 Literature revieweventua lly , they b ec om e the dominant s pe c ie s in the up perpart of the GI tract.(Apajalahti et al . 2004).

Bacter ia l act iv i ty takes place mainly to the crop and caecum and to a lesserextent in the smal l intest ine. The bacter ia l populat ion is d iverse, especial ly inthe c ae c um , wh ere the s lo w turnover of the con tents (1 to 2 t imes a day) resul tsin the development of more and different types of bacter ia (Apajalaht i et a l .2 0 0 4 ) . The i leum contains 10^ bacter ia per gram of contents and th is consistsmainly of facul tat ive anaerobes, predominant ly lactobaci l l i , but a lso enteroccocia n d co l ifo rms. Th e c ae cu m has 10^^ bacter ia per gram of contents an d has lessfacul tat ive than strict anaerobes (Apa ja laht i e t a l . 2004) . Recent molecu lar


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Cha pter 1 .. Literature reviewresul ts in mo re ef fic ient fee d con vers ion (J in et a l . 1997). T he usu al prac t ise inm o d e r n , in tens ive, poul t ry product ion is to keep poul t ry incubators and hatch ingrooms as s ter i le as poss ib le. However, th is a f fects negat ive ly the developmentof the ch icke n in test ina l microf lora (Apa ja laht i and Ke t tunen 2006 ). Ad di t ional ly ,s t ressfu l condi t ions such as h igh temperature, h igh humid i ty , change of feed

and t ransportat ion, admin is t rat ion of ant ib io t ics may upset the balance of gutmicrof lora. M oreo ver, oppo rtunis t ic bacter ia l invad ers of the env i ronment m ayp o s e a con t inua l cha l lenge to the ch icke n . T he p ro tec t ive m ec han ism s o f thehost , such as the low gastr ic pH (2.6 for g izzard) ,that wil l result in a 10 to 100fo ld reduct ion of bacter ia num bers in d ige sted food or water , and the produ ct ionof volati le fatty a c i d s , that are know n to su pp res s the populat ion ofenteropathogens, can par t ly protect the host (J in et a l . 1997; E d e n s 2 0 0 3 ) . A lso ,


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Chapter 1 Literature reviewa l. (1996a) reported on twelve Lactobacillus s t ra ins iso lated from ch i ckenintest ine, having inhibi tory act iv i ty against f ive strains of Salmonella and threesero types o f Esch. coli. S u c h an t im ic rob ia l subs tances inc lude bac te r ioc ins(ac idol in , bu igar ic in , n is in , reuter in) , bacter ioc in- l ike substances (Mahajan andS a h o o 1998), sor t chain vo lat i lefatty acids ( lact ic, propionic, butyr ic, and acet ic)

and hydrogen perox ides (Fu l le r 1989; J in e t a l . 1997; Ma ha jan and S ah oo 1998;G h a d b a n 200 2; C urb elo et al . 20 05 ). In part icular, lactoba ci l l i are know n toprodu ce bac ter ioc ins, wh ich are def ined as pept ides, prote ins, or prote inac eou scompounds and most o f them have an inh ib i tory act ion against Gram pos i t iveor /and Gram negat i ve bac te r ia . For example , Lact. acidophilus has beenreported to produce a large number of bacter ioc inswith an inhibitory effect onpathogen ic bac te r ia such as Salmonella, Co l i fo rms and Campylobacter (Tahara


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Chapter 1 Literature review1992 ; c i ted by Cu rbe lo et a l . 200 5). In other words hydrog en p erox ide inh ib i tsthe growth of pathogens through i ts strong oxidiz ing effect on the bacterial ce l ls ,or through the destruct ion of bas i c molecular s t ructures of nuc le ic ac id and ce l lproteins (J in et al . 1996a)

The bacter ic ida l e f fect o f organic ac ids ( lact ic , acet ic , butyr ic and propionic)p roduced by Lactobacillus, Enterococcus a n d Bifidobacterium, has beenattributed to the i r capaci ty to reduce in test ina l pH (J in et a l . 1997). Organicac i ds , which are major end products of the metabol ism of lact ic ac id bacter ias u c h a s Lactobacillus, Enterococcus a n d Bifidobacterium s t ra ins, haveantagonis t ic act iv i ty against pathogens. The ac id ic env i ronment of the


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Chapter 1 Literature revieweff ic iency of compet i t ive exclusion for tiie cont ro l o f pathogens such asSalmonella, Esch. coli a n d Campylobacter. Un def ine d cul tures were found to beef fect ive against pathogens in vitro,how ever , invivo s tud ies were more var iab le(S tavr i c and D 'aoust 1993) . The same s tudy conc ludedthat def ined cul tures areless ef fect ive than undef ined cul tures in vitro. Fu l le r (1989) suggests that

compet i t ion between ex is t ing micro f lo ra and pathogens for s i tes o f adherenceon the in tes t ina l sur face is another mechanism by which benef i c ia l o rgan ismsprevent co lon isat ion by patho gen s.

1.3.2.4Adhesion to epithelialcellsA number of bacter ia stay in constant contactwith the mobi le v iscous layer of


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Chapter 1 Literature reviewev idence tliat th e growth of enteropathogens in mucus contr ibutes to theirpa thogenes is (Wadoll


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Chapter 1 Literature reviewfrom ch i cken and i s an adhes ive Lactobacillus strain that ef f ic ient ly preve ntsp a t h og e n a d h e r e n c e invitro.

A d h e s i o n is cons idered to be a complex but essent ia l mechanism for bacter ia lco lon isa t ion . In the c a s e o f probiot ics , adhesion is a mul t is tep process involv ingthe c lose associat ion of the potent ia l probiot ic lact ic ac id bacter ia with theintest ina l mucosa and the epi the l ia l ce l ls . G u s i l s e t a l . (2002b) suggest thata d h e s i o n might be a prerequis i te of compet i t ive exc lus io n; as the L A B grow,attach to the gut wal l and further co lonise and inoculate the luminal contentsespec ia l l y i n the c rop and caecum o f ch ickens . Some LAB have the ab i l i t y toat tach to the sur face adhesion receptors of the gut , thereby exc luding the


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Chapter Literature reviewpili whic h ca n be desc r ibed as prote ina ceo us p ro ject ions. O n' the other han d,lac tobac il ii use ex t race llu la r s ubs tanc es , con ta in ing m uc opo lys acc har ide s ,prote ins (Conway and Kje l leberg 1989), l ip ids and l ipote ichoic ac ids (Shermanand Savage 1986) to at tach to the epi the l ium cel ls . Granato et a l . (1999)reported that Lact. johnsonii Lai uses l i po te i cho ic ac id as an adhes ionm e c h a n i s m . Severa l researchers such as Jakava-V i l janen e t a l . (2002) , Lorcaet a l . (2002) and Rojas et a l . (2002) have managed to iso late and character ise-adhesion-promot ing prote ins on the sur face of Lact. fermentum a nd Lact.brevis. Gus i ls (2002a) reported the presence of lect inl ike proteic structures onthe sur face of severa l lactobaci l l i {Lact. animalis, Lact. fermentum andpart icular ly Lact. fermentum spp.. Cellobiosus w as found to have h ighhydrophobic i ty va lue s. Lec t in l ike s tructures a nd (or) po lys ac ch ar ide s tend to link


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Chapter 1 Literature review(2002) obta ined resul ts from their work with potent ial probiot ic lact ic acidbacter ia of duck or ig in , showing that the strains with high hydrophobici ty alsoshowed s t rong adhes ion to c rop ce l l s and Hep2 ce l ls .

I t is wel l documented in severa l invitroadh es ion m ode ls , us ing C a c o - ce l ls ,thatlac tobaci l l i are able to compet i t ive ly exc lude enteropathogens (Tuomola andS a l m i n e n 1 998; T uo m ola et a l . 1999 ; L ee et a l . 200 3). G ene ra l ly , lactobaci l l iob ta ined from ch ickens have the abi l i ty to adhere to severa l ep i the l ia l ce l ls suchas c rop, smal l and large in test ines, though adhesion to the ce l ls o f smal lin test ine is weaker. G u s i l s e t a l . (2002b) and J in et a l . (1996b; 1996c)demons t ra ted that some lactobaci l l i were able to adhere s t rongly to ch icken i lea l


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Chapter 1 Literature reviewGIT o f m ice in vivo. Lact. casei rhamnosus GG, Lact. Johnsonii Lai, Lact.salivarius and Lact.plantarum s t ra in 299v co lo nise d a l l the segm ents of the gutof gnotobiot ic mice.

It is general ly bel ieved that w he n potent ial pro biot ic ba cteria hav e the abi l ity toadh ere to in test ina l m uc os al ce l ls the m aximu m probiotic e f fect is ac hie ve d.However , Serv in and Cocon ie r (2003) s ta ted that there some ev idence thatexogenously admin is tered organisms can have a probiot ic e f fect , even thoughthe o rgan isms pass th rough into t h e f a e c e s without hav ing adhered ormul t ip l ied. Fol lowing c l in ica l pharmacokinet ic s tudies Bezckorovainy (2001)s u g g e s t e dthat in order to have a cont inuing probiot ic effect the probiot ic cul ture


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Chapter 1 Literature review1.3.2.5Autoggregation and coaggregation withipathogensT h e aggregat ion or 'c lumping' of bacter ia l cel ls belonging to the same(autoaggregat ion) or to d i f ferent (coaggregat ion) bacter ia l cel ls is an example ofbacter ia l interact ions and comprises a part of the general research on screeninglact ic ac id bacter ia o f ch icken or ig in (Roos e t a l . 1999; Ko lenbrander 2000) .S e v e r a l res ea rch ers hav e reported the i r iiportance of aggrega t ion abi li ty oflactobaci l l i in enabl ing colonisat ion of the oral cavi ty , the urogeni ta l tract and ingenet i c exchange v ia con jugat ion ( G a s s o n e t a l . 1992 ; S en s ing and Dun ny1993 ; M astrom arino et a l . 20 02 ). Mo jgani et a l . (2007) hav e studied them e c h a n i s m of autoaggregat ion of Lactobacillus s t ra ins as potent ia l probiot icsa n d sugges tedthat cel l aggregat ion is probably due to the proteins present inthe cul ture supernatant and proteins or l ipoproteins, located on the cel l surface.


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Chapter 1 Literature reviewa g g r e g a t e s (Kmet and Lucchini 1999). The aggregat ion abi l i ty of the lactobaci l l ima y be contr ibuted by a pro te in known a s A P F (Aggrega t ion-Promot ing Fac tor ) ,w h i c h , wh en s ec rete d, ac ts as an aggregat ion media tor between two bacter ia lcel ls in many lactobaci l l i ofdifferent or ig in (Kmet and Lucchini 1999; Styr iak eta l . 2 0 0 1 )

T h e antagonist ic act iv i ty of lactobaci l l i could also be associated with their abil ityto coag graga te . A s the lac tobac i l li coa ggreg atewith the pathogens, they createlarge contact areas between them, which enable the so cal led ' inhibi torymicroenvi ronment ' , which is based actual ly on the inhibi tory act iv i ty of certa inmetabol i testhat a re concent ra ted in these a r eas . Addi t ional ly , a cont inui ty zone


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Chapter 1 Literature reviewT h e mechanism of the 'compet i t ion for essent ia l nutr ients ' is the bas is ofprebiot ics , which are def ined as non-d igest ib le carbohydrate f ract ions fed ind iets , that are benefic ial to the host by st imulat ing the growth o f one or morebacter ia in the Gltract (Dun k ley e t a l . 200 9) . F ruc too l igosac cha r ides have b eenshown to impact bacter ia l populat ions by promot ing the growth o f Lactobacillusa n d Bifidobacterium sp p . (Dunk ley e t a l . 2009 ) . O y a r z a b a l .a n d C o n n e r ( 1 99 5 )s u g g e s t e d that f ruc too l igosacchar ides a re no t d iges ted by in tes t ina l enzymesand can be used by lactobaci l l i and b i f idobacter ia to sat is fy the i r growthrequi rements for energy. At the same time the pH leve ls decreased because o fthe p roduc t ion o f ac id s . C ons equ ent l y , the growth o f Gram negat i ve bac te r ia ,s u c h a s Salmonella a n d Esch. coll, is rest ra ined as they are not ab le to usef ruc too l igosacch ar ides and they a re sens i t i ve to low pH .


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(P


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1.3.5 Othermodes ofactionofprobiotics

1.3.5.1Enterotoxin neutralisationProbiot ics have been found to have an ant i -enterotoxin act iv i ty (J in et al . 1997),but the ev idence for that is st i l l l imited. A commercial ly avai lable lactobaci l l ipreparat ion {Lact. bulgaricus) .was shown to neutra l ise. Esc /? , coli enterotoxinboth invitro(Mi tchel l and Kenw orth 197 6) and invivo (Fos ter e t a l . 198 0).

1.3.5.2Stimulation ofimmuneresponse in thedigestivetractof the chick

1.3.5.2.1A via n intest inal imm unityT h G ltract prevents enter ic in fect ions by non- immunologica l and immunologica lm e c h a n i s m s . Intest inal peristals is, that reduc es the in teract ions of pa thoge ns



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diver t i cu lum, P e y e r ' s pa t ches and l ymphocy tes (Bar -S h i ra and F r i edm an 2006) .T h e defence sys tem in the d iges t ivetract of the chick is immature at hatch andthe posthach per iod is o f major immunolog ica l impor tance (Revo l ledo e t a l .2006) . Im media te ly a f ter hatch the c h icks are ex po se d to env i ronme nta lant igens. The in tes t ina l l ymphoides t i ssues deve lop in the first 4 pos thatchday s , though the G A L T that conta ins T l ymphocytes , which prov ide the majorp layers in ce l l -media ted imm une res po nse s, and B lympho cytes , whichsyn thes ise and secrete ant ibod ies (Dav ison 2003) and on ant ibod ies , i sobserved on ly in the second posthatch week and maturat ion occurs la ter .Th erefore , the hatch ing ch ic ks are suscep t ib le to pathogen s imm edia te lypos tha tch (S k l an 2005) . S ubseq uen t l y , G A L T p lays an important role on thehost defence aga ins t pathogens and on the pro tec t ion o f the mucous



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a nd sub-mucosa (Madara 1997). Shin et al. (2002) report that one of theLactobacillus strains they studied,Lact. Fermentum Y L - 3 ,managed to attach toc a e c a l epithelial cells adjacent to intracrypts and the pericryptal region ands u g g e s t e d that this may be the cause of a transient translocation of smalln u m b e r s of bacteria via M cells of the P ey e r ' s patches and other G A L Tsurfaces.

Ev i d e n c eis accumulatingthatsuggeststhatprobiotics exert an essential role instimulating the immune system in avian species (Jin et al. 1997). Nahashon eta l. (1996 ) found that Lactobacillus supplementation of layer diets increasedcellularity of P ey e r ' s patches in the ileum indicating a stimulation of the



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{Primalac, S t a r - L a b s / F o r a g e R e s e a r c h , Inc ., C lark sd a le , M O ) on cy tok inelevels and on the local cel l -mediated immuni ty inpoultry w h e n c h a l le n g e dwithEimeria acervulina. An o ther com me rc i a l p robi ot ic (Protexin/Boost) provide d tobroi lers, prom oted s igni f icant ant ibody pro duct ion (Ka bir et a l . 200 4).

1.3.5.3Histological alterationsof the gastrointestinaltractT h e intest ine plays an important ro le in the digest ion and absorpt ion of theingested feed ingred ients (Yamauchi 2007) . The av ian crypt area has few ce l l sat hatch that a re fo rmed comp l e te l y within 48 hours pos thatch and matureswithin 5 days pos tha tch (Kou tsos and A r i as 2006) . Intestinal villi are theextension of the lamina propr ia into the in tes t ina l lumen (Yamauchi 2007) .



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per fo rmance o f t he an i ma l . T hough , the re i s some ev i dence that probiot icsimprove the in tes t ina l m uc os a deve lopm ent . P e l i c an o e t a l . (2005) found thatbirds fed Bacillus subtilis-hased probiot ic sh ow ed s igni f icant ly (P


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T a b l e2.P e r f o r m a n c eofprobiotico rgan i sms u s e dinc h i c k e nproductionReference Organism used Effectsin performance (difference fromcontrol)Kabiretal.(2004) Protexin/Boost 2mg/10L''drinking 15% significantly(p< 0.01)higher liveweightgainsGilde los

santosetal.(2005)

Saccharomyces boulardii orBaciiius cereusvar. toyoiisupplemented in feed

60 and 75% significantly(p< 0.01)higher liveweightgains and improvedfeed efficiency by10and 12% forSac.Bouiardii andBac.cereus, respecteively

Khaksefidi andGhoorchi(2006)

50 mg kg"' Baciiius subtilissupplemented in feed

4 and6%significantly(p< 0.01)higherliveweightgains and improved feedconversionby6%for1-21and22-42days, respectively

Timmemnanetal.(2006)

Amultispecies (i\/lSPB-differentprobiotic species of human origin)andachicken-specific( C S P B -7Lactobacillus speciesisolatedfrom chickens) probioticpreparation, 10-10^ CFU mf indrinkingwater

MSPBtreatmentresulted inaslightincrease(by1.84%)in broiler productivity basedonanindex takinginto account dailyweightgain, feedefficiency, andmortality

O'Deaetal.(2006)

Pr1:Lact acidophilus, Lact.bifidus,andStreptococcus faecaiis,indrinkingwateror spray, Pr2: Lact.acidophiius,E.faecalis, and bifidobacteriain

6,6.5and7%significantly(p< 0.01)higher liveweightgainsforPri inwater,Pr inspray and Pr2 in feed,respectively,atday36to42



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gain and feed c onv ers ion we re s imi lar between the prob io t ics a nd the pos i t ivecont ro l(with antimicrobial) both in the ini t ial and the total per iods .

The e f fec ts on per formance o f ch ickens prov ided prob io t i cs , repor ted in thel ite ra ture , are not c onc lus ive . S ix o f the sev en pa pers rev iew ed, as wel l as thesys temat i c rev iew and meta-ana lys is o f 27 Braz i l ian s tud ies (Far ia F i lho e t a l .2006) have repor ted increased body weight ga in and feed in take o f ch ickensprov idedwith p rob io t i cs e i ther by feed (Gi l de los santo s e t a l . 20 05 ; Kh aks ef id i

a n d G h o o r c h i 2 0 0 6 ; O ' D e a e t a l . 2006; Opal insk i e t a l . 2007; Apata 2008) ordr ink ing w ater (K abi r et a i . 20 04 ; T imm erm an et a l . 200 4) , dur ing e i ther thewh ole p er iod of s tudy or a part of it. H ow eve r, O pa l ins ki et a l . (2007) reported



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E u r o p e w as Sa/mone/Za-posi ti ve (Eu ropea n Fo od Safe ty Author ity 200 7) .H ow ever , Salm. enteritidis a n d Salm. Typhimurium, which are the two mostcom mo nly reported S a lm on el la types in human c a s e s in the E U , we re identi fiedin only 40% of Salmonella posi t ive f loc ks.

Salmonella se ro types that colonise a part icular host s p e c i e s , such as S .gallinarium in poultry, are ca l led hos t-restr ic ted. Salmonella se ro types , suc h asSalm. enteritidis an d Salm. T yph i mur i um that a re assoc i a tedwith a broader

range of unre la ted host sp ec ies and ca n induce sys tem ic d ise ase in them , arereferred to as unrestr ic ted or broad -rang e sero type s (Uz za u et a l . 20 00 ).



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the intest inal epi thel ium, contacts the lamina propr ia, where i t can mul t ip ly , orenter into d e e p e r t i ssues , an d af ter re ac hing the bloo d strea m , it infects internalo rgans su c h as the l iver an d sp lee n (Van He mer t e t a l . 200 7) .

Invasion of epi thel ia l cel ls by Salmonella pla ys a cri t ical role in Salmonellap a t h o g e n e s i s . Ac co rd i ng to Z h ou and G a l an (2001), upon con tac twith intest inalepi thel ia l cel ls . Salmonella enterica serovar spp. injects a set of bacterialproteins into host cel ls v ia the b acte r ia l pathogen ic i ty Is land 1 (S PI-1 ) type III

sec re t i on sys tem. Salmonella us es the type III sec ret ion sy ste m , wh ich is aneedle- l i l


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1.4.2.2Effectiveness ofprobiotics againstSalmonellaAl though severa l mechanisms by which prob io t i c bacter ia cou ld inhibit theco lon isat ion o f invaded organ isms have been prop ose d, the exact me ch anisminvolved in the exclusion of Salmonella in ch icks has to be eluc idate d.Compet i t ion for receptor s i tes of the caecal epi thel ium, antagonist ic act iv i tythrough product ion of bacter iocins, product ion of volat i le fatty ac ids by obl igatea n a e r o b e s in the caecum have been suggested (Vanbel le e t a l . .1990; J ih e t a l .1997; Gh ad ba n 2002 ; Na va et a l . 200 5 ; Revo l ledo et a l . 2006) . Va n Immersee let a l . (2006b) reviewed the use of organic acids to combat Salmonella inpoul t ry . The ant ibacter ia l capaci ty of medium-chain fatty a c ids (C 6 to C I 2 ,capro ic , capry l ic , capr ic and laur ic acid) against Salmonella is gre ater thanshor t -cha infatty ac ids ( formic, acet ic, propionic and butyr ic acid) .

Chap ter 1 Literature review


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(2003)Studiedthe e f fec t o f a com m erc ia l compe t it ive e xc lu s ion product B ro i lac fon the co lon izat ion o f S .Infantis in day-old pheasant chicl


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vacc i na ted a t tlie l i a t chery and g i ven Av i an P ac P lus fo r tiiefirst 3 days af terplacement, as wel l as at 10 and 14 days, 2 days pr ior to vaccinat ion and 2 dayspostvacc inat ion. S ix hours a f ter p lacement , the ch icks were cha l lenged with 10^C F U ml'"' Salm. enteritidis. T he probiot ic- t reated group ha d a s igni f icant ly lowe rconcent ra t ion o f Salm. enteritidis c a e c a l colonizat ion at days 3, 7, 10, 17, 24,3 1 , 3 8 , and 41 w hen comparedwith the non-treated, control group (P < 0.05).S imi lar ly , there was a s igni f icant d i f ference (P < 0.05) in the f requency ofisolat ion of Salm. enteritidis f rom the internal organs ( l iver and spleen) whenprob io t ic - t reated and nonprob io t ic - t reated groups were co m par ed. T he resu ltsobta ined in T e l lez and c o-worke r ' s s tudy are in agreem ent with those f romP r o m s o p o n e e t a l . (1998) s tudy . C h ic ke ns were ad min is tered Avian Pac Plus byspra y at the ha tchery, in the wa ter for two da ys and were c ha l len ge d, s ix ho urs



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S e v e r a l rese arch ers we re u nable to dem onst ra te the abi li ty o f mo nog ene r icpreparat ions to protectpoultry aga i ns t Salivonella infect ion (Stav r ic and D'aoust1993) . L a Ra g i one et a l . (2004) sho w edthat a s ingle oral do se of 10^ C F U ml'""Lact. Johnsonii F1 97 58 w as not suf fi c ient to supp res s Salm. enteritidis.H o w e v e r , there are some o lder s tud ies , and some very promis ing recent ones,in whic h com pet i t ive iso lates for reduc t ion of Salmonella car r iage in ch icke nshave b een se lec ted . So er jad i e t a l . (1981) show edthat nat ive avian lactobaci l l ir e d u c e d the number o f sa lmonel lae adher ing to the crop mucosa o f ch ickens by1 to 2 log uni ts . However, t reatments with lactobaci l l i d id not lower the numbero f ch i cke ns shedd i ng sa l mon e l l ae , o r reduce the numb er o f sa l mone l l aeadher ing to the mu co sa o f the c ae cu m . W atk ins and M i lle r (1983) treated atotalo f 205 ch i ckswithLact. acidophilus an d found a s igni f ican t (P


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laboratory studies. However, in commercial product ion i t is not pract ical andprobiot ics are usu al ly adm inistered ei ther in the drinl


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W hen L A B a re suspend ed in w a te r the ph enomen on o f osmo s i s is observed(Garbut t 1997; Tor tora e t a l . 1998; Adams and M o s s 2003). The di f ference inthe mineral concentrat ion ins ide and outs ide the cel l resul ts in a net movementof water through ce l l wa l l , wh ich is semi -permeable , and c a u s e s the disruptionof the cel l .

Chlor inat ion of water is a common pract ice to avoid t ransmission of harmfulbacter ia through water consumpt ion. When chlor ine is used as a dis infectant inpiped distr ibution sy st em , a free c hlorin e res idua l of 0.2-0 .5m g L""* throug hout isdesi rable to reduce the r isk of microbial growth (World Heal th Organizat ion1997). Chlor inat ion u sing an in- line proport ioner (a de vice for a cc urately


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2007) h ave d es cr ib ed , th is i s s tored in atanl


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F L F has been demonstrated to be ef fect ive against several food-bornepathogens (Brooks 2008). In order to reduce Salmonella and En terobacter iaca enum bers in F L F , a conc entrat ion of aroun d 10^ C F U g'"* L A B and m ore than 150mm ol of lact ic acid and a p H be low 4.5 is ne ce ssa ry. A s far as pignutrition isc o n c e r n e d a low concentrat ion of acet ic acid (


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F ermented feed has h igh num bers o f lac t ic ac id bacter ia , so m e of which mighthave potent ia l p rob io ti c proper t ies . S po nta ne ou s fermentat ion o f LF has bee nc o n s i d e r e d as unrel iable and fa i led to give consistent good resul ts .B a c k s l o p p i n g is usual ly pract iced in p igger ies. This is the pract ice ofmaintain ing a cont inuous fermentat ion by retain ing a proport ion of a previouss uc c es s f u l fermentat ion and using th is as an inoculum for the next batch.Ho we v e r , th is has proved to be r isky, as yeasts eventual ly dominate theresident microf lora and negatively affect palatabi l i ty and the nutri t ional value ofthe feed. In addi t ion, S F L P often fai ls to generate suff icient lact ic a c i d . Inc onc l us i on , p roduct ion o f IFLF us ing carefu l l y se lec ted inocu lum organ ismsthatrapidly produce high concentrat ion of lact ic acid is desi rable. I t is advantageousi f the selected stra in also has probiot ic propert ies such as being to lerant of the

Ch apter 1 Literature review


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s teep ing t ime and enzyme supplementat ion on t ine per formance o f weaner p igsfound that a liquid : fee d rat io of 2 : 1 and 3: 1 ha d s imi lar d igest ib le e nerg y( DE) con tent to the control d iet , thoug h the 4 : 1 d iet had s igni f icant ly lowe r D E .T h e y c o n c l u d e d that grain pro ce ss ing had a greater ef fect on nutrientavai labi l i ty than the water to feed rat io. Brooks (2008) reviewed the ef fect ofs teep ing in l iqu id feed ing sys tems and suggested that s teep ing feed for 8-16hact iva tes the phytase that occurs natural ly in the per icarp of some grains andalso inc rea ses the b ioava i lab i li ty o f nutr ients su ch as ph os ph om s, ca lc ium ,magnes ium and copper in the gra in , and subsequent ly a l lows the d ie taryinclusion o f exog eno us minera ls to be lowered. Ch oc t (2004) repor tedthat in hisexper iment s teep ing may have ac t iva ted the natura l l y occur r ing p-g lucanaseand xy la na se , ca us in g part ia l p o lymer isat ion o f the N S P s , and conseq uen t l y



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early a c c e s s to semi -mois t d ie ts for day o ld ch icks can s t imula te thedevelopment of thei r gastrointest inal t ract and prevents them from dehydrat iondur ing t ransport . C h i c k s are often left wi thout any feed or water for an extendedper iod of t ime dur ing t ransportat ion f rom the hatchery to the farm. The ear lyprovis ion of energy and water in the first 48h af ter hatching resul ted to chickswith s igni f icant ly (P


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F e e d i wa t e rratio o f F L F prov ided to feed range s betwe en 1 :2 an d 1 :4 (P lum ed -Ferrer a nd Vo n W r igh t 20 09) . H eres et a l . (2002 ; 20 03 a ; 2003 b ; 2003 c ; 2004 ;2004b) sugges t a feed :w a te r ratio of 1:1.4 of the ferm ente d feed provided toc h i c k e n s , which could be considered as moist rather than l iquid.

F e e d i n g F L F has a l so been co r re la tedwith a lower preva lence o f Salmonella inc h i c k e n husb and ry . A redu ced probabil it y o f Salmonella co lonisa t ion inc h i c k e n s fe d F L F . was reported by Heres (2004). In h is study, 14 out of 18 pensof ch ick ens fed dry feed , but on ly 4 out o f 18 pen s o f ch icke ns fed F L F , werefound to be Salmonella posi t ive fo l lowing inocu lat ionwith a dose of 10 C F U ml '

Salm. enteritidis. T he pro tec t ive e f fec t o f FL F aga ins t Salmonella w a s a l so



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conve nt iona l dry fee d. Ho wev er , there wa s no s ign i fi cant reduct ion o fSalmonella in the low er intest inaltract (Heres e t a l . 2003c) . Th is may have beenb e c a u s e the lactob aci l l i prese nt in the sp ec i f ic F L F did not have probiot icpropert ies against Salmonella.

I n some s tud i es , L F , N F L F o r F L F , have been reported to improve feed intakea n d protein digest ib i l i ty (Brooks 2008). C h a h et a l . (1975) reported s igni f icant ly(P


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c o n v e r s i o n by 6 .3% (P


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products of lower cost . Modi fy ing raw mater ia ls pr ior to feeding through s implesteeping in water for a suf f ic ient per iod of t ime can act ivate natural ly occurr inge n z y m e s , which produces a possibi l i ty of improving the feed ut i l isat ionef f i c iency . For example Yamomoto e t a l . (2007) used a t rad i t iona l J a p a n e s ef eed , ca l led Ko j i - feedthat i s p roduced by w hea t b ran a n d S h o c h u d is ti ll ery by product , fermented with Aspergillus awamori, to examine i ts ef fect on thegrowth p erform anc e and nutr ient avai labi l ity in broi lers. R es ul ts sh ow ed thatbro ile rs fed the F L F had an improved per forman ce (0 .05% , P


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wal l and to the al tered microbial populat ion.

1.6 Selectioncriteria fo r probioticsM a n y researchers have been work ing on se lec t ing prob io t i c s t ra ins froma n i m a l s (O 'Su l l i van 20 01 ; M ojgan i e t a l . 2007) a nd p lants (C h iu et a l . 2008 ) foran ima l use , or hum ans use (J ac ob se n e t a l . 1999) . S e lec t ion cr ite ria u s e d , forpotent ia l probiot ics for anim al use in order to ac hiev e we l l esta bl ish ed andposit ive probiot ic effects vary in the l i terature. E d e n s (2003) suggested anumber of desi rable probiot ic character is t ics and funct ions for potent ia lprobiot ics (Table 3), co nsid er in g safety for the host an d the cap ac i ty of the

Chapter 1 Literature reviewC o l l ado e t a l . 200 7) an d 4) ant imicrobia l act iv i ty a gains t potent ia lly path oge nic


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bac te r ia (Tsa i et a l . 20 05; O lnood e t a l . 2007) .

European regu la t ion about feed add i t i ves , regards Nurmis and Ranta la (1973)proposed the pract ice of dosing chicl


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stated that the ileum conta ins a much larger population of bacter iathan otherpartsof the sma l lintestine.

T a b l e3.Des i rab le Prob io t i c Charac te r i s ti csand Func t ions

Desirable Probiotic Characteristics: Normal inhabitant of the host or host adapted Non-toxic and non pathogenic Accurate taxonomic identification Tolerateprocessing,storage and delivery Capable ofsurvival,proliferation and metabolic activity in thetargetsite, which implies:

o Resistance to gastric acids and bile saltso Readily bind to epithelium and mucuso Persistent viability in the gastrointestinaltracto Ability to competewiththe resident flora

Produce inhibitorysubstancesagainst other bacteria Alter microbial activity Modulate immune responses Actively competes for receptor sites

Chapter 1 Literature review1.6.1 Aggregation experiments


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Autoag gregat ion o f lac t i c ac id bacter ia may be ne ce ssa ry for adh es ion tointestinal epithel ial cel ls and with the addi t ion of thei r potent ia l coaggegat ionabi l i ty they may form a barrier that prevents colonizat ion of pathogenicm icroo rgan isms ( Ko s et a l . 200 3) by providing a com pet i t ive adv anta ge in thein tes tina l microb io ta (G had ban 2002) . Sp en ce r and C h e s s o n (1994) suggestedthat coag gregat ion between lac t i c ac id bacter ia and enteropathogens has adi rect ef fect in exc luding the pa thoge nic b acter ia f rom the gastrointest inal t ract.

There are few studies analys ing the aggregat ion abi l i ty of lact ic acid bacter iawith pathogenic bac ter ia and com par ing d if fe rent method olog ies to me asure

Chapter 1 Literature reviewA s impler , sa fer and more cos t e f fec t i ve method o f measur ing bacter ia l


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aggregat ion suggested by severa l authors (Kmet and Lucch in i 1997; Kmet andLu c c h i n i 19 99), previous ly us ed suc ce ssfu l ly in the labora tor ies of the U nivers i tyo f P lymouth (D em ec kov a 200 3) m ea sur es the bac ter ia l aggrega t ion in tens ity inrelat ion to the time needed for the bacter ia to form aggrega tes that can beo b s e r v e d to thebottom o f the tubes. Sc an ning e lec t ron m icros cop y (Drago e t a l .1997) can be also used to conf i rm the bacter ia l aggregat ion.

1.6.2 AntagonisticactivityT here a re many in vitro methods for measur ing the antagonist ic act ion ofprob io t i c microorganisms aga ins t pathogenic bacter ia . For example, c r i t i ca l

Chapter 1 Literature review1.6.3 Acidic pl-l andbile saltresistance


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The v iabi l i ty of the potent ia l probiot ic s t ra ins under the physical and chemicals t ress ing condit ions of acid and bi le salt in the Gl tract was cons idered byG i b s o n et a l (2000) as one of the desi rable character is t ics for probiot ics toprovide heal th benef i ts . The fact that the avian gastrointest inal tract is shorterthan the mammal ian one and that the feed requires only 2.5 hours to passthrough the Gl t ract , suggests that the use of res istance to low pH as aselec t ion cr i ter ion for potent ia l probiot ics for av ian sp ec ies is not as cru cial asfor mammal ian s p e c i e s . O n the o ther han d, the pH of the ch icken G ltract cou ldbe as ac id ic as 2, which m ay be an o bsta cle for the su rv ival of lactob aci l l i .

Chapter 1 Literature review(Larb ier and Lec lercq 1994) . B i le secre tedinto the d uod ena l sect ion o f the sm al l


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in test ine h as bee n foun d to reduc e the surv ival of bacte r ia. T his is bel iev ed tobe due to the cel l wal ls of the bacter ia consist ing of l ip ids and fatty a c i ds , w h i chare broken d own by the b i le sa l ts (J in e t a l . 1998 a) . S eve ra l rese arc hers suc ha s F loch e t a l . (1972) , Gi l l i l and and Speck (1977) , Tannock e t a l . (1989)c o n s i d e r e d re s ista nc e to low p H and bi le sal ts a s cr iter ia for selec t ing potent ia lprobiot ics and l inked i t to the abi l i ty of lactobaci l l i part icularly to de-conjugateb ile ac ids .

R e c e n t l y , severa l researchers (L i ong and S hah 2005 ; Mo jgan i e t a l . 2007)s u g g e s t that taurocho l i c a c i d , which is conjugated bi le, is more inhibi tory than

Chapter 1 Literature reviewthe same t ime. Fu l le r (1973; 1989) and J in e t a l . (1997) suggested that


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lactob aci l l i co lon isin g the cro p ca n eff iciently con trol the Esch. coli populat ion.L ight and e lec t ron microscopy revea led that the adherent bacter ia of theintest inal microf lora are connected by f ibres to the mucosal surface (J in et a l .1996b).

A l though adhes ion is often considered as a select ion cr i ter ion for potent ia lprobiot ics, there is no c lear evidence of the importance of adherence of lact icac id bacter ia as far as the colonisat ion to the Gltract i s concerned . C h a n et al.(1985), Bruce et a l . (1988) and R e i d et a l . (1985; 1987) have been the pioneersin demonstrat ing by in vitro an d in vivo s tud ies that severa l se lec ted

Chapter 1 Literature reviewgenera l l y accep tedthat adhesion propert ies also contr ibute to the modulat ion of


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the immune sys tem (Sch i f f r in e t a l . 1997; Sa lminen e t a l . 1998) and thetherapeut i c management o f the enter i c microb io ta in par t i cu lar (Co l lado e t a l .2 0 0 5 a ) , and the e f f i cacy o f prob io t i c s t ra ins in genera l (Serv in and Coconnier2003) .

Th ere are seve ra l wa ys o f s tudy ing the a dhe s ion o f LA B exper imenta l l y . Invivos tud ies are genera l l y co ns id ere d to be the mos t re l iab le . Howe ver , thecomplexi ty of the gastrointest inal tract and i ts microf lora mal


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sho we d negl ig ib le adhe sio n of th is part icular s t ra in to lE C .

Ano ther d i sadvan tage o f in vivo s tudies is that they are time consumi ng ande x p e n s i v e . T he com pl ica t ions invo lved in s tudy ing bacter ia l adh es ion in vivo,have led to the development of in vitromodel sy s tem s for the initial select ion ofpotent ia l ly adhe rent s t ra ins (C ol lado et a l . 20 05 a). S a v a g e (1983) examinedbacter ia l adhesion using isolated epi thel ia l surface intact in l iv ing animals andwith mucosa l ce l l s or t i ssue removed from an imals and main ta ined in vitro.Recent l y , various permanent intest inal cel l l ines l ike C a c o - 2 a nd H T - 2 9 , isolatedext race llu lar matr ix (E C M ) pro te ins , Basem ent M em bran e M at r ige l (B M M )

Chapter 1 Literature review1.6.5 Hydrophobicity


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T h e sm al l sur fac e are a o f bacter ia ma ke s shor t range in terac t ions pos s ib le . T heapproach o f two hydrophobic sur faces leads to the d is locat ion o f waterfrom thes y s t e m , benef i t ing the interact ions between the surfaces (Der jaguin, L andau ,averw ey, Ov erb eek (D LV O ) Th eory) . S o , whe n a potentia l p rob io t ic i s se le c te d,hydrophobic i ty shou ld be taken into con s iderat ion. T he mutua l a t trac t ionbetween the probiot ic bacter ium and the epi thel ia l cel l prevents i ts removal bythe normal intest inal f low.

T h e h igher af f in i t ies of lactobaci i l i for nonpolar solvents, such as xy lene,s u g g e s t that these bacter ia p o s s e s s a hydrophobic rather than hydrophi l ic

Chapter 1 Literature review( R o s e n b e r g et a l . 1980) is considerd as a s imple, re l iable, economical and wel l


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es tab l i shed as sa y us ed for s tudying the hydro pho bic interact ions of ce l ls(Gusi ls e t a l . 2002a ) .

1.6.6 Homo/heterofermentation testLac t i c ac id bacter ia are separated into two groups: the homofermentat ive L A B ,which convert sugars ent i re ly , or a lmost ent i re ly , to lact ic a c i d , andhetero fermentat ive L A B , which produce more than one major end product sucha s lact ic a c i d , acet i c a c i d , e tha nol , manni to l and C O2 f rom he xos es (M ul ler1990). Hom oferm entat ion is important if L A B are to be used to ferme nt fee d. Ith a s been sugges ted that hom oferme ntat ion rather than heterofermentat ion

Chapter 1 Literature reviewthe source mater ia lfrom donor b i rds , andthat the b acter ia l com pos i t ion and the


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ef f i cacy cou ld not be s tandard ised, that made the deve lopment o f prob io t i cproducts o f known bacter ia l compos i t ion a demand (S tavr i c and D 'aoust 1993) .

O n the o ther hand deve lop ing prob io t i cs based on def ined cu l tures isproblem at ic too. F i rst of a i l , there is s t il l lac k of a soun d scien t i fic ba sis for these lec t ion pro ce ss o f potent ia l p rob io t ic s t ra ins . W et m icrob io logy me thods haveproved to be inadequate and unre l iab le and on the o ther hand molecu larmethods are cos t ly . Add i t iona l ly , there is ev idencethat probiot ic mixed cul turespotency de cr ea se dur ing co ld s torage and repeated laboratory manipu la t ion(S tavr ic and D 'aoust 1993) . R e l iab le d iagno st i c sc he m es for identi fi ca tion o f

Chapter 1 Literature reviewof probiot ics intiie dr ink ing water does not guarantee the intake of an adequate


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inoculum by al l b i rds. T he m ethod of adm inistrat ion by ferme nted l iquid feed toc h i c k e n s is s t il l develop ing a nd is fac ed with d iscred i t . European Unionlegis lat ion controls the technology of fermented l iquid feed on commercial uni ts ,as o rg an ism s use d for fermentat ion have to be reg is tered. Th is might be a riskfor co nsu m ers and producers to benef it from the po tential po sit ive effects ofusing lact ic acid bacter ia and fermented feed (Brooks et a l . 2003b).

L iqu id feed ing ra ises one more prob lem, as the wet feed s tays in the trough fora long t ime, spo i lage bacter ia and molds can accumulate , wh ich i f consumedmight be r isky for animal heal th. Addi t ion of >300 ppm of chlor ine dioxide, a


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Cha pter 1 Literature review to dete rmin e the in vivo effect of tiie s e l e c t e d Lactobacillus s t ra in on


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microb ia l , immunolog ica l and h is to log ica l parameters in the ch i cken .

Chapter 2 Screening of LA B ofchicl


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Selection, characterisation and screening oflacticacidbacteriaof chickenoriginthathaveprobioticproperties2.1 IntroductionM a n y researchers have a t tempted to se lec t prob io t i c s t ra ins f rom an imals(O 'Su l l i van 2 00 1; Mojgan i e t a l . 200 7) and p lants (C h iu e t a l . 2008 ) for u se inan ima ls , o r hum ans ( Ja co bse n e t a l . 1999) . S e l ec t i on and sc re en i ng o fLactobacillus s t ra ins is w id es pre ad , as they are co m mo nly known for the i rprob io t i c proper t ies (Nemcova 1997; Garr iga e t a l . 1998; Gus i l s e t a l . 1999a;Gusi l s e t a l . 1999b ; E h rman n et a l . 200 2 ; G us i l s e t a l . 2002a ; Gus i l s et al .

Chap ter 2 Screening of LA B ofchicl


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f inding a potential bacterial strain that i s non- tox ic , non pathogenic and o fc h i c k e n o r ig in , to ensure co lon isat ion o f the ch icken Gl tract. A l though severa lauthors (Hautefort and H i n ton 2000 ; Bezkorova i ny 2001) d i d no t cons i de rs pec i es and locat ion speci f ic i ty an essent ia l select ion character is t ic , v iabi l i tyand biological act iv i ty within t he ac i d i c ch i cken G l tract was inc luded as ase lec t ion cr i te r ia in the present scre en in g progra mm e. A dh es ion to m uc us andepi thel ia l ce l ls w a s co ns ide red a prerequis i te for the se lect io n of a probiot icstrain.

In vitro me thods are co m mo nly us ed for scre en ing potentia l p rob io t ic s t ra insb e c a u s e they are inexpens ive and more rap id than in vivo me thods . H ow e ver ,

Chapter 2 Screening ofU\B ofchiclien origin

Sampling


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Isolationof lactobacilli

Gram staining and catalasetest

-0-Homo/heterofermentation

AggregationtestAutoaggregationCoaggregationwithpathogens

Identificationof strainsAPICHL kit16SrDNA sequencing

Chapter 2 Screening of LA B of chicl


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sc reen i ng p r o c e s sis shown inT a b l e4.

T ab l e 4. The number of strains subjected to e a c h test during the s c reen i ngp r o c e s sand the number of isolates p r o c e s s e dto the next s tage.Procedure Strains Organismsto thenextstageIsolationof lactobacilli 111 111Gram staining 111 109 positive rodsCatalasetest 111 111

Fridge damage - 58survived and had goodgrowth

Autoaggregationtest 58 25 good and nonnal activityCoaggregationtestwithpathogens 25

19 good aggregationwithpathogens

Identification(API CHL kit) 19 2 representative from eachspecies,8 intotalAntagonistic activity 8 (Nos 3. 6. 10,15,16.18,22,24) All

8 (Nos 3, 6, 10,15,16,18,22,

Chapter 2 Screening of M S ofchicl


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remo ved asep t i ca l l y , and sm al l in tes tine an d crop ep i the l ia l ce l l s were taken bys c r a p i n g the epi thel ium with a s l ide . A i l sa m ple s we re di luted in 10 miphospha te -bu ffe red sa l i ne (P B S ) (O x o i d , E n g l and) and p l ated in de M anR o g o s a a n d S h a r p e ( M R S , O x o i d , E n g l a n d ) a n d R o g o s a (O xo i d , E ng l and)agar. The streak plate method of isolat ion was used to obtain pure cul tures f romthe mixed cul ture of L A B . A f ter se lec t ion , the pure co lon ies we re subc u l tured fora 2"^ t ime o n M R S aga r p la tes and incubated in an aero b ic ja rs ( to prevent anyoxygen f rom comi ng into con tac twith samples of cul tures) at 37 C, in 5% CO2

atmosphere , for 72 h.

Chapter 2 Screening of LAB of chici


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production ofselectedlactobacilliO n e hun dred a nd e leve n lac t i c ac id bacter ia were iso la ted on M R S ( iso la tionmed i um fo r Lactobacillus a n d Pediococcus s t ra ins) an d R o g o s a agar( lactobaci l l i ) and maintained at -80 C in 1ml a liquots of M R S broth (Ox o id ,En g l a n d )with 20 % v /v g lyce ro l . S tock cu l tures we re react iva ted by subc u l ture inM R S broth and incubation at 37 C, for 24 h, before theirfurther exper imenta lus e . Lactobaci l l i cul tures were maintained in a f r idge (4 C) and weresubc u l tured on M R S ag ar p la tes onc e every we ek to re ta in v iab il it y , main ta in ingthem in the f ridge ag a in . T he gra m s ta in and the ca ta lase tes t were us ed toconf i rm that the orga n ism s we re lac tobac i l li (g ram pos i ti ve and cata lasenegat ive rods) . T he pre sen ce o f the cata lase en zym e wa s detec ted us ing

Chap ter 2 Screening of LA B ofchicl


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T h e remain ingfifty e ight lactobaci l l i were tested for aggregat ing abi l i ty , and 25that showed good and normal aggregat ing ab i l i t y were tes ted for coaggregat ingabil ity with p a t h o g e n s . Salmonella enterica enteritidis ( N C T C 5188) obta inedfrom t he Na t i ona l Co l l ec t i on o f T ype C u l tu res , Ce n t ra l Pub l i c Heal th Laboratory ,a n d Clostridium perfringens ( N C I B M 8693) obta ined from the Univers i ty ofP lymou th s tock c u l tures , were use d in the coag gregat ion tes ts .

Prior to u se , the stra ins were su bc ul ture d twice innutrient broth at 37 C, for 24hours and C los t r id ia lbroth (Oxo id , UK) a t 37 C for 24 hours , under anaerob iccondi t ions , resp ect ive ly .


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Ch apter 2 Screening ofU\Bof chiciien originpart icles, (formed bytiie aggregated cel ls) gravi tated to the bot tom of the tubes,


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leaving a c lear supernatant f lu id,within 2h. The react ion t ime was recorded for

the mea sure m ent of agg rega t ion intensi ty : and c lass i f ied as maxim um (4) 90min (Kmet and Lucch in i 1997; Kmet and Lucch in i 1999) .

Coaggrega t i on w as a l so a s s e s s e d us ing the method of Drago et . a l . (1997).Salmonella spp., a n d Esch. coli were grown at 37 C innutrient and clostr idialbro th respect ive ly , fo r 24h , in 5% C O2 atmo sphere , and under ana erob iccondi t ions, respect ively, and Lactobacilli were grown at 37 C , in M R S broth, for2 4 h , in 5% C O2 atm osph ere. 500p l o f each Lactobacillus susp ens i on w as mi xed

Chap ter 2 Screening of M S ofchicl


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fo r obse rvat ion by sc an nin g e lec t ron mic ros co pe, as fo l lows. T en p i of ea ch

Lactobacillus su sp en s ion we re p laced onto a s l ide (The rman ox, Nu nc , Inc) , a i rdr ied, f ixedwith 2 .5% g lu tara ldeyde for 2h a t room temperature , washed threet imes in 0 .1 % (w/v) sod ium c aco dy la te buf fer pH 7. S am ple s we re dehyd ratedthrough a graded ser ies o f e thanol and acetone mix tures , c r i t i ca l point dried at3 5 C an d 125 0ps i fo r 15m in, coa tedwith go l d and observed by S E M , underh i gh vacuumwith a J E O L 5 6 0 0 L o w V a c u u m S E M . S ix randomized f ie lds wereeva l ua ted in e ac h sam p l e .

O f 58 lactobaci l l ithat were tes ted for the i r capac i ty to aggre gate , 23 were found


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Chapter 2 Screening of LAB of chicken originT h e 25 lactobaci l l i that showed normal and rapid aggregat ive act iv i ty werefurther tested for their abi l i ty to aggregate with Salm. enteritidis and CI.


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perfringens. O n e Lactobacillus s t ra in sho we d ma x imum aggregat ion (scored as4)with Salm. enteritidis ,two sh ow ed marl


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niXi3om-1Origin D)O)a2

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Selection of a Chicken Lactobacillus Strain With Probiotic Properties and Its Application in Poultry Production